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• 1.1: Introduction - Basic Biology The most obvious thing about living organisms is their astounding diversity. Estimates put the number of eukaryotic species at about 8.7 million, while bacteria account for anywhere between 107 and 109 different species. The number of species of archaea is still uncertain, but is expected to be very large. These organisms, representing the three great domains of life, together occupy every environmental niche imaginable. • 1.2: Introduction - Basic Chemistry To understand biochemistry, one must possess at least a basic understanding of organic and general chemistry. In this brief section, we will provide a rapid review of the simple concepts necessary to understand cellular chemistry. Chemistry is chemistry, whether in a cell or outside it, but biological chemistry is a particular subset of organic chemistry that often involves enormous macromolecules, and that happens in the aqueous environment of the cell. • 1.3: Introduction - Water and Buffers When it comes to water, we’re literally drowning in it, as water is by far the most abundant component of every cell. To understand life, we begin the discussion with the basics of water, because everything that happens in cells, even reactions buried deep inside enzymes, away from water, is influenced by water’s chemistry. 01: In The Beginning Figure 1.2 Slices of cork as seen by Hooke YouTube Lectures by Kevin HERE & HERE Graphic images in this book were products of the work of several talented students. Links to their Web pages are below Click HERE for Martha Baker’s Web Page Click HERE for Pehr Jacobson’s Web Page Click HERE for Aleia Kim’s Web Page Click HERE for Penelope Irving’s Web Page Problem set related to this section HERE Point by Point summary of this section HERE To get a certificate for mastering this section of the book, click HERE Kevin Ahern’s free iTunes U Courses - Basic / Med School / Advanced Biochemistry Free & Easy (our other book) HERE / Facebook Page Kevin and Indira’s Guide to Getting into Medical School - iTunes U Course / Book To see Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 To register for Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 Biochemistry Free For All Facebook Page (please like us) Kevin Ahern’s Web Page / Facebook Page / Taralyn Tan’s Web Page Kevin Ahern’s free downloads HERE OSU’s Biochemistry/Biophysics program HERE OSU’s College of Science HERE Oregon State University HERE Email Kevin Ahern / Indira Rajagopal / Taralyn Tan 1.02: Introduction - Basic Chemistry Source: BiochemFFA_1_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy “Organic chemistry is the chemistry of carbon compounds. Biochemistry is the chemistry of carbon compounds that crawl” -Michael Adams. To understand biochemistry, one must possess at least a basic understanding of organic and general chemistry. In this brief section, we will provide a rapid review of the simple concepts necessary to understand cellular chemistry. Chemistry is chemistry, whether in a cell or outside it, but biological chemistry is a particular subset of organic chemistry that often involves enormous macromolecules, and that happens in the aqueous environment of the cell. Covalent bonds, as you know, are the result of sharing of electrons between two atoms. Ionic bonds, by contrast, are formed when one atom donates an electron to another, such as in the formation of sodium chloride. Single covalent bonds can rotate freely, but double bonds cannot. Single bonds around a carbon atom are arranged in a tetrahedron with bond angles of 109.5° relative to each other, with the carbon at the center (Figure 1.19). Double bonded carbons create a planar structure with bond angles typically of about 120°. Electronegativity Electronegativity is a measure of the affinity a nucleus has for outer shell electrons (Table 1.2). High electronegativity corresponds to high affinity. Electrons in a covalent bond are held closer to the nucleus with a greater electronegativity compared to a nucleus with lower electronegativity. Table 1.2 Image by Aleia Kim For example, in a molecule of water, with hydrogen covalently bonded to oxygen, the electrons are “pulled” toward the oxygen, which is more electronegative. Because of this, there is a slightly greater negative charge near the oxygen atom of water, compared to the hydrogen (which, correspondingly has a slightly higher positive charge). This unequal charge distribution sets up a dipole, with one side being somewhat negative and the other somewhat positive. Because of this, the molecule is described as polar. Hydrogen bonds between water molecules are the result of the attraction of the partial positive and partial negative charges on different water molecules (Figure 1.20). Hydrogen bonds can also form between hydrogens with a partial positive charge and other strongly electronegative atoms, like nitrogen, with a partial negative charge. It is important to remember that hydrogen bonds are interactions between molecules (or parts of molecules) and are not bonds between atoms, like covalent or ionic bonds. Bonds between hydrogen and carbon do not form significant partial charges because the electronegativities of the two atoms are similar. Consequently, molecules containing many carbon-hydrogen bonds will not form hydrogen bonds and therefore, do not mix well with water. Such molecules are called hydrophobic. Other compounds with the ability to make hydrogen bonds are polar and can dissolve in water. They are called hydrophilic. Molecules possessing both characteristics are called amphiphilic. Weak interactions
Hydrogen bonds are one kind of electrostatic (i.e., based on charge) interaction between dipoles. Other forms of electrostatic interactions that are important in biochemistry include weak interactions between a polar molecule and a transient dipole, or between two temporary dipoles. These temporary dipoles result from the movement of electrons in a molecule. As electrons move around, the place where they are, at a given time, becomes temporarily more negatively charged and could now attract a temporary positive charge on another molecule. Since electrons don’t stay put, these dipoles are very short-lived. Thus, the attraction that depends on these dipoles fluctuates and is very weak. Weak interactions like these are sometimes called van der Waals forces. Many molecular interactions in cells depend on weak interactions. Although the individual hydrogen bonds or other dipole-dipole interactions are weak, because of their large numbers, they can result in quite strong interactions between molecules. Oxidation/reduction Oxidation involves loss of electrons and reduction results in gain of electrons. For every biological oxidation, there is a corresponding reduction - one molecule loses electrons to another molecule. Oxidation reactions tend to release energy and are a source of bioenergy for chemotrophic cells. Ionization Ionization of biomolecules, by contrast does not involve oxidation/reduction. In ionization, a hydrogen ion (H+) leaves behind its electron as it exits (leaving behind a negative charge) or joins a group (adding a positive charge). Biological ionizations typically involve carboxyl groups or amines, though phosphates or sulfates can also be ionized. A carboxyl group can have two ionization states - a charge of -1 corresponds to the carboxyl without its proton and a charge of zero corresponds to the charge of the carboxyl with its proton on. An amine also has two ionization states. A charge of zero corresponds to a nitrogen with three covalent bonds (usually in the form of C-NH2) and a charge of +1 corresponds to a nitrogen making four covalent bonds (usually X-NH3 +). Stereochemistry A carbon has the ability to make four single bonds (forming a tetrahedral structure) and if it bonds to four different chemical groups, their atoms can be arranged around the carbon in two different ways, giving rise to stereochemical “handedness” (Figure 1.21). Each carbon with such a property is referred to as an asymmetric center. The property of handedness only occurs when a carbon has four different groups bonded to it. Enzymes have very specific 3-D structures, so for biological molecules that can exist in different stereoisomeric forms, an enzyme that synthesizes it would make only one of the possible isomers. By contrast, the same molecules made chemically (not using enzymes) end up with equal amounts of both isomers, called a racemic mix. Gibbs free energy The Gibbs free energy calculation allows us to determine whether a reaction will be spontaneous, by taking into consideration two factors, change in enthalpy (ΔH) and change in entropy (ΔS). The free energy content of a system is given by the Gibbs free energy ($G$) and is equal to the enthalpy ($H$) for a process minus the absolute temperature (T) times the entropy (S) $G = H = TS$ For a process, the change in the Gibbs free energy ΔG is given by $ΔG = ΔH - TΔS$ A negative $ΔG$ corresponds to release of free energy. Reactions that release energy are exergonic, whereas those that absorb energy are called endergonic. The biological standard Gibbs free energy change (ΔG°’) corresponds to the ΔG for a process under standard conditions of temperature, pressure, and at pH = 7. For a reaction $aA + bB \rightleftharpoons cC + dD,$ the equilibrium constant, $K_{eq}$ is equal to $K_{eq} = \dfrac{ [C]^c_{eq} [D]^d_{eq}}{[A]^a_{eq} [B]^b_{eq}}$ where $a$, $b$, $c$, and $d$ are integers in the balanced equation. Large values of $K_{eq}$ correspond to favorable reactions (more C and D produced than A and B) and small values of $K_{eq}$ mean the opposite. At equilibrium, $ΔG^{o\prime} = -RT \ln K_{eq}$ If a process has a $ΔG = Z$ and a second process has a $ΔG = Y$, then if the two processes are linked, $ΔG$ and $ΔG^{o \prime}$ values for the overall reaction will be the sum of the individual ΔG and ΔG°’ values. $ΔG_{total} = ΔG_1+ ΔG_2 = Z + Y$ $ΔG^{o \prime }_{total} = ΔG_1^{o \prime}+ ΔG_2^{o\prime}$ Catalysis Catalysis is an increase in the rate of a reaction induced by a substance that is, itself, unchanged by the reaction. Because catalysts remain unchanged at the end of a reaction, a single catalyst molecule can be reused for many reaction cycles. Proteins that catalyze reactions in cells are called enzymes, while ribozymes are RNA molecules that act as catalysts.
Source: BiochemFFA_1_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy When it comes to water, we’re literally drowning in it, as water is by far the most abundant component of every cell. To understand life, we begin the discussion with the basics of water, because everything that happens in cells, even reactions buried deep inside enzymes, away from water, is influenced by water’s chemistry. The water molecule has wide ‘V’ shape (the HO-H angle is 104°) with uneven sharing of electrons between the oxygen and the hydrogen atoms (Figure 1.23). Oxygen, with its higher electronegativity, holds electrons closer to itself than the hydrogens do. The hydrogens, as a result, are described as having a partial positive charge (typically designated as δ+) and the oxygen has a partial negative charge (written as δ- ). Thus, water is a polar molecule because charges are distributed around it unevenly, not symmetrically. Water as a solvent Water (Figure 1.23) is described as a solvent because of its ability to solvate (dissolve) many, but not all, molecules. Molecules that are ionic or polar dissolve readily in water, but non-polar substances dissolve poorly in water, if at all. Oil, for example, which is non-polar, separates from water when mixed with it. On the other hand, sodium chloride, which ionizes, and ethanol, which is polar, are able to form hydrogen bonds, so both dissolve in water. Ethanol’s solubility in water is crucial for brewers, winemakers, and distillers – but for this property, there would be no wine, beer or spirits. As explained in an earlier section, we use the term hydrophilic to describe substances that interact well with water and dissolve in it and the term hydrophobic to refer to materials that are non-polar and do not dissolve in water. Table 1.3 illustrates some polar and non-polar substances. A third term, amphiphilic, refers to compounds that have both properties. Soaps, for example are amphiphilic, containing a long, non-polar aliphatic tail and a head that ionizes. Table 1.3 Image by Aleia Kim Solubility The solubility of materials in water is based in free energy changes, as measured by ΔG. Remember, from chemistry, that H is the enthalpy (heat at constant pressure) and S is entropy. Given this, \[ΔG = ΔH - TΔS\] where T is the temperature in Kelvin. For a process to be favorable, the ΔG for it must be less than zero. From the equation, lowered ΔG values will be favored with decreases in enthalpy and/or increases in entropy. Let us first consider why non-polar materials do not dissolve in water. We could imagine a situation where the process of dissolving involves the “surrounding” of each molecule of the nonpolar solute in water, just like each sodium and each chloride ion gets surrounded by water molecules as salt dissolves. Water organization There is a significant difference, though between surrounding a non-polar molecule with water molecules and surrounding ions (or polar compounds) with water molecules. The difference is that since non-polar molecules don’t really interact with water, the water behaves very differently than it does with ions or molecules that form hydrogen bonds. In fact, around each non-polar molecule, water gets very organized, aligning itself regularly. As any freshman chemistry student probably remembers, entropy is a measure of disorder, so when something becomes ordered, entropy decreases, meaning the ΔS is negative, so the TΔS term in the equation is positive (negative of a negative). Since mixing a non-polar substance with water doesn’t generally have any significant heat component, the ΔG is positive. This means, then, that dissolving a non-polar compound in water is not favorable and does not occur to any significant extent. Further, when the non-polar material associates with itself and not water, then the water molecules are free to mix, without being ordered, resulting in an increase of entropy. Entropy therefore drives the separation of non-polar substances from aqueous solutions. Amphiphilic substances Next, we consider mixing of an amphiphilic substance, such as a soap, with water (Figure 1.24). The sodium ions attached to the fatty acids in soap readily come off in aqueous solution, leaving behind a negatively charged molecule at one end and a non-polar region at the other end. The ionization of the soap causes in an increase in entropy - two particles instead of one. The non-polar portion of the negatively charged soap ion is problematic - if exposed to water, it will cause water to organize and result in a decrease of entropy and a positive ΔG. Since we know fatty acids dissolve in water, there must be something else at play. There is. Just like the non-polar molecules in the first example associated with each other and not water, so too do the non-polar portions of the soap ions associate with each other and exclude water. The result is that the soap ions arrange themselves as micelles (Figure 1.25) with the non-polar portions on the interior of the structure away from water and the polar portions on the outside interacting with water. The interaction of the polar heads with water returns the water to its more disordered state. This increase in disorder, or entropy, drives the formation of micelles. As will be seen in the discussion of the lipid bilayer, the same forces drive glycerophospholipids and sphingolipids to spontaneously form bilayers where the non-polar portions of the molecules interact with each other to exclude water and the polar portions arrange themselves on the outsides of the bilayer (Figure 1.28). Yet another example is seen in the folding of globular proteins in the cytoplasm. Nonpolar amino acids are found in the interior portion of the protein (water excluded). Interaction of the non-polar amino acids turns out to be a driving force for the folding of proteins as they are being made in an aqueous solution. Hydrogen bonds The importance of hydrogen bonds in biochemistry (Figure 1.30) is hard to overstate. Linus Pauling himself said, “ . . . . I believe that as the methods of structural chemistry are further applied to physiological problems it will be found that the significance of the hydrogen bond for physiology is greater than that of any other single structural feature.” In 2011, an IUPAC task group gave an evidence-based definition of hydrogen bonding that states,
“The hydrogen bond is an attractive interaction between a hydrogen atom from a molecule or a molecular fragment X–H in which X is more electronegative than H, and an atom or a group of atoms in the same or a different molecule, in which there is evidence of bond formation.” Partial Charges The difference in electronegativity between hydrogen and the molecule to which it is covalently bound give rise to partial charges as described above. These tiny charges (δ+ and δ- ) result in formation of hydrogen bonds, which occur when the partial positive charge of a hydrogen atom is attracted to the partial negative of another molecule. In water, that means the hydrogen of one water molecule is attracted to the oxygen of another (Figure 1.31). Since water is an asymmetrical molecule, it means also that the charges are asymmetrical. Such an uneven distribution is what makes a dipole. Dipolar molecules are important for interactions with other dipolar molecules and for dissolving ionic substances (Figure 1.32). Hydrogen bonds are not exclusive to water. In fact, they are important forces holding together macromolecules that include proteins and nucleic acids. Hydrogen bonds occur within and between macromolecules. The complementary pairing that occurs between bases in opposite strands of DNA, for example, is based on hydrogen bonds. Each hydrogen bond is relatively weak (compared to a covalent bond, for example - Table 1.4), but collectively they can be quite strong. Table 1.4 Image by Aleia Kim Benefits of weak interactions Their weakness, however, is actually quite beneficial for cells, particularly as regards nucleic acids (Figure 1.33). The strands of DNA, for example, must be separated over short stretches in the processes of replication and the synthesis of RNA. Since only a few base pairs at a time need to be separated, the energy required to do this is small and the enzymes involved in the processes can readily take them apart, as needed. Hydrogen bonds also play roles in binding of substrates to enzymes, catalysis, and protein-protein interaction, as well as other kinds of binding, such as protein-DNA, or antibody-antigen. As noted, hydrogen bonds are weaker than covalent bonds (Table 1.4) and their strength varies form very weak (1-2 kJ/mol) to fairly strong (29 kJ/mol). Hydrogen bonds only occur over relatively short distances (2.2 to 4.0 Å). The farther apart the hydrogen bond distance is, the weaker the bond is. The strength of the bond in kJ/mol represents the amount of heat that must be put into the system to break the bond - the larger the number, the greater the strength of the bond. Hydrogen bonds are readily broken using heat. The boiling of water, for example, requires breaking of H-bonds. When a biological structure, such as a protein or a DNA molecule, is stabilized by hydrogen bonds, breaking those bonds destabilizes the structure and can result in denaturation of the substance - loss of structure. It is partly for this reason that most proteins and all DNAs lose their native, or folded, structures when heated to boiling. Image by Aleia Kim Table 1.5 For DNA molecules, denaturation results in complete separation of the strands from each other. For most proteins, this means loss of their characteristic three-dimensional structure and with it, loss of the function they performed. Though a few proteins can readily reassume their original structure when the solution they are in is cooled, most can’t. This is one of the reasons that we cook our food. Proteins are essential for life, so denaturation of bacterial proteins results in death of any microorganisms contaminating the food. The importance of buffers Water can ionize to a slight extent (10-7 M) to form H+ (proton) and OH- (hydroxide). We measure the proton concentration of a solution with pH, which is the negative log of the proton concentration. pH = -Log[H+] If the proton concentration, [H+]= 10-7 M, then the pH is 7. We could just as easily measure the hydroxide concentration with the pOH by the parallel equation, pOH = -Log[OH- ] In pure water, dissociation of a proton simultaneously creates a hydroxide, so the pOH of pure water is 7, as well. This also means that pH + pOH = 14 Now, because protons and hydroxides can combine to form water, a large amount of one will cause there to be a small amount of the other. Why is this the case? In simple terms, if I dump 0.1 moles of H+ into a pure water solution, the high proton concentration will react with the relatively small amount of hydroxides to create water, thus reducing hydroxide concentration. Similarly, if I dump excess hydroxide (as NaOH, for example) into pure water, the proton concentration falls for the same reason. Acids vs bases Chemists use the term “acid” to refer to a substance which has protons that can dissociate (come off) when dissolved in water. They use the term “base” to refer to a substance that can absorb protons when dissolved in water. Both acids and bases come in strong and weak forms. (Examples of weak acids are shown in Table 1.5.) Strong acids, such as HCl, dissociate completely in water. If we add 0.1 moles (6.02x1022 molecules) of HCl to a solution to make a liter, it will have 0.1 moles of H+ and 0.1 moles of Cl- or 6.02x1022 molecules of each . There will be no remaining HCl when this happens. A strong base like NaOH also dissociates completely into Na+ and OH- . Weak Acids Weak acids and bases differ from their strong counterparts. When you put one mole of acetic acid (HAc) into pure water, only a tiny percentage of the HAc molecules dissociate into H+ and Ac- . Clearly, weak acids are very different from strong acids. Weak bases behave similarly, except that they accept protons, rather than donate them. Since we can view everything as a form of a weak acid, we will not use the term weak base here. Students are often puzzled and expect that [H+] = [A- ] because the dissociation equation shows one of each from HA. This is, in fact, true ONLY when HA is allowed to dissociate in pure water. Usually the HA is placed into solution that has protons and hydroxides to affect things. Those protons and /or hydroxides change the H+ and Aconcentration unequally, since A- can absorb some of the protons and/or HA can release H+ when influenced by the OH- in the solution. Therefore, one must calculate the proton concentration from the pH using the Henderson Hasselbalch equation. \[pH = pKa + log ([Ac- ]/[HAc])\]
Image by Aleia Kim Table 1.6 You may wonder why we care about weak acids. You may never have thought much of weak acids when you were in General Chemistry. Your instructor described them as buffers and you probably dutifully memorized the fact that “buffers are substances that resist change in pH” without really learning what Clearing Confusion - this meant. Buffers are much too important to be thought of in this way. UPS Weak acids are critical for life because their affinity for protons causes them to behave like a UPS. We’re not referring to the UPS that is the United Parcel Service, but instead, to the encased battery backup systems for computers called Uninterruptible Power Supplies that kick on to keep a computer running during a power failure. The battery in a laptop computer is a UPS, for example. We can think of weak acids as Uninterruptible Proton Suppliers within certain pH ranges, providing (or absorbing) protons as needed. Weak acids thus help to keep the H+ concentration (and thus the pH) of the solution they are in relatively constant. Consider the bicarbonate/carbonic acid system. Figure 1.35 shows what happens when H2CO3dissociates. Adding hydroxide ions (by adding a strong base like NaOH) to the solution causes the H+ ions to react with OH- ions to make water. Consequently, the concentration of H+ ions would go down and the pH would go up. However, in contrast to the situation with a solution of pure water, there is a backup source of H+ available in the form of H2CO3. Here is where the UPS function kicks in. As protons are taken away by the added hydroxyl ions (making water), they are partly replaced by protons from the H2CO3. This is why a weak acid is a buffer. It resists changes in pH by releasing protons to compensate for those “used up” in reacting with the hydroxyl ions. Henderson-Hasselbalch It is useful to be able to predict the response of the H2CO3 system to changes in H+ concentration. The Henderson-Hasselbalch equation defines the relationship between pH and the ratio of HCO3 - and H2CO3. It is pH = pKa + log ([HCO3- ]/ [H2CO3]) This simple equation defines the relationship between the pH of a solution and the ratio of HCO3- and H2CO3 in it. The new term, called the pKa, is defined as pKa = -Log Ka, just as pH = -Log [H+]. The Ka is the acid dissociation constant and is a measure of the strength of an acid. For a general acid, HA, which dissociates as HA ⇄ H+ + A -, Ka = [H+][A- ]/[HA] Thus, the stronger the acid, the more protons that will dissociate from it when added to water and the larger the value its Ka will have. Large values of Ka translate to lower values of pKa. As a result, the lower the pKa value is for a given acid, the stronger the weak acid is. Constant pKa Please note that pKa is a constant for a given acid. The pKa for carbonic acid is 6.37. By comparison, the pKa for formic acid is 3.75. Formic acid is therefore a stronger acid than acetic acid. A stronger acid will have more protons dissociated at a given pH than a weaker acid. Now, how does this translate into stabilizing pH? Figure 1.35 shows a titration curve. In this curve, the titration begins with the conditions at the lower left (very low pH). At this pH, the H2CO3 form predominates, but as more and more OH- is added (moving to the 45 Why do we care about pH? Because biological molecules can, in some cases, be exquisitely sensitive to changes in it. As the pH of a solution changes, the charges of molecules in the solution can change, as you will see. Changing charges on biological molecules, especially proteins, can drastically affect how they work and even whether they work at all right), the pH goes up, the amount of HCO3- goes up and (correspondingly), the amount of H2CO3 goes down. Notice that the curve “flattens” near the pKa (6.37). Buffering region Flattening of the curve tells us is that the pH is not changing much (not going up as fast) as it did earlier when the same amount of hydroxide was added. The system is resisting a change in pH (not stopping the change, but slowing it) in the region of about one pH unit above and one pH unit below the pKa. Thus, the buffering region of the carbonic acid/ bicarbonate buffer is from about 5.37 to 7.37. It is maximally strong at a pH of 6.37. Now it starts to become apparent how the buffer works. HA can donate protons when extras are needed (such as when OH- is added to the solution by the addition of NaOH). Similarly, A- can accept protons when extra H+ are added to the solution (adding HCl, for example). The maximum ability to donate or accept protons comes when [A- ] = [HA] This is consistent with the Henderson Hasselbalch equation and the titration curve. When [A- ] = [HA], pH = 6.37 + Log(1). Since Log(1) = 0, pH = 6.37 = pKa for carbonic acid. Thus for any buffer, the buffer will have maximum strength and display flattening of its titration curve when [A- ] = [HA] and when pH = pKa. If a buffer has more than one pKa (Figure 1.36), then each pKa region will display the behavior. Buffered vs non-buffered To understand how well a buffer protects against changes in pH, consider the effect of adding .01 moles of HCl to 1.0 liter of pure water (no volume change) at pH 7, compared to adding it to 1.0 liter of a 1M acetate buffer at pH 4.76. Since HCl completely dissociates, in 0.01M (10-2 M) HCl you will have 0.01M H+. For the pure water, the pH drops from 7.0 down to 2.0 (pH = -log(0.01M)). By contrast, the acetate buffer’s pH after adding the same amount of HCl is 4.74. Thus, the pure water solution sees its pH fall from 7 to 2 (5 pH units), whereas the buffered solution saw its pH drop from 4.76 to 4.74 (0.02 pH units). Clearly, the buffer minimizes the impact of the added protons compared to the pure water. Buffer capacity It is important to note that buffers have capacities limited by their concentration. Let’s imagine that in the previous paragraph, we had added the 0.01 moles HCl to an acetate buffer that had a concentration of 0.01M and equal amounts of Ac- and HAc. When we try to do the math in parallel to the previous calculation, we see that there are 0.01M protons, but only 0.005M A- to absorb them. We could imagine that 0.005M of the protons would be absorbed, but that would still leave 0.005M of protons unbuffered. Thus, the pH of this solution would be approximately pH = -log(0.005M) = 2.30
Exceeding buffer capacity dropped the pH significantly compared to adding the same amount of protons to a 1M acetate buffer. Consequently, when considering buffers, it is important to recognize that their concentration sets their limits. Another limit is the pH range in which one hopes to control proton concentration. Multiple ionizable groups Now, what happens if a molecule has two (or more) ionizable groups? It turns out, not surprisingly, that each group will have its own pKa and, as a consequence, will have multiple regions of buffering. Figure 1.36 shows the titration curve for the amino acid aspartic acid. Note that in- stead of a single flattening of the curve, as was seen for acetic acid, aspartic acid’s titration curve displays three such regions. These are individual buffering regions, each centered on the respective pKa values for the carboxyl group and the amine group. Aspartic acid has four possible charges: +1 (α-carboxyl group, α-amino group, and Rgroup carboxyl each has a proton), 0 (α- carboxyl group missing proton, α- amino group has a proton, R-group carboxyl has a proton), -1 (α-carboxyl group and R-group carboxyl each lack a proton, α-amino group retains a proton), -2 (α-carboxyl, R-group carboxyl, and α-amino groups all lack extra proton). Prediction How does one predict the charge for an amino acid at a given pH? A good rule of thumb for estimating charge is that if the pH is more than one unit below the pKa for a group (carboxyl or amino), the proton is on. If the pH is more than one unit above the pKa for the group, the proton is off. If the pH is NOT more than one or less than one pH unit from the pKa, this simple assumption will not work. Further, it is important to recognize that these rules of thumb are estimates only. The pI (pH at which the charge of a molecule is zero) is an exact value calculated as the average of the two pKa values on either side of the zero region. It is calculated at the average of the two pKa values around the point where the charge of the molecule is zero. For aspartic acid, this corresponds to pKa1and pKa2.
Thumbanil: Structure of human hemoglobin. The proteins α and βsubunits are in red and blue, and the iron-containing hemegroups in green. Image used with permission (CC BY-SA 3.0; Richard Wheeler). 02: Structure and Function Source: BiochemFFA_2_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy "The man who does not read good books has no advantage over the man who cannot read them." Mark Twain In this chapter, we will examine the structures of the major classes of biomolecules, with an eye to understanding how these structures relate to function. As noted earlier, water is the most abundant molecule in cells, and provides the aqueous environment in which cellular chemistry happens. Dissolved in this water are inorganic ions like sodium, potassium and calcium. But the distinctiveness of biochemistry derives from the vast numbers of complex, large, carbon compounds, that are made by living cells. You have probably learned that the major classes of biological molecules are proteins, nucleic acids, carbohydrates and lipids. The first three of these major groups are macromolecules that are built as long polymers made up of smaller subunits or monomers, like strings of beads. The lipids, while not chains of monomers, also have smaller subunits that are assembled in various ways to make the lipid components of cells, including membranes. The chemical properties and three dimensional conformations of these molecules determine all the molecular interactions upon which life depends. Whether building structures within cells, transferring information, or catalyzing reactions, the activities of biomolecules are governed by their structures. The properties and shapes of macromolecules, in turn, depend on the subunits of which they are built. Interactive 2.1: The enzyme Hexokinase: as for all enzymes, the activity of hexokinase depends on its structure. Protein Database (PDB) We will next examine the major groups of biological macromolecules: proteins, polysaccharides, nucleic acids, and lipids. The building blocks of the first three, respectively, are amino acids, monosaccharides (sugars), and nucleotides. Acetyl-CoA is the most common building block of lipids. 2.04: Structure and Function- Proteins II Source: BiochemFFA_2_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy In this section, we hope to bring to life the connection between structure and function of proteins. So far, we have described notable features of the four elements (primary, secondary, tertiary, and quaternary) of protein structure and discussed example proteins/motifs exhibiting them. In this section, we will examine from a functional perspective a few proteins/domains whose function relies on secondary, tertiary, or quaternary structure. It is, of course a bit of a narrow focus to ascribe protein function to any one component of structure, but our hope is by presenting these examples, we can bring to life the way in which a protein’s secondary, tertiary, and quarternary structure lead to the functions it has. Hemoglobin Wikipedia Fibrous proteins - secondary structure Proteins whose cellular or extracellular roles have a strong structural component are composed primarily of primary and second structure, with little folding of the chains. Thus, they have very little tertiary structure and are fibrous in nature. Proteins exhibiting these traits are commonly insoluble in water and are referred to as fibrous proteins (also called scleroproteins). The examples described in this category are found exclusively in animals where they serve roles in flesh, connective tissues and hardened external structures, such as hair. They also contain the three common fibrous protein structures α -helices (keratins), β-strands/sheets (fibroin & elastin) and triple helices (collagen). The fibrous proteins have some commonality of amino acid sequence. Each possesses an abundance of repeating sequences of amino acids with small, non-reactive side groups. Many contain short repeats of sequences, often with glycine. Keratins The keratins are a family of related animal proteins that take numerous forms. α-keratins are structural components of the outer layer of human skin and are integral to hair, nails, claws, feathers, beaks, scales, and hooves. Keratins provide strength to tissues, such as the tongue, and over 50 different keratins are encoded in the human genome. At a cellular level, keratins comprise the intermediate filaments of the cytoskeleton. α- keratins primarily contain α-helices, but can also have β-strand/sheet structures. Individual α-helices are often intertwined to form coils of coiled structures and these strands can also be further joined together by disulfide bonds, increasing structural strength considerably. This is particularly relevant for α-keratin in hair, which contains about 14% cysteine. The odor of burned hair and that of the chemicals used to curl/uncurl hair (breaking/re-making disulfide bonds) arise from their sulfurous components. β-keratins are comprised of β-sheets, as their name implies. Fibroin An insoluble fibrous protein that is a component of the silk of spiders and the larvae of moths and other insects, fibroin is comprised of antiparallel β-strands tightly packed together to form β- sheets. The primary structure of fibroin is a short repeating sequence with glycine at every other residue (Figure 2.57). The small R-groups of the glycine and alanine in the repeating sequence allows for the tight packing characteristic of the fibers of silk. Wikipedia link HERE Elastin As suggested by its name, elastin is a protein with elastic characteristics that functions in many tissues of the body to allow them to resume their shapes after expanding or contracting. The protein is rich in glycine and proline and can comprise over 50% of the weight of dry, defatted arteries. Elastin is made by linking tropoelastin proteins together through lysine residues to make a durable complex crosslinked by desmosine. In arteries, elastin helps with pressure wave propagation for facilitating blood flow. Collagen
Collagen is the most abundant protein in mammals, occupying up to a third of the total mass. There are at least 16 types of collagen. Its fibers are a major component of tendons and they are also found abundantly in skin. Collagen is also prominent in cornea, cartilage, bone, blood vessels and the gut. Collagen’s structure is an example of a helix of helices, being composed of three lefthanded helical chains that each are coiled together in a right-handed fashion to make the collagen fiber (Figure 2.60). Each helix is stretched out more than an α-helix, giving it an extended appearance. On the inside of the triple helical structure, only residues of glycine are found, since the side chains of other amino acids are too bulky. Collagen chains have the repeating structure glycinem-n where m is often proline and n is often hydroxyproline (Figure 2.61). Collagen is synthesized in a pre-procollagen form. Processing of the pre-procollagen in the endoplasmic reticulum results in glycosylation, removal of the ‘pre’ sequence, and hydroxylation of lysine and proline residues (see below). The hydroxides can form covalent cross-links with each other, strengthening the collagen fibers. As pro-collagen is exported out of the cell, proteases trim it, resulting in a final form of collagen called tropocollagen. Hydroxylation Hydroxylation of proline and lysine side chains occurs post-translationally in a reaction catalyzed by prolyl-4-hydroxylase and lysyl-hydroxylase (lysyl oxidase), respectively. The reaction requires vitamin C. Since hydroxylation of these residues is essential for formation of stable triple helices at body temperature, vitamin C deficiency results in weak, unstable collagen and, consequently, weakened connective tissues. It is the cause of the disease known as scurvy. Hydrolyzed collagen is used to make gelatin, which is important in the food industry. collagens. Wikipedia link HERE Lamins Lamins are fibrous proteins that provide structure in the cell nucleus and play a role in transcription regulation. They are similar to proteins making up the intermediate filaments, but have extra amino acids in one coil of the protein. Lamins help to form the nuclear lamin in the interior of the nuclear envelope and play important roles in assembling and disassembling the latter in the process of mitosis. They also help to position nuclear pores. In the process of mitosis, disassembly of the nuclear envelope is promoted by phosphorylation of lamins by a protein called mitosis promoting factor and assembly is favored by reversing the reaction (dephosphorylation). Structural domains - tertiary structure Every globular protein relies on its tertiary structure to perform its function, so rather than trying to find representative proteins for tertiary structure (an almost impossible task!), we focus here on a few elements of tertiary structure that are common to many proteins. These are the structural domains and they differ from the structural motifs of supersecondary structure by being larger (25-500 amino acids), having a conserved amino acid sequence, and a history of evolving and functioning independently of the protein chains they are found in. Structural domains are fundamental units of tertiary structure and are found in more than one protein. A structural domain is selfstabilizing and often folds independently of the rest of the protein chain. Leucine zipper A common feature of many eukaryotic DNA binding proteins, leucine zippers are characterized by a repeating set of leucine residues in a protein that interact like a zipper to favor dimerization. Another part of the domain has amino acids (commonly arginine and lysine) that allow it to interact with the DNA double helix (Figure 2.63). Transcription factors that contain leucine zippers include Jun-B, CREB, and AP-1 fos/ jun. Zinc fingers The shortest structural domains are the zinc fingers, which get their name from the fact that one or more coordinated zinc ions stabilize their finger-like structure. Despite their name, some zinc fingers do not bind zinc. There are many structural domains classified as zinc fingers and these are grouped into different families. Zinc fingers were first identified as components of DNA binding transcription factors, but others are now known to bind RNA, protein, and even lipid structures. Cysteine and histidine side chains commonly play roles in coordinating the zinc. Src SH2 domain The Src oncoprotein contains a conserved SH2structural domain that recognizes and binds phosphorylated tyrosine side chains in other proteins (Figure 2.65). Phosphorylation is a fundamental activity in signaling and phosphorylation of tyrosine and interaction between proteins carrying signals is critically needed for cellular communication. The SH2domain is found in over 100 human proteins. Helix-turn-helix domain Helix-turn-helix is a common domain found in DNA binding proteins, consisting of two α-helices separated by a small number of amino acids. As seen in Figure 2.66, the helix parts of the structural domain interact with the bases in the major groove of DNA. Individual α-helices in a protein are part of a helix-turn-helix structure, where the turn separates the individual helices. Pleckstrin homology domain Pleckstrin Homology (PH) domains are protein domains with important functions in the process of signaling. This arises partly from the affinity for binding phosphorylated inositides, such as PIP2 and PIP3, found in Figure 2.66 - Helix-Turn-Helix Domain of a Protein Bound to DNA Wikipedia Figure 2.65 - SH2 Domain Wikipedia biological membranes. PH domains can also bind to G-proteins and protein kinase C. The domain spans about amino acids and is found in numerous signaling proteins. These include Akt/Rac Serine/ Threonine Protein Kinases, Btk/ltk/Tec tyrosine protein kinases, insulin receptor substrate (IRS-1), Phosphatidylinositolspecific phospholipase C, and several yeast proteins involved in cell cycle regulation. Structural globular proteins
Enzymes catalyze reactions and proteins such as hemoglobin perform important specialized functions. Evolutionary selection has reduced and eliminated waste so that we can be sure every protein in a cell has a function, even though in some cases we may not know what it is. Sometimes the structure of the proFigure 2.68 - Relationship of basement membrane to epithelium, endothelium, and connective tissue tein is its primary function because the structure provides stability, organization, connections other important properties. It is with this in mind that we present the following proteins. Basement membrane The basement membrane is a layered extracellular matrix of tissue comprised of protein fibers (type IV collagen) and glycosaminoglycans that separates the epithelium from other tissues (Figure 2.68). More importantly, the basement membrane acts like a glue to hold tissues together. The skin, for example, is anchored to the rest of the body by the basement membrane. Basement membranes provide an interface of interaction between cells and the environment around them, thus facilitating signaling processes. They play roles in differentiation during embryogenesis and also in maintenance of function in adult organisms. Actin Actin is the most abundant globular protein found in most types of eukaryotic cells, comprising as much as 20% of the weight of muscle cells. Similar proteins have been identified in bacteria (MreB) and archaeons (Ta0583). Actin is a monomeric subunit able to polymerize readily into two different types of filaments. Microfilaments are major component of the cytoskeleton and are acted on by myosin in the contraction of muscle cells (See HERE). Actin will be discussed in more detail in the next section HERE. Intermediate Filaments Intermediate filaments are a part of the cytoskeleton in many animal cells and are comprised of over 70 different proteins. They are called intermediate because their size (average diameter = 10 nm) is between that of the microfilaments (7 nm) and the microtubules (25 nm). The intermediate filament components include fibrous proteins, such as the keratins and the lamins, which are nuclear, as well as cytoplasmic forms. Intermediate filaments give flexibility to cells because of their own physical properties. They can, for example, be stretched to several times of their original length. Six types There are six different types of intermediate filaments. Type I and II are acidic or basic and attract each other to make larger filaments. They include epithelial keratins and trichocytic keratins (hair components). Type III proteins include four structural proteins - desmin, GFAP (glial fibrillary acidic protein), peripherin, and vimentin. Type IV also is a grouping of three proteins and one multiprotein structure (neurofilaments). The three proteins are α-internexin, synemin, and syncoilin. Type V intermediate filaments encompass the lamins, which give structure to the nucleus. Phosphorylation of lamins leads to their disassembly and this is important in the process of mitosis. The Type VI category includes only a single protein known as nestin. Tubulin A third type of filament found in cells is that of the microbutules. Comprised of a polymer of two units of a globular protein called tubulin, microtubules provide “rails” for motor proteins to move organelles and other “cargo” from one part of a cell to another. Microtubules and tubulin are discussed in more detail HERE. Vimentin Vimentin (Figure 2.70) is the most widely distributed protein of the intermediate filaments. It is expressed in fibroblasts, leukocytes, and blood vessel endothelial cells. The protein has a significant role maintaining the position of organelles in the cytoplasm, with attachments to the nucleus, mitochondria, and endoplasmic reticulum (Figure 2.70). Vimentin provides elasticity to cells and resilience that does not arise from the microtubules or microfilaments. Wounded mice that lack the vimentin gene survive, but take longer to heal wounds than wild type mice. Vimentin also controls the movement of cholesterol from lysosomes to the site of esterification. The result is a reduction in the amount of cholesterol stored inside of cells and has implications for adrenal cells, which must have esters of cholesterol. Mucin Mucins are a group of proteins found in animal epithelial tissue that have many glycosyl residues on them and typically are of high molecular weight (1 to 10 million Da). They are gel-like in their character and are often used for lubrication. Mucus is comprised of mucins. In addition to lubrication, mucins also help to control mineralization, such as bone formation in vertebrate organisms and calcification in echinoderms. They also play roles in the immune system by helping to bind pathogens. Mucins are commonly secreted onto mucosal surfaces (nostrils, eyes, mouth, ears, stomach, genitals, anus) or into fluids, such as saliva. Because of their extensive mucosylation, mucins hold a considerable amount of water (giving them the “slimy” feel) and are resistant to proteolysis. Vinculin Vinculin (Figure 2.72) is a membrane cytoskeletal protein found in the focal adhesion structures of mammalian cells. It is found at cell-cell and cell-matrix junctions and interacts with integrins, talin, paxillins and F-actin. Vinculin is thought to assist (along with other proteins) in anchoring actin microfilaments to the membrane (Figure 2.71). Binding of vinculin to actin and to talin is regulating by polyphosphoinositides and can be inhibited by acidic phospholipids. Syndecans Syndecans are transmembrane proteins that make a single pass with a long amino acid chain (24-25 residues) through plasma membranes and facilitate G proteincoupled receptors’ interaction with Figure 2.71 - Actin filaments (green) attached to vinculin in focal adhesion (red) Wikipedia ligands, such as growth factors, fibronectin, collagens (I, III, and IV) and antithrombin-1. Syndecans typically have 3-5 heparan sulfate and chondroitin sulfate chains attached to them.
Heparan sulfate can be cleaved at the site of a wound and stimulate action of fibroblast growth factor in the healing process. The role of syndecans in cell-cell adhesion is shown in mutant cells lacking syndecan I that do not adhere well to each other. Syndecan 4 is also known to adhere to integrin. Syndecans can also inhibit the spread of tumors by the ability of the syndecan 1 ectodomain to suppress growth of tumor cells without affecting normal epithelial cells. Defensin Defensins (Figure 2.73) are a group of small cationic proteins (rich in cysteine residues) that serve as host defense peptides in vertebrate and invertebrate organisms. They protect against infection by various bacteria, fungi, and viruses. Defensins contain between 18 and 45 amino acids with (typically) about 6- 8 cysteine residues. In the immune system, defensins help to kill bacteria engulfed by phagocytosis by epithelial cells and neutrophils. They kill 120 Figure 2.72 - Vinculin Wikipedia bacteria by acting like ionophores - binding the membrane and opening pore-like structures to release ions and nutrients from the cells. Focal adhesions In the cell, focal adhesions are structures containing multiple proteins that mechanically link cytoskeletal structures (actin bundles) with the extracellular matrix. They are dynamic, with proteins bringing and leaving with signals regarding the cell cycle, cell motility, and more almost constantly. Focal adhesions serve as anchors and as a signaling hub at cellular locations where integrins bind molecules and where membrane clustering events occur. Over 100 different proteins are found in focal adhesions. Focal adhesions communicate important messages to cells, acting as sensors to update information about the status of the extracellular matrix, which, in turn, adjusts/ affects their actions. In sedentary cells, they are stabler than in cells in motion because when cells move, focal adhesion contacts are established at the “front” and removed at the rear as motion progresses. This can be very important in white blood cells’ ability to find tissue damage. Ankyrin Ankyrins (Figure 2.74) are a family of membrane adaptor proteins serving as “anchors” to interconnect integral membrane proteins to the spectrin-actin membrane cytoskeleton. Ankyrins are anchored to the plasma membrane by covalently linked palmitoyl-CoA. They bind to the β subunit of spectrin and at least a dozen groups of integral membrane proteins. The ankyrin proteins contain four functional domains: an N-terminal region with 24 tandem ankyrin repeats, a central spectrin-binding domain, a “death domain” interacting with apoptotic proteins, and a C-terminal regulatory domain that is highly varied significantly among different ankyrins. Spectrin Spectrin (Figures 2.75 & 2.76) is a protein of the cellular cytoskeleton that plays an important role in maintaining its structure and the integrity of the plasma membrane. In animals, spectrin gives red blood cells their shape. Spectrin is located inside the inner layer of the eukaryotic plasma membrane where it forms a network of pentagonal or hexagonal arrangements. Spectrin fibers collect together at junctional complexes of actin and is also attached to ankyrin for stability, as well as numerous integral membrane proteins, such as glycophorin. Integrins In multicellular organisms, cells need connections, both to each other and to the extracellular matrix. Facilitating these attachments at the cellular end are transmembrane proteins known as integrins (Figure 2.77). Integrins are found in all metazoan cells. Ligands for the integrins include collagen, fibronectin, laminin, and vitronectin. Integrins function not only in attachment, but also in communication, cell migration, virus linkages (adenovirus, for example), and blood clotting. Integrins are able to sense chemical and mechanical signals about the extracellular matrix and move that information to intracellular domains as part of the process of signal transduction. Inside the cells, responses to the signals affect cell shape, regulation of the cell cycle, movement, or changes in other cell receptors in the membrane. The process is dynamic and allows for rapid responses as may be necessary, for example in the process of blood clotting, where the integrin known as GPIbIIIa (on the surface of blood platelets) attaches to fibrin in a clot as it develops. Integrins work along with other receptors, including immunoglobulins, other cell adhesion molecules, cadherins, selectins, and syndecans. In mammals the proteins have a large number of subunits - 18 α- and 8 β-chains. They are a bridge between its links outside the cell to the extracellular matrix (ECM) and its links inside the cell to the cytoskeleton. Integrins play central role in formation and stability of focal adhesions. These are large molecular complexes arising from clustering of integrin-ECM connections. In the process of cellular movement, integrins at the “front” of the cell (in the direction of the movement), make new attachments to substrate and release connections to substrate in the back of the cell. These latter integrins are then endocytosed and reused. Integrins also help to modulate signal transduction through tyrosine kinase receptors in the cell membrane by regulating movement of adapters to the plasma membrane. β1c integrin, for example, recruits the Shp2 phosphatase to the insulin growth factor receptor to cause it to become dephosphorylated, thus turning off the signal it communicates. Integrins can also help to recruit signaling molecules inside of the cell to activated tyrosine kinases to help them to communicate their signals. Cadherins Cadherins (Figure 2.78) constitute a type-1 class of transmembrane proteins playing important roles in cell adhesion. They require calcium ions to function, forming adherens junctions that hold tissues together (See Figure 2.69). Cells of a specific cadherin type will preferentially cluster with each other in preference to associating with cells containing a different cadherin type. Caderins are both receptors and places for ligands to attach. They assist in the proper positioning of cells in development, separation of different tissue layers, and cell migration. Selectins
Selectins (Figure 2.79) are cell adhesion glycoproteins that bind to sugar molecules. As such, they are a type of lectin - proteins that bind sugar polymers (see HERE also). All selectins have an N-terminal calcium-dependent lectin domain, a single transmembrane domain, and an intracellular cytoplasmic tail. There are three different types of selectins, 1) E-selectin (endothelial); 2) L (lymphocytic; and 3) P (platelets and endothelial cells. Selectins function in lymphocyte homing (adhesion of blood lymphocytes to cells in lymphoid organs), in inflammation processes, and in cancer metastasis. Near the site of inflammation, P-selectin on the surface of blood capillary cells interacts with glycoproteins on leukocyte cell surfaces. This has the effect of slowing the movement of the leukocyte. At the target site of inflammation, E- selectin on the endothelial cells of the blood vessel and L-selectin on the surface of the leukocyte bind to their respective carbohydrates, stopping the leukocyte movement. The leukocyte then crosses the wall of the capillary and begins the immune response. Selectins are involved in the inflammatory processes of asthma, psoriasis, multiple scleroris, and rheumatoid arthritis. Laminins Laminins are extracellular matrix glycoproteins that a major components of the basal lamina and affect cell differentiation, migration, and adhesion. They are secreted into the extracellular matrix where they are incorporated and are essential for tissue maintenance and survival. When laminins are defective, muscles may not form properly and give rise to muscular dystrophy. Laminins are associated with fibronectin, entactin, and perlecan proteins in type IV collagen networks and bind to integrin receptors in the plasma membrane. As a consequence, laminins contribute to cellular attachment, differentiation, shape, and movement. The proteins are trimeric in structure, having one α-chain, a β-chain, and a γ-chain. Fifteen combinations of different chains are known. Vitronectin Vitronectin is a glycoprotein (75kDa) found in blood serum (platelets), the extracellular matrix, and in bone. It promotes the process of cell adhesion and spreading and binds to several protease inhibitors (serpins). It is secreted from cells and is believed to play roles in blood clotting and the malignancy of tumors. One domain of vitronectin binds to plasminogen activator inhibitor and acts to stabilize it. Another domain of the protein binds to cellular integrin proteins, such as the vitronectin receptor that anchors cells to the extracellular matrix. Catenins Catenins are a family of proteins interacting with cadherin proteins in cell adhesion (Figure 2.69). Four main types of catenins are known, α-, β-, γ-, and δ-catenin. Catenins play roles in cellular organization before development occurs and help to regulate cellular growth. α-catenin and β-catenin are found at adherens junctions with cadherin and help cells to maintain epithelial layers. Cadherins are connected to actin filaments of the cytoskeleton and catenins play the critical role. Catenins are important for the process whereby cellular division is inhibited when cells come in contact with each other (contact inhibition). When catenin genes are mutated, cadherin cell adhesions can disappear and tumorigenesis may result. Catenins have been found to be associated with colorectal and numerous other forms of cancer. Glycophorins All of the membrane proteins described so far are notable for the connections they make to other proteins and cellular structures. Some membrane proteins, though, are designed to reduce cellular connections to proteins of other cells. This is particularly important for blood cells where “stickiness” is undesirable except where clotting is concerned. Glycophorins (Figure 2.80) are membrane-spanning sialoglycoproteins of red blood cells. They are heavily glycosylated (60%).and rich in sialic acid, giving the cells a very hydrophilic (and negatively charged) coat, which enables them to circulate in the bloodstream without adhering to other cells or the vessel walls. Five glycophorins have been identified - four (A,B,C,and D) from isolated membranes and a fifth form (E) from coding in the human genome. The proteins are abundant, forming about 2% of the total membrane proteins in these cells. Glycophorins have important roles in regulating RBC membrane mechanical properties and shape. Because some glycophorins can be expressed in various nonerythroid tissues (particularly Glycophorin C), the importance of their interactions with the membrane skeleton may have a considerable biological significance. Cooperativity and allosterism - quaternary structure Quaternary structure, of course describes the interactions of individual subunits of a multi-subunit protein (Figure 2.81). The result of these interactions can give rise to important biological phenomena, such as cooperative binding of substrates to a protein and allosteric effects on the action of an enzyme. Allosteric effects can occur by a series of mechanisms, but a common feature is that binding of an effector to an enzyme subunit causes (or locks) the enzyme in either a Tstate (less activity) or an R-state (more activity). Effectors can be enzyme substrates (homotropic effectors) or non-substrates (heterotropic effectors). Allosterism will be covered in more depth in the Catalysis chapter HERE. We begin our consideration of quaternary structure with a discussion of cooperativity, how it arises in the multi-subunit protein hemoglobin and how its properties contrast with those of the related, single subunit protein myoglobin. Cooperativity Cooperativity is defined as the phenomenon where binding of one ligand molecule by a protein favors the binding of additional molecules of the same type. Hemoglobin, for example, exhibits cooperativity when the binding of an oxygen molecule by the iron of the heme group in one of the four subunits causes a slight conformation change in the subunit. This happens because the heme iron is attached to a histidine side chain and binding of oxygen ‘lifts’ the iron along with the histidine ring (also known as the imidazole ring). Movie 2.3 - Hemoglobin’s structural changes on binding oxygen Wikipedia
Since each hemoglobin subunit interacts with and influences the other subunits, they too are induced to change shape slightly when the first subunit binds to oxygen (a transition described as going from the T-state to the R-state). These shape changes favor each of the remaining subunits binding oxygen, as well. This is very important in the lungs where oxygen is picked up by hemoglobin, because the binding of the first oxygen molecule facilitates the rapid uptake of more oxygen molecules. In the tissues, where the oxygen concentration is lower, the oxygen leaves hemoglobin and the proteins flips from the R-state back to the Tstate. CO2 transport Cooperativity is only one of many fascinating structural aspects of hemoglobin that help the body to receive oxygen where it is needed and pick it up where it is abundant. Hemoglobin also assists in the transport of the product of cellular respiration (carbon dioxide) from the tissues producing it to the lungs where it is exhaled. Like the binding of oxygen to hemoglobin, binding of other molecules to hemoglobin affects its affinity for oxygen. The effect is particularly pronounced when comparing the oxygen binding characteristics of hemoglobin’s four subunits with the oxygen binding of the related protein myoglobin’s single subunit (Figure 2.83). Different oxygen binding Like hemoglobin, myoglobin contains an iron in a heme group that binds to oxygen. The structure of the globin protein in myoglobin is very similar to the structure of the globins in hemoglobin and hemoglobin is thought to have evolved from myoglobin in evolutionary history. As seen in Figure 2.83, the binding curve of hemoglobin for oxygen is S-shaped (sigmoidal), whereas the binding curve for myoglobin is hyperbolic. What this tells us is that hemoglobin’s affinity for oxygen is low at a low concentration oxygen, but increases as the oxygen concentration increases. Since myoglobin very quickly saturates with oxygen, even under low oxygen concentrations, it says that its affinity for oxygen is high and doesn’t change. Because myoglobin has only a single subunit, binding of oxygen by that subunit can’t affect any other subunits, since there are no other subunits to affect. Consequently, cooperativity requires more than one subunit. Therefore, hemoglobin can exhibit cooperativity, but myoglobin can’t. It is worth noting that simply having multiple subunits does not mean cooperativity will exist. Hemoglobin is one protein that exhibits the characteristic, but many multisubunit proteins do not. Interactive 2.2 - Hemoglobin in the presence (top) and absence (bottom) of oxygen Storage vs. delivery The lack of ability of myoglobin to adjust its affinity for oxygen according to the oxygen concentration (low affinity at low oxygen concentration, such as in tissues and high affinity at high oxygen concentration, such as in the lungs) means it is better suited for storing oxygen than for delivering it according to the varying oxygen needs of and animal body. As we shall see, besides cooperativity, hemoglobin has other structural features that allow it to deliver oxygen precisely where it is needed most in the body. Bohr effect The Bohr Effect was first described over 100 years ago by Christian Bohr, father of the famous physicist, Niels Bohr. Shown graphically (Figures 2.86, 2.87, and 2.88), the observed effect is that hemoglobin’s affinity for oxygen decreases as the pH decreases and as the concentration of carbon dioxide increases. Binding of the protons and carbon dioxide by amino Figure 2.85 - Sequential model of binding. The sequential model is one way to explain hemoglobin’s cooperativity. Squares represent no oxygen bound. Circles represent subunits bound with oxygen and rounded subunits correspond to units whose affinity for oxygen increases by interacting with a subunit that has bound oxygen. Image by Aleia Kim acid side chains in the globin proteins helps to facilitate structural changes in them. Most commonly, the amino acid affected by protons is histidine #146 of the β strands. When this happens, the ionized histidine can form an ionic bond with the side chain of aspartic acid #94, which has the effect of stabilizing the T-state (reduced oxygen binding state) and releasing oxygen. Other histidines and the amine of the amino terminal amino acids in the α-chains are also binding sites for protons. 2,3-BPG Another molecule favoring the release of oxygen by hemoglobin is 2,3- bisphosphoglycerate (also called 2,3-BPG or just BPG - Figure 2.89). Like protons and carbon dioxide, 2,3-BPG is produced by actively respiring tissues, as a byproduct of glucose metabolism. The 2,3-BPG mole cule fits into the ‘hole of the donut’ of adult hemoglobin (Figure 2.89). Such binding of 2,3-BPG favors the T-state (tight - low oxygen binding) of hemoglobin, which has a reduced affinity for oxygen. In the absence of 2,3-BPG, hemoglobin can more easily exist in the R-state (relaxed - higher oxygen binding), which has a high affinity for oxygen. Smokers Notably, the blood of smokers is higher in the concentration of 2,3-BPG than non-smokers, so more of their hemoglobin remains in the T-state and thus the oxygen carrying capacity of smokers is lower than non-smokers.Another reason why smokers’ oxygen carrying capacity is lower than that of non-smokers is that cigarette smoke contains carbon monoxide and this molecule, which has almost identical dimensions to molecular oxygen, effectively outcompetes with oxygen for binding to the iron atom of heme (Figure 2.90). Part of carbon monoxide’s toxicity is due to its ability to bind hemoglobin and prevent oxygen from binding. Carbon dioxide Carbon dioxide binds to form a carbamate when binding the α-amine of each globin chain. The process of forming this structure releases a proton, which helps to further enhance the Bohr effect. Physiologically, the binding of CO2 and H+ has significance because actively respiring tissues (such as contracting muscles) require oxygen and release protons and carbon dioxide. The higher the concentration of protons and carbon dioxide, the more oxygen is released to feed the tissues that need it most.
About 40% of the released protons and about 20% of the carbon dioxide are carried back to the lungs by hemoglobin. The remainder travel as part of the bicarbonate buffering system or as dissolved CO2. In the lungs, the process reverses itself. The lungs have a higher pH than respiring tissues, so protons are released from hemoglobin and CO2 too is freed to be exhaled. Fetal hemoglobin Adult hemoglobin releases oxygen when it binds 2,3- BPG. This is in contrast to fetal hemoglobin, which has a slightly different configuration (α2γ2) than adult hemoglobin (α2β2). Fetal hemoglobin has a greater affinity for oxygen than maternal hemoglobin, allowing the fetus to obtain oxygen effectively from the mother’s blood. Part of the reason for fetal hemoglobin’s greater affinity for oxygen is that it doesn’t bind 2,3-BPG. Consequently, fetal hemoglobin remains in the R-state much more than adult hemoglobin and because of this, fetal hemoglobin has greater affinity for oxygen than adult hemoglobin and can take oxygen away from adult hemoglobin. Thus, the fetus can get oxygen from the mother. Sickle cell disease Mutations to the globin genes coding for hemoglobin can sometimes have deleterious consequences. Sickle cell disease (also called sickle cell anemia) is a genetically transmitted disease that arises from such mutations. There are different forms of the disease. It is a recessive trait, meaning that to be afflicted with it, an individual must inherit two copies of the mutated gene. The predominant form of hemoglobin in adults is hemoglobin A, designated HbA (two α chains and two β chains). The mutant form is known as HbS. The most common mutation is an A to T mutation in the middle of the codon for the seventh amino acid (some counting schemes call it the sixth amino acid) of the β-chain. This results in conversion of a GAG codon to GTG and thus changes the amino acid specified at that position from a glutamic acid to a valine. This minor change places a small hydrophobic patch of amino acids on the surface of the β-globin chains. Polymerization Under conditions of low oxygen, these hydrophobic patches will associate with each other to make long polymers of hemoglobin molecules. The result is that the red blood cells containing them will change shape from being rounded to forming the shape of a sickle (Figure 2.94). Rounded red blood cells readily make it through tiny capillaries, but sickleshaped cells do not. Worse, they block the flow of other blood cells. Tissues where these blockages occur are already low in oxygen, so stopping the flow of blood through them causes them to go quickly anaerobic, causing pain and, in some cases, death of tissue. In severe circumstances, sickled red blood cells death may result. The disease is referred to as an anemia because the sickling of the red blood cells targets them for removal by the blood monitoring system of the body, so a person with the disease has chronically reduced numbers of red blood cells. Heterozygote advantage Interestingly, there appears to be a selective advantage to people who are heterozygous for the disease in areas where malaria is prominent. Heterozygotes do not suffer obvious ill effects of the disease, but their red blood cells appear to be more susceptible to rupture when infected. As a consequence, the parasite gets less of a chance to reproduce and the infected person has a greater chance of survival. The protective effect of the mutant gene, though, does not extend to people who suffer the full blown disease (homozygotes for the mutant gene). Treatments for the disease include transfusion, pain management, and avoidance of heavy exertion. The drug hydroxyurea has been linked to reduction in number and severity of attacks, as well as an increase in survival time1,2. It appears to work by reactivating expression of the fetal hemoglobin gene, which typically is not synthesized to any significant extent normally after about 6 weeks of age. Oxygen binding Animals have needs for oxygen that differ from all other organisms. Oxygen, of course, is the terminal electron acceptor in animals and is necessary for electron transport to work. When electron transport is functioning, ATP generation by cells is many times more efficient than when it is absent. Since abundant ATP is essential for muscular contraction and animals move around a lot - to catch prey, to exercise, to escape danger, etc., having an abundant supply of oxygen is important. This is particularly a concern deep inside tissues where diffusion of oxygen alone (as occurs in insects) does not deliver sufficient quantities necessary for long term survival. The issue is not a problem for plants since, for the most part, their motions are largely related to growth and thus don’t have rapidly changing needs/demands for oxygen that animals have. Unicellular organisms have a variety of mechanisms for obtaining oxygen and surviving without it. Two other important oxygen binding proteins besides hemoglobin are myoglobin and hemocyanin. Myoglobin Myoglobin is the primary oxygen-storage protein found in animal muscle tissues. In contrast to hemoglobin, which circulates throughout the body, myoglobin protein is only found in muscle tissue and appears in the blood only after injury. Like hemoglobin, myoglobin binds oxygen at a prosthetic heme group it contains. The red color of meat arises from the heme of myoglobin and the browning of meat by cooking it comes from oxidation of the ferrous (Fe++) ion of myoglobin’s heme to the ferric (Fe+++) ion via oxidation in the cooking process. As meat sits in our atmosphere (an oxygen-rich environment), oxidation of Fe++ to Fe+++ occurs, leaving the brown color noted above. If meat is stored in a carbon monoxide (CO) environment, CO binds to the heme group and reduces the amount of oxidation, keeping meat looking red for a longer period of time. High affinity
Myoglobin (Figure 2.97) displays higher affinity for oxygen at low oxygen concentrations than hemoglobin and is therefore able to absorb oxygen delivered by hemoglobin under these conditions. Myoglobin’s high affinity for oxygen makes it better suited for oxygen storage than delivery. The protein exists as a single subunit of globin (in contrast to hemoglobin, which contains four subunits) and is related to the subunits found in hemoglobin. Mammals that dive deeply in the ocean, such as whales and seals, have muscles with particularly high abundance of myoglobin. When oxygen concentration in muscles falls to low levels, myoglobin releases its oxygen, thus functioning as an oxygen “battery” that delivers oxygen fuel when needed and holding onto it under all other conditions. Myoglobin holds the distinction of being the first protein for which the 3D structure was determined by X-ray crystallography by John Kendrew in 1958, an achievement for which he later won the Nobel Prize. Hemocyanin Hemocyanin is the protein transporting oxygen in the bodies of molluscs and arthropods. It is a coppercontaining protein found not within blood cells of these organisms, but rather is suspended in the circulating hemolymph they possess. The oxygen binding site of hemocyanin contains a pair of copper(I) cations directly coordinated to the protein by the imidazole rings of six histidine side chains. Most, but not all hemocyanins bind oxygen non-cooperatively and are less efficient than hemoglobin at transporting oxygen. Notably, the hemocyanins of horseshoe crabs and some other arthropods do, in fact, bind oxygen cooperatively. Hemocyanin contains many subunit proteins, each with two copper atoms that can bind one oxygen molecule (O2). Subunit proteins have atomic masses of about 75 kilodaltons (kDa). These may be arranged in dimers or hexamers depending on species. Superstructures comprised of dimer or hexamer complexes are arranged in chains or clusters and have molecular weights of over 1500 kDa.
Source: BiochemFFA_2_4.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy To this point, the proteins we have discussed have not been catalysts (enzymes). The majority of proteins in cells, however, catalyze reactions. In this section we begin our discussion of a subclass of proteins that catalyze reactions releasing energy and convert it into mechanical force. These operate at the cellular and organismal level and are known as motor proteins. Motor proteins rely on globular structural proteins, so it is important that we describe how these cellular “railways” are assembled before discussing the motor proteins themselves. There are two relevant fibrous structures serving as rails for motor proteins. They are: 1. microfilaments (composed of an actin polymer) and 2. microtubules (composed of a polymer of tubulin. Actin The monomeric unit of actin is called G-actin (globular actin) and the polymer is known as F-actin (filamentous actin). Filaments of Factin comprise the smallest filaments of cells known as microfilaments (Figure 2.101). Actin is essential for muscular contraction and also has diverse roles in cellular signaling and maintenance of cell junctions. In conjunction with other proteins, actin has numerous interactions with the cell membrane. The β- and γ-forms of actin are components of the cytoskeleton and facilitate motility inside of cells. α-actin is important in muscle tissues, where it is used by myosin in the mechanical process of contraction (See HERE). Monomeric and polymeric forms of actin play roles in cellular activities relating to motion. Two parallel F-actin strands can pair with each other and create a double helical structure with 2.17 subunits per turn of the helix. Helical F-actin in muscles contains tropomyosin, which covers the actin binding sites for myosin in resting muscles to prevent contraction. Other proteins bound to actin muscle filaments include the troponins (I, T, and C). Actin Cellular Action Examples of actin action at the cellular level include cell motility, cytokinesis, intracellular transport of vesicles and organelles, and cell shape. Each actin monomer is bound to a molecule of ATP or ADP and the presence of one of these is essential for proper G-actin functioning. The role of ATP In the monomer, actin is more commonly bound to ATP, whereas in the filaments, it is typically bound to ADP. Actin is an inefficient ATPase, breaking the molecule down slowly, but the catalysis speeds up as much as 40,000 fold when the monomer begins to polymerize. Actin also has a binding site for divalent cations - either calcium or magnesium. F- Actin binds to structural proteins at the adherens junction (Figure 2.102). These include α-actinin, vinculin (provides a membrane connection and connections to the catenins and cadherin). Polymerization Polymerization of actin begins with a nucleating event (Figure 2.103). One factor known to affect the process is known as the Arp 2/3 complex. It does this by mimicking an actin dimer, starting an autocatalytic process of actin assembly. The Arp 2/3 complex plays roles both in the initiation of polymerization of new actin filaments as well as the formation of branches in the filaments. Two proteins play roles in modulating polymer growth. Thymosin functions on the end of actin filaments to control growth. Profilin works on G-actin monomers exchanging ADP for ATP, promoting addition of monomers to a growing chain. F-actin filaments are held together by relatively weak bonds compared to the covalent bonds of the monomers of nucleic acids, thus allowing for easier disassembly when desired. Actin’s amino acid sequence is optimized, having diverged only a relatively small amount (20%) between algae and humans. Mutations in the actin gene result in muscular diseases and/or deafness. Tubulin Tubulin proteins are the monomeric building blocks of eukaryotic microtubules (Figure 2.104 & 2.105). Bacterial (TubZ) and archaeon (FtsZ) equivalents are known. The α-tubulin and β-tubulin proteins polymerize to make microtubule structures in the cytoplasm of cells. Microtubules are major components of the cytoskeleton of eukaryotic cells, providing structural support, transport within the cell, and functions necessary for segregation of DNAs during cell division. Dimerization of the α-tubulin and β-tubulin proteins is necessary for polymerization and requires that the subunits bind to GTP. Microtubules only grow in one direction. β- tubulin is found on the plus end of the tubule (growth end = plus end) and α-tubulin is exposed on the other end (non-growth end = minus end). Dimers of α-tubulin/β-tubulin are incorporated into growing microtubules in this orientation. If a dimer is bound to GDP instead of GTP, it tends to be unstable and fall apart, whereas those bound to GTP stably assemble into microtubules. Microtubules Microtubules, along with microfilaments and intermediate filaments (see HERE) constitute the cytoskeleton of cells. Found in the cytoplasm, they are found in eukaryotic cells, as well as some bacteria. Microtubules help to give cells structure. They comprise the inner structure of flagella and cilia and provide rail-like surfaces for the transport of materials within cells. Polymerization of α- tubulin and β-tubulin to form microtubules occurs after a nucleating event. Individual units get arranged in microtubule organizing centers (MTOCs), an example of which is the centrosome. Centrosomes are focal points of connection of microtubules. Basal bodies of cilia and flagella also help to organize microtubules. Motor proteins From the transport of materials within a cell to the process of cytokinesis where one cell splits into two in mitosis, a cell has needs for motion at the molecular level. Secretory vesicles and organelles must be transported. Chromosomes must be separated in mitosis and meiosis. The proteins dynein and kinesin (Figure 2.106) are necessary for intracellular movement. These motor proteins facilitate the movement of materials inside of cells along microtubule “rails”. These motor proteins are able to move along a portion of the cytoskeleton by converting chemical energy into motion with the hydrolysis of ATP. An exception is flagellar rotation, which uses energy provided from a gradient created by a proton pump. Kinesins and dyneins
As noted, kinesins and dyneins navigate in cells on microtubule tracks (Figure 2.108 & Movie 2.4). Most kinesins move in the direction of the synthesis of the microtubule (+ end movement), which is generally away from the cell center and the opposite direction of movement of dyneins, which are said to do retrograde transport toward the cell center. Both proteins provide movement functions necessary for the processes of mitosis and meiosis. These include spindle formation, chromosome separation, and shuttling of organelles, such as the mitochondria, Golgi apparatuses, and vesicles. Kinesins are comprised of two heavy chains and two light chains. The head motor domains of heavy chains (in the feet) use energy of ATP hydrolysis to do mechanical work for the movement along the microtubules. There are at least fourteen distinct kinesin families and probably many related ones in addition. Dyneins are placed into two groups - cytoplasmic and axonemal (also called ciliary or flagellar dyneins - Figure 2.109). Dyneins are more complex in structure than kinesins with many small polypeptide units. Notably, plants do not have dynein motor proteins, but do contain kinesins. Movie 2.4 The motor protein kinesin walking down a microtubule. Image used with permission (Public Domain; zp706). Myosin An important group of motor proteins in the cell is the myosins. Like kinesins and dyneins, myosins use energy from hydrolysis of ATP for movement. In this case, the movement is mostly not along microtubules, but rather along microfilaments comprised of a polymer of actin (F-actin). Movement of myosin on actin is best known as the driving force for muscular contraction. Myosins are a huge family of proteins, all of which bind to actin and all of which involve motion. Eighteen different classes of myosin proteins are known. Myosin II is the form responsible for generating muscle contraction. It is an elongated protein formed from two heavy chains with motor heads and two light chains. Each myosin motor head binds actin and has an ATP binding site. The myosin heads bind and hydrolyze ATP. This hydrolysis produces the energy necessary for myosin to walk toward the plus end of an actin filament. Non-muscle myosin IIs provide contraction needed to power the action of cytokinesis. Other myosin proteins are involved in movement of non-muscle cells. Myosin I is involved in intracellular organization. Myosin V performs vesicle and organelle transport. Myosin XI provides movement along cellular microfilament networks to facilitate organelle and cytoplasmic streaming in a particular direction. Structure Myosins have six subunits, two heavy chains and four light chains. Myosin proteins have domains frequently described as a head and a tail (Figure 2.111). Some also describe an intermediate hinge region as a neck. The head portion of myosin is the part that binds to actin. It uses energy from ATP hydrolysis to move along the actin filaments. In muscles, myosin proteins form aggregated structures referred to as thick filaments. Movements are directional. Structural considerations of muscular contraction Before we discuss the steps in the process of muscular contraction, it is important to describe anatomical aspects of muscles and nomenclature. There are three types of muscle tissue - skeletal (striated), smooth, and (in vertebrates) cardiac. We shall concern ourselves mostly here with skeletal muscle tissue. Muscles may be activated by the central nervous system or, in the case of smooth and cardiac muscles, may contract involuntarily. Skeletal muscles may be slow twitch or fast twitch. Sarcomeres Sarcomeres are described as the basic units comprising striated muscles and are comprised of thick (myosin) and thin (actin) filaments and a protein called titin. The filaments slide past each other in muscular contraction and then backwards in muscular relaxation. They are not found in smooth muscles. Under the microscope, a sarcomere is the region between two Z-lines of striated muscle tissue (Figure 2.112). The Z-line is the distinct, narrow, dark region in the middle of an I-band. Within the sarcomere is an entire Aband with its central H-zone. Within the Hzone are located tails of myosin fibers, with the head pointed outwards from there projecting all the way to the I-band. The outside of the Aband is the darkest and it gets lighter moving towards the center. Within the Iband are located thin filaments that are not occupied with thick myosin filaments. The Aband contains intact thick filaments overlaying thin filaments except in the central H zone, which contains only thick filaments. In the center of the H-zone is a line, known as the M-line. It contains connecting elements of the cellular cytoskeleton. In muscular contraction, myosin heads walk along pulling their tails over the actin thin filaments, using energy from hydrolysis of ATP and pulling them towards the center of the sarcomere. Sarcolemma The sarcolemma (also known as the myolemma) is to muscle cells what the plasma membrane is to other eukaryotic cells - a barrier between inside and outside. It contains a lipid bilayer and a glycocalyx on the outside of it. The glyocalyx contains polysaccharides and connects with the basement membrane. The basement membrane serves a s scaffolding to connect muscle fibers to. This connection is made by transmembrane proteins bridging the actin cytoskeleton on the inside of the cell with the basement membrane on the outside. On the ends of the muscle fibers, each sarcolemma fuses with a tendon fiber and these, in turn, adhere to bones. Sarcoplasmic reticulum The sarcoplasmic reticulum (Figure 2.114) is a name for the structure found within muscle cells that is similar to the smooth endoplasmic reticulum found in other cells. It contains a specialized set of proteins to meet needs unique to muscle cells. The organelle largely serves as a calcium “battery,” releasing stored calcium to initiate muscular contraction when stimulated and taking up calcium when signaled at the end of the contraction cycle. It accomplishes these tasks using calcium ion channels for release of the ion and specific calcium ion pumps to take it up. Movement direction
All myosins but myosin VI move towards the + end (the growing end) of the microfilament. The neck portion serves to link the head and the tail. It also a binding site for myosin light chain proteins that form part of a macromolecular complex with regulatory functions. The tail is the point of attachment of molecules or other “cargo” being moved. It can also connect with other myosin subunits and may have a role to play in controlling movement. Muscular contraction The sliding filament model has been proposed to describe the process of muscular tension/contraction. In this process a repeating set of actions slide a thin actin filament over a thick myosin filament as a means of creating tension/ shortening of the muscle fiber. Steps in the process occur as follows: A. A signal from the central nervous system (action potential) arrives at a motor neuron, which it transmits towards the neuromuscular junction (see more on the neurotransmission part of the process HERE) B. At the end of the axon, the nerve signal stimulates the opening of calcium channels at the axon terminus causing calcium to flow into the terminal. C. Movement of calcium into the axon of the nerve causes acetylcholine (a neurotransmitter) in synaptic vesicles to fuse with the plasma membrane. This causes the acetylcholine to be expelled into the synaptic cleft between the axon and the adjacent skeletal muscle fiber. D. Acetylcholine diffuses across the synapse and then binds to nicotinic acetylcholine receptors on the neuromuscular junction, activating them. E. Activation of the receptor stimulates opening gates of sodium and potassium channels, allowing sodium to move into the cell and potassium to exit. The polarity of the membrane of the muscle cell (called a sarcolemma - Figure 2.111) changes rapidly (called the end plate potential). F. Change in the end plate potential results in opening of voltage sensitive ion channels specific for sodium or potassium only to Figure 2.117 - 3. ATP cleavage by myosin allows actin attachment (J) Wikipediaopen, creating an action potential (voltage change) that spreads throughout the cell in all directions. G. The spreading action potential depolarizes the inner muscle fiber and opens calcium channels on the sarcoplasmic reticulum (Figure 2.115). H. Calcium released from the sarcoplasmic reticulum binds to troponin on the actin filaments (Figure 2.115). I. Troponin alters the structure of the tropomyosin to which is it bound. This causes tropomyosin to move slightly, allowing access to myosin binding sites on the microfilament (also called thin filament) that it was covering (Figure 2.116). J. Myosin (bound to ATP) cleaves the ATP to ADP and Pi, which it holds onto in its head region and then attaches itself to the exposed binding sites on the thin filaments causing inorganic phosphate to be released from the myosin followed by ADP (Figure 2.117). K. Release of ADP and Pi is tightly coupled to a bending of the myosin hinge, resulting in what is called the power stroke. This causes the thin filament to move relative to the thick fibers of myosin (Figures 2.118 & 2.119). L. Such movement of the thin filaments causes the Z lines to be pulled closer to each other. This results in shortening of the sarcomere as a whole (Figure 2.122) and narrowing of the I band and the H zones (Figure 2.123). M. If ATP is available, it binds to myosin, allowing it to let go of the actin (Figures 2.120 & 2.121). If ATP is not available, the muscle will remain locked in this state. This is the cause of rigor mortis in death - contraction without release of muscles . Figure 2.120 - When ATP is present, it binds to myosin (M). Wikipedia N. After myosin has bound the ATP, it hydrolyzes it, producing ADP and Pi, which are held by the head. Hydrolysis of ATP resets the hinge region to its original state, unbending it. This unbent state is also referred to as the cocked position. O.If tropomyosin is still permitting access to binding sites on actin, the process repeats so long as ATP is available and calcium remains at a high enough concentration to permit it to bond to troponin. Relaxation of the muscle tension occurs as the action potential in the muscle cell dissipates. This happens because all of the following things happen 1) the nerve signal stops; 2) the neurotransmitter is degraded by the enzyme acetylcholinesterase; and 3) the calcium concentration declines because it is taken up by the sarcoplasmic reticulum. It should be noted that the sarcoplasmic reticulum is always taking up calcium. Only when its calcium gates are opened by the action potential is it unable to reduce cellular calcium concentration. As the action potential decreases, then the calcium gates close and the sarcoplasmic reticulum “catches up” and cellular calcium concentrations fall. At that point troponin releases calcium, tropomyosin goes back to covering myosin binding sites on actin, myosin loses its attachment to actin and the thin filaments slide back to their original positions relative to the myosin thick filaments. Tropomyosin Tropomyosins are proteins that interact with actin thin filaments to help regulate their roles in movement, both in muscle cells and non-muscle cells (Figure 2.124). Tropomyosins interact to form head-to-toe dimers and perch along the α-helical groove of an actin filament. The isoforms of tropomyosin that are in muscle cells control interactions between myosin and the actin filament within the sarcomere and help to regulate contraction of the muscle. In other cells, nonmuscle tropomyosins help to regulate the cytoskeleton’s functions. The interactions of tropomyosin with the cytoskeleton are considerably more complicated than what occurs in muscle cells. Muscle cells have five tropomyosin isoforms, but in the cytoskeleton of non-muscle cells, there are over 40 tropomyosins. Troponin The troponins involved in muscular contraction are actually a complex of three proteins known as troponin I, troponin C, and troponin T (Figure 2.125). They associate with each other and with tropomyosin on actin filaments to help regulate the process of muscular contraction. Troponin I prevents binding of myosin’s head to actin and thus prevents the most important step in contraction.
Troponin C is a unit that binds to calcium ions. Troponin T is responsible for binding all three proteins to tropomyosin. Troponins in the bloodstream are indicative of heart disorders. Elevation of troponins in the blood occurs after a myocardial infarction and can remain high for up to two weeks. Actinin Actinin is a skeletal muscle protein that attaches filaments of actin to Z-lines of skeletal muscle cells. In smooth muscle cells, it also connects actin to dense bodies. Titin Titin (also known as connectin) is the molecular equivalent of a spring that provides striated muscle cells with elasticity. It is the third most abundant protein in muscle cells. The protein is enormous, with 244 folded individual protein domains spread across 363 exons (largest known number), with the largest known exon (17,106 base pairs long), and it is the largest protein known (27,000 to 33,000 amino acids, depending on splicing). Unstructured sequences The folded protein domains are linked together by unstructured sequences. The unstructured regions of the protein allow for unfolding when stretching occurs and refolding upon relaxation. Titin connects the M and Z lines in the sarcomere (Figure 2.123). Tension created in titin serves to limit the range of motion of the sarcomere, giving rise to what is called passive stiffness. Skeletal and cardiac muscles have slight amino acid sequence variations in their ti tin proteins and these appear to relate to differences in the mechanical characteristics of each muscle. Energy backup for muscle energy Myoglobin was described as a molecular batter for oxygen. Muscle cells have a better of their own for ATP. The is important for animals, but not for plants because a plant’s need for energy is different than an animal’s. Plants do not need to access energy sources as rapidly as animals do, nor do they have to maintain a constant internal temperature. Plants can neither flee predators, nor chase prey. These needs of animals are much more immediate and require that energy stores be accessible on demand. Muscles, of course, enable the motion of animals and the energy required for muscle contraction is ATP. To have stores of energy readily available, muscles have, in addition to ATP, creatine phosphate for energy and glycogen for quick release of glucose to make more energy. The synthesis of creatine phosphate is a prime example of the effects of concentration on the synthesis of high energy molecules. For example, creatine phosphate has an energy of hydrolysis of -43.1 kJ/mol whereas ATP has an energy of hydrolysis of -30.5 kJ/mol. Creatine phosphate, however, is made from creatine and ATP in the reaction shown in Figure 2.126. How is this possible? The ∆G°’ of this reaction is +12.6 kJ/mol, reflecting the energies noted above. In a resting muscle cell, ATP is abundant and ADP is low, driving the reaction downward, creating creatine phosphate. When muscular contraction commences, ATP levels fall and ADP levels climb. The above reaction then reverses and proceeds to synthesize ATP immediately. Thus, creatine phosphate acts like a battery, storing energy when ATP levels are high and releasing it almost instantaneously to create ATP when its levels fall.
Source: BiochemFFA_2_5.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy The nucleic acids, DNA and RNA, may be thought of as the information molecules of the cell. In this section, we will examine the structures of DNA and RNA, and how these structures are related to the functions these molecules perform. We will begin with DNA, which is the hereditary information in every cell, that is copied and passed on from generation to generation. The race to elucidate the structure of DNA was one of the greatest stories of 20th century science. Discovered in 1869 by Friedrich Miescher, DNA was identified as the genetic material in experiments in the 1940s led by Oswald Avery, Colin MacLeod, and Maclyn McCarty. X-ray diffraction work of Rosalind Franklin and the observations of Erwin Chargaff were combined by James Watson and Francis Crick to form a model of DNA that we are familiar with today. Their famous paper, in the April 25, 1953 issue of Nature, opened the modern era of molecular biology. Arguably, that one-page paper has had more scientific impact per word than any other research article ever published. Today, every high school biology student is familiar with the double helical structure of DNA and knows that G pairs with C and A with T. The double helix, made up of a pair of DNA strands, has at its core, bases joined by hydrogen bonds to form base pairs - adenine always paired with thymine, and guanine invariably paired with cytosine. Two hydrogen bonds are formed between adenine and thymine, but three hydrogen bonds hold together guanine and cytosine (Figure 2.127). The complementary structure immediately suggested to Watson and Crick how DNA might be (and in fact, is) replicated and it further explains how information is DNA is transmitted to RNA for the synthesis of proteins. In addition to the hydrogen bonds between bases of each strand, the double helix is held together by hydrophobic interactions of the stacked, non-polar bases. Crucially, the sequence of the bases in DNA carry the information for making proteins. Read in groups of three, the sequence of the bases directly specifies the sequence of the amino acids in the encoded protein. Structure A DNA strand is a polymer of nucleoside monophosphates held together by phosphodiester bonds. Two such paired strands make up the DNA molecule, which is then twisted into a helix. In the most common Bform, the DNA helix has a repeat of 10.5 base pairs per turn, with sugars and phosphate forming the covalent phosphodiester “backbone” of the molecule and the adenine, guanine, cytosine, and thymine bases oriented in the middle where they form the now familiar base pairs that look like the rungs of a ladder. Building blocks The term nucleotide refers to the building blocks of both DNA (deoxyribonucleoside triphosphates, dNTPs) and RNA (ribonucleoside triphosphates, NTPs). In order to discuss this important group of molecules, it is necessary to define some terms. Nucleotides contain three primary structural components. These are a nitrogenous base, a pentose sugar, and at least one phosphate. Molecules that contain only a sugar and a nitrogenous base (no phosphate) are called nucleosides. The nitrogenous bases found in nucleic acids include adenine and guanine (called purines) and cytosine, uracil, or thymine (called pyrimidines). There are two sugars found in nucleotides - deoxyribose and ribose (Figure 2.128). By convention, the carbons on these sugars are labeled 1’ to 5’. (This is to distinguish the carbons on the sugars from those on the bases, which have their carbons simply labeled as 1, 2, 3, etc.) Deoxyribose differs from ribose at the 2’ position, with ribose having an OH group, where deoxyribose has H. Nucleotides containing deoxyribose are called deoxyribonucleotides and are the forms found in DNA. Nucleotides containing ribose are called ribonucleotides and are found in RNA. Both DNA and RNA contain nucleotides with adenine, guanine, and cytosine, but with very minor exceptions, RNA contains uracil nucleotides, whereas DNA contains thymine nucleotides. When a base is attached to a sugar, the product, a nucleoside, gains a new name. • uracil-containing = uridine (attached to ribose) / deoxyuridine (attached to deoxyribose) • thymine-containing = ribothymidine (attached to ribose) / thymidine (attached to deoxyribose) • cytosine-containing = cytidine (attached to ribose - Figure 2.129) / deoxycytidine (attached to deoxyribose) • guanine-containing = guanosine (attached to ribose) / deoxyguanosine (attached to deoxyribose) • adenine-containing = adenosine (attached to ribose) / deoxyadenosine (attached to deoxyribose) Of these, deoxyuridine and ribothymidine are the least common. The addition of one or more phosphates to a nucleoside makes it a nucleotide. Nucleotides are often referred to as nucleoside phosphates, for this reason. The number of phosphates in the nucleotide is indicated by the appropriate prefixes (mono, di or tri). Thus, cytidine, for example, refers to a nucleoside (no phosphate), but cytidine monophosphate refers to a nucleotide (with one phosphate). Addition of second and third phosphates to a nucleoside monophosphate requires energy, due to the repulsion of negatively charged phosphates and this chemical energy is the basis of the high energy triphosphate nucleotides (such as ATP) that fuel cells. Note: Ribonucleotides as Energy Sources Though ATP is the most common and best known cellular energy source, each of the four ribonucleotides plays important roles in providing energy. GTP, for example, is the energy source for protein synthesis (translation) as well as for a handful of metabolic reactions. A bond between UDP and glucose makes UDP-glucose, the building block for making glycogen. CDP is similarly linked to several different molecular building blocks important for glycerophospholipid synthesis (such as CDP-diacylglycerol).
The bulk of ATP made in cells is not from directly coupled biochemical metabolism, but rather by the combined processes of electron transport and oxidative phosphorylation in mitochondria and/or photophosphorylation that occurs in the chloroplasts of photosynthetic organisms. Triphosphate energy in ATP is transferred to the other nucleosides/nucleotides by action of enzymes called kinases. For example, nucleoside diphosphokinase (NDPK) catalyzes the following reaction \[\ce{ATP + NDP <-> ADP + NTP}\] where ‘N’ of “NDP” and “NTP corresponds to any base. Other kinases can put single phosphates onto nucleosides or onto nucleoside monophosphates using energy from ATP. Deoxyribonucleotides Individual deoxyribonucleotides are derived from corresponding ribonucleoside diphosphates via catalysis by the enzyme known as ribonucleotide reductase (RNR). The deoxyribonucleoside diphosphates are then converted to the corresponding triphosphates (dNTPs) by the addition of a phosphate group. Synthesis of nucleotides containing thymine is distinct from synthesis of all of the other nucleotides and will be discussed later. Building DNA strands Each DNA strand is built from dNTPs by the formation of a phosphodiester bond, catalyzed by DNA polymerase, between the 3’OH of one nucleotide and the 5’ phosphate of the next. The result of this directional growth of the strand is that the one end of the strand has a free 5’ phosphate and the other a free 3’ hydroxyl group (Figure 2.130). These are designated as the 5’ and 3’ ends of the strand. Figure 2.131 shows two strands of DNA (left and right). The strand on the left, from 5’ to 3’ reads T-C-G-A, whereas the strand on the right, reading from 5’ to 3’ is T-C-G-A. The strands in a double-stranded DNA are arranged in an anti-parallel fashion with the 5’ end of one strand across from the 3’ end of the other. Hydrogen bonds Hydrogen bonds between the base pairs hold a nucleic acid duplex together, with two hydrogen bonds per A-T pair (or per A-U pair in RNA) and three hydrogen bonds per G-C pair. The B-form of DNA has a prominent major groove and a minor groove tracing the path of the helix (Figure 2.132). Proteins, such as transcription factors bind in these grooves and access the hydrogen bonds of the base pairs to “read” the sequence therein. Other forms of DNA besides the B-form (Movie 2.5) are known (Figure 2.133). One of these, the A-form, was identified by Rosalind Franklin in the same issue of Nature as Watson and Crick’s paper. Though the A-form structure is a relatively minor form of DNA and resembles the B-form, it turns out to be important in the duplex form of RNA and in RNA-DNA hybrids. Both the A form and the B-form of DNA have the helix oriented in what is termed the right-handed form. Movie 2.5 - B-form DNA duplex rotating in space Wikipedia Z-DNA The A-form and the B-form stand in contrast to another form of DNA, known as the Z-form. ZDNA, as it is known, has the same base-pairing rules as the B and A forms, but instead has the helices twisted in the opposite direction, making a left-handed helix (Figure 2.133). The Z-form has a sort of zig-zag shape, giving rise to the name Z-DNA. In addition, the helix is rather stretched out compared to the A- and B-forms. Why are there different topological forms of DNA? The answer relates to both superhelical tension and sequence bias. Sequence bias means that certain sequences tend to favor the “flipping” of Bform DNA into other forms. ZDNA forms are favored by long stretches of alternating Gs and Cs. Superhelical tension is discussed below. Superhelicity Short stretches of linear DNA duplexes exist in the B-form and have 10.5 base pairs per turn. Double helices of DNA in the cell can vary in the number of base pairs per turn they contain. There are several reasons for this. For example, during DNA replication, strands of DNA at the site of replication get unwound at the rate of 6000 rpm by an enzyme called helicase. The effect of such local unwinding at one place in a DNA has the effect increasing the winding ahead of it. Unrelieved, such ‘tension’ in a DNA duplex can result in structural obstacles to replication. Such adjustments can occur in three ways. First, tension can provide the energy for ‘flipping’ DNA structure. Z-DNA can arise as a means of relieving the tension. Second, DNA can ‘supercoil’ to relieve the tension (Figures 2.134 & 2.135). In this method, the duplex crosses over itself repeatedly, much like a rubber band will coil up if one holds one section in place and twists another part of it. Third, enzymes called topoisomerases can act to relieve or, in some cases, increase the tension by adding or removing twists in the DNA. Topological isomers As noted, so-called “relaxed” DNA has 10.5 base pairs per turn. Each turn corresponds to one twist of the DNA. Using enzymes, it is possible to change the number of base pairs per turn. In either the case of increasing or decreasing the twists per turn, tension is introduced into the DNA structure. If the tension cannot be relieved, the DNA duplex will act to relieve the strain, as noted. This is most easily visualized for circular DNA, though long linear DNA (such as found in eukaryotic chromosomes) or DNAs constrained in other ways will exhibit the same behavior. Parameters To understand topologies, we introduce the concepts of ‘writhe’ and ‘linking number’. First, imagine either opening a closed circle of DNA and either removing one twist or adding one twist and then re-forming the circle. Since the strands have no free ends, they cannot relieve the induced tension by re-adding or removing the twists at their ends, respectively. Instead, the tension is relieved by “superhelices” that form with crossing of the double strands over each other (figure 8 structures in Figure 2.136). Though it is not apparent to visualize, each crossing of the double strands in this way allows twists to be increased or decreased correspondingly. Thus, superhelicity allows the double helix to reassume 10.5 base pairs per turn by adding or subtracting twists as necessary and replacing them with writhes.
We write the equation L= T + W where T is the number of twists in a DNA, W is the number of writhes, and L is the linking number. The linking number is therefore the sum of the twists and writhes. Interestingly, inside of cells, DNAs typically are in a supercoiled form. Supercoiling affects the size of the DNA (compacts it) and also the expression of genes within the DNA, some having enhanced expression and some having reduced expression when supercoiling is present. Enzymes called topoisomerases alter the superhelical density of DNAs and play roles in DNA replication, transcription, and control of gene expression. They work by making cuts in one strand (Type I topoisomerases) or both strands (Type II topoisomerases) and then add or subtract twists as appropriate to the target DNA. After that process is complete, the topoisomerase re-ligates the nick/cut it had made in the DNA in the first step. Topoisomerases may be the targets of antibiotics. The family of antibiotics known as fluoroquinolones work by interfering with the action of bacterial type II topoisomerases. Ciprofloxacin also preferentially targets bacterial type II topoisomerases. Other topoisomerase inhibitors target eukaryotic topoisomerases and are used in anti-cancer treatments. RNA The structure of RNA (Figure 2.137) is very similar to that of a single strand of DNA. Built of ribonucleotides, joined together by the same sort of phosphodiester bonds as in DNA, RNA uses uracil in place of thymine. In cells, RNA is assembled by RNA polymerases, which copy a DNA template in the much same way that DNA polymerases replicate a parental strand. During the synthesis of RNA, uracil is used across from an adenine in the DNA template. The building of messenger RNAs by copying a DNA template is a crucial step in the transfer of the information in DNA to a form that directs the synthesis of protein. Additionally, ribosomal and transfer RNAs serve important roles in “reading” the information in the mRNA codons and in polypeptide synthesis. RNAs are also known to play important roles in the regulation of gene expression. RNA world The discovery, in 1990, that RNAs could play a role in catalysis, a function once thought to be solely the domain of proteins, was followed by the discovery of many more so-called ribozymes- RNAs that functioned as enzymes. This suggested the answer to a long-standing chicken or egg puzzle - if DNA encodes proteins, but the replication of DNA requires proteins, how did a replicating system come into being? This problem could be solved if the first replicator was RNA, a molecule that can both encode information and carry out catalysis. This idea, called the “RNA world” hypothesis, suggests that DNA as genetic material and proteins as catalysts arose later, and eventually prevailed because of the advantages they offer. The lack of a 2’OH on deoxyribose makes DNA more stable than RNA. The double-stranded structure of DNA also provides an elegant way to easily replicate it. RNA catalysts, however, remain, as remnants of that early world. In fact, the formation of peptide bonds, essential for the synthesis of proteins, is catalyzed by RNA. Secondary structure With respect to structure, RNAs are more varied than their DNA cousin. Created by copying regions of DNA, cellular RNAs are synthesized as single strands, but they often have self-complementary regions leading to “foldbacks” containing duplex regions. These are most easily visualized in the ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) (Figure 2.138), though other RNAs, including messenger RNAs (mRNAs), small nuclear RNAs (snRNAs), microRNAs (Figure 2.139), and small interfering RNAs (siRNAs) may each have double helical regions as well. Base pairing Base pairing in RNA is slightly different than DNA. This is due to the presence of the base uracil in RNA in place of thymine in DNA. Like thymine, uracil forms base pairs with adenine, but unlike thymine, uracil can, to a limited extent, also base pair with guanine, giving rise to many more possibilities for pairing within a single strand of RNA. These additional base pairing possibilities mean that RNA has many ways it can fold upon itself that single-stranded DNA cannot. Folding, of course, is critical for protein function, and we now know that, like proteins, some RNAs in their folded form can catalyze reactions just like enzymes. Such RNAs are referred to as ribozymes. It is for this reason scientists think that RNA was the first genetic material, because it could not only carry information, but also catalyze reactions. Such a scheme might allow certain RNAs to make copies of themselves, which would, in turn, make more copies of themselves, providing a positive selection. Stability RNA is less chemically stable than DNA. The presence of the 2’ hydroxyl on ribose makes RNA much more prone to hydrolysis than DNA, which has a hydrogen instead of a hydroxyl. Further, RNA has uracil instead of thymine. It turns out that cytosine is the least chemically stable base in nucleic acids. It can spontaneously deaminate and in turn is converted to a uracil. This reaction occurs in both DNA and RNA, but since DNA normally has thymine instead of uracil, the presence of uracil in DNA indicates that deamination of cytosine has occurred and that the uracil needs to be replaced with a cytosine. Such an event occurring in RNA would be essentially undetectable, since uracil is a normal component of RNA. Mutations in RNA have much fewer consequences than mutations in DNA because they are not passed between cells in division. Catalysis RNA structure, like protein structure, has importance, in some cases, for catalytic function. Like random coils in proteins that give rise to tertiary structure, single-stranded regions of RNA that link duplex regions give these molecules a tertiary structure, as well. Catalytic RNAs, called ribozymes, catalyze important cellular reactions, including the formation of peptide bonds in ribosomes (Figure 2.114). DNA, which is usually present in cells in strictly duplex forms (no tertiary structure, per se), is not known to be involved in catalysis.
RNA structures are important for reasons other than catalysis. The 3D arrangement of tRNAs is necessary for enzymes that attach amino acids to them to do so properly. Further, small RNAs called siRNAs found in the nucleus of cells appear to play roles in both gene regulation and in cellular defenses against viruses. The key to the mechanisms of these actions is the formation of short foldback RNA structures that are recognized by cellular proteins and then chopped into smaller units. One strand is copied and used to base pair with specific mRNAs to prevent the synthesis of proteins from them. Denaturing nucleic acids Like proteins, nucleic acids can be denatured. Forces holding duplexes together include hydrogen bonds between the bases of each strand that, like the hydrogen bonds in proteins, can be broken with heat or urea. (Another important stabilizing force for DNA arises from the stacking interactions between the bases in a strand.) Single strands absorb light at 260 nm more strongly than double strands. This is known as the hyperchromic effect (Figure 2.141)and is a consequence of the disruption of interactions among the stacked bases. The changes in absorbance allow one to easily follow the course of DNA denaturation. Denatured duplexes can readily renature when the temperature is lowered below the “melting temperature” or Tm, the temperature at which half of the DNA strands are in duplex form. Under such conditions, the two strands can re-form hydrogen bonds between the complementary sequences, returning the duplex to its original state. For DNA, strand separation and rehybridization are important for the technique known as the polymerase chain reaction (PCR). Strand separation of DNA duplexes is accomplished in the method by heating them to boiling. Hybridization is an important aspect of the method that requires single stranded primers to “find” matching sequences on the template DNA and form a duplex. Considerations for efficient hybridization (also called annealing) include temperature, salt concentration, strand concentration, and magnesium ion levels (for more on PCR, see HERE). DNA packaging DNA is easily the largest macromolecule in a cell. The single chromosome in small bacterial cells, for example, can have a molecular weight of over 1 billion Daltons. If one were to take all of the DNA of human chromosomes from a single cell and lay them end to end, they would be over 7 feet long. Such an enormous molecule demands careful packaging to fit within the confines of a nucleus (eukaryotes) or a tiny cell (bacteria). The chromatin system of eukaryotes is the best known, but bacteria, too, have a system for compacting DNA. DNA in Bacteria In bacteria, there is no nucleus for the DNA. Instead, DNA is contained in a structure called a nucleoid (Figure 2.142). It contains about 60% DNA with much of the remainder comprised of RNAs and transcription factors. Bacteria do not have histone proteins that DNA wrap around, but they do have proteins that help organize the DNA in the cell - mostly by making looping structures. These proteins are known as Nucleoid Associated Proteins and include ones named HU, H-NS, Fis, CbpA, and Dps. Of these, HU most resembles eukaryotic histone H2B and binds to DNA non-specifically. The proteins associate with the DNA and can also cluster, which may be the origin of the loops. It is likely these proteins play a role in helping to regulate transcription and respond to DNA damage. They may also be involved in recombination. Eukaryotes The method eukaryotes use for compacting DNA in the nucleus is considerably different, and with good reason - eukaryotic DNAs are typically much larger than prokaryotic DNAs, but must fit into a nucleus that is not much bigger than a prokaryotic cell. Human DNA, for example, is about 1000 times longer than c DNA. The strategy employed in eukaryotic cells is that of spooling - DNA is coiled around positively charged proteins called histones. These proteins, whose sequence is very similar in cells as diverse as yeast and humans, come in four types, dubbed H1, H2a, H2b, H3, and H4. A sixth type, referred to as H5 is actually an isoform of H1 and is rare. Two each of H2a, H2b, H3, and H4 are found in the core structure of what is called the fundamental unit of chromatin - the nucleosome (Figure 2.143). Octamer The core of 8 proteins is called an octamer. The stretch of DNA wrapped around the octamer totals about 147 base pairs and makes 1 2/3 turns around it. This complex is referred to as a core particle (Figure 2.144). A linker region of about 50-80 base pairs separate core particles. The term nucleosome then refers to a a core particle plus a linker region (Figure 2.143). Histone H1 sits near the junction of the incoming DNA and the histone core. It is often referred to as the linker histone. In the absence of H1, non-condensed nucleosomes resemble “beads on a string” when viewed in an electron microscope. Histones Histone proteins are similar in structure and are rich in basic amino acids, such as lysine and arginine (Figure 2.145). These amino acids are positively charged at physiological pH, with enables them to form tight ionic bonds with the negatively charged phosphate backbone of DNA. For DNA, compression comes at different levels (Figure 2.146). The first level is at the nucleosomal level. Nucleosomes are stacked and coiled into higher order structures. 10 nm fibers are the simplest higher order structure (beads on a string) and they grow in complexity. 30 nm fibers consist of stacked nucleosomes and they are packed tightly. Higher level packing produces the metaphase chromosome found in meiosis and mitosis. The chromatin complex is a logistical concern for the processes of DNA replication and (particularly) gene expression where specific regions of DNA must be transcribed. Altering chromatin structure is therefore an essential function for transcriptional activation in eukaryotes. One strategy involves adding acetyl groups to the positively charged lysine side chains to “loosen their grip” on the negatively charged DNA, thus allowing greater access of proteins involved in activating transcription to gain access to the DNA. The mechanisms involved in eukaryotic gene expression are Ames test
The Ames test (Figure 2.147) is an analytical method that allows one to determine whether a compound causes mutations in DNA (is mutagenic) or not. The test is named for Dr. Bruce Ames, a UC Berkeley emeritus professor who was instrumental in creating it. In the procedure, a single base pair of a selectable marker of an organism is mutated in a plasmid to render it nonfunctional. In the example, a strain of Salmonella is created that lacks the ability to grow in the absence of histidine. Without histidine, the organism will not grow, but if that one base in the plasmid’s histidine gene gets changed back to its original base, a functional gene will be made and the organism will be able to grow without histidine. A culture of the bacterium lacking the functional gene is grown with the supply of histidine it requires. It is split into two vials. To one of the vials, a compound that one wants to test the mutagenicity of is added. To the other vial, nothing is added. The bacteria in each vial are spread onto plates lacking histidine. In the absence of mutation, no bacteria will grow. The more colonies of bacteria that grow, the more mutation happened. Note that even the vial without the possible mutagenic compound will have a few colonies grow, as a result of mutations unlinked to the potential mutagen. Mutation happens in all cells at a low level. If the plate with the cells from the vial with the compound has more colonies than the cells from the control vial (no compound), then that would be evidence that the compound causes more mutations than would normally occur and it is therefore a mutagen. On the other hand, if there was no significant difference in the number of colonies on each plate, then that would suggest it is not mutagenic. The test is not perfect - it identifies about 90% of known mutagens - but its simplicity and inexpensive design make it an excellent choice for an initial screen of a compound. 2.07: Structure and Function- Carbohydrates Endogenous glycation, on the other hand, arises with a frequency that is proportional to the concentration of free sugar in the body. These occur most frequently with fructose, galactose, and glucose in that decreasing order and are detected in the bloodstream. Both proteins and lipids can be glycated and the accumulation of endogenous advanced glycation endproducts (AGEs) is associated with Type 2 diabetes, as well as in increases in cardiovascular disease (damage to endothelium, cartilage, and fibrinogen), peripheral neuropathy (attack of myelin sheath), and deafness (loss of myelin sheath). The formation of AGEs increases oxidative stress, but is also thought to be exacerbated by it. Increased oxidative stress, in turn causes additional harm. Damage to collagen in blood cells causes them to stiffen and weaken and is a factor in hardening of the arteries and formation of aneurysms, respectively. One indicator of diabetes is increased glycation of hemoglobin in red blood cells, since circulating sugar concentration are high in the blood of diabetics. Hemoglobin glycation is measured in testing for blood glucose control in diabetic patients. Homopolymer Monomeric Unit Glycogen Glucose Cellulose Glucose Amylose Glucose Callose Glucose Chitin N-acetylglucosamine Xylan Xylose Mannan Mannose Chrysolaminarin Glucose Function in skin Hyaluronic acid is a major component of skin and has functions in tissue repair. With exposure to excess UVB radiation, cells in the dermis produce less hyaluronan and increase its degradation. For some cancers the plasma level of hyaluronic acid correlates with malignancy. Hyaluronic acid levels have been used as a marker for prostate and breast cancer and to follow disease progression. The compound can to used to induce healing after cataract surgery. Hyaluronic acid is also abundant in the granulation tissue matrix that replaces a fibrin clot during the healing of wounds. In wound healing, it is thought that large polymers of hyaluronic acid appear early and they physically make room for white blood cells to mediate an immune response. Breakdown Breakdown of hyaluronic acid is catalyzed by enzymes known as hyaluronidases. Humans have seven types of such enzymes, some of which act as tumor suppressors. Smaller hyaluronan fragments can induce inflammatory response in macrophages and dendritic cells after tissue damage. They can also perform proangiogenic functions. Proteoglycans Glycosaminoglycans are commonly found attached to proteins and these are referred to as proteoglycans. Linkage between the protein and the glycosaminoglycan is made through a serine side-chain. Proteoglycans are made by glycosylation of target proteins in the Golgi apparatus.
Source: BiochemFFA_2_7.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Lipids are a diverse group of molecules that all share the characteristic that at least a portion of them is hydrophobic. Lipids play many roles in cells, including serving as energy storage (fats/oils), constituents of membranes (glycerophospholipids, sphingolipids, cholesterol), hormones (steroids), vitamins (fat soluble), oxygen/ electron carriers (heme), among others. For lipids that are very hydrophobic, such as fats/ oils, movement and storage in the aqueous environment of the body requires special structures. Other, amphipathic lipids, such as glycerophospholipids and sphingolipids spontaneously organize themselves into lipid bilayers when placed in water. Interestingly, major parts of many lipids can be derived from acetyl-CoA. Fatty acids The most ubiquitous lipids in cells are the fatty acids. Found in fats, glycerophospholipids, sphingolipids and serving as as membrane anchors for proteins and other biomolecules, fatty acids are important for energy storage, membrane structure, and as precursors of most classes of lipids. Fatty acids, as can be seen from Figure 2.190 are characterized by a polar head group and a long hydrocarbon tail. Fatty acids with hydrocarbon tails that lack any double bonds are described as saturated, while those with one or more double bonds in their tails are known as unsaturated fatty acids. The effect of double bonds on the fatty acid tail is to introduce a kink, or bend, in the tail, as shown for oleic acid. Stearic acid, a saturated fatty acid, by contrast has a straight hydrocarbon tail. Figures 2.190-2.194 show the most common saturated and unsaturated fatty acids. Fatty acids with unsaturated tails have a lower melting temperature than those with saturated tails of the same length. Shorter tails also decrease melting temperature. These properties carry over to the fats/oils containing them. Fatty acids with more than one double bond are called polyunsaturated. Plants are excellent sources of unsaturated and polyunsaturated fatty acids. The position of the double bond(s) in fatty acids has important considerations both for their synthesis and for their actions in the body. Biochemically, the double bonds found in fatty acids are predominantly in the cis configuration. So-called trans fats arise as a chemical by-product of partial hydrogenation of vegetable oil. In humans, consumption of trans fat raises low density lipoprotein (LDL) levels and lowers high density lipoprotein (HDL) levels. Each is thought to contribute to the risk of developing coronary artery disease. The most Figure 2.194 - Fatty acid models. Carboxyl end labeled in red Wikipedia common fatty acids in our body include palmitate, stearate, oleate, linolenate, linoleate, and arachidonate. Two notable shorter fatty acids are nonanoic (9 carbons) and decanoic acid (10 carbons), both of which appear to have anti-seizure effects. Decanoic acid directly inhibits excitatory neurotransmission in the brain and may contribute to the anticonvulsant effect of the ketogenic diet. Numbering Figure 2.195 shows two different systems for locating double bonds in a fatty acid. The ω system counts carbons starting with the methyl end (shown in red) while the Δ system counts from the carboxyl end (shown in blue). For example, an ω-3 (omega 3) fatty acid would have a double bond at the third carbon from the methyl end. In the Δ system, a fatty acid that has a cis double bond at carbon 6, counting from the carboxyl end, would be written as cis-Δ6. Fatty acids are described as essential fatty acids if they must be in the diet (can’t be synthesized by the organism) and nonessential fatty acids if the organism can synthesize them. Humans and other animals lack the desaturase enzymes necessary to make double bonds at positions greater than Δ-9, so fatty acids with double bonds beyond this position must be obtained in the diet. Linoleic acid and linolenic acid, both fall in this category. Related unsaturated fatty acids can be made from these fatty acids, so the presence of linoleic and linolenic acids in the diet eliminates the need to have all unsaturated fatty acids in the diet. Both linoleic and linolenic acid contain 18 carbons, but linoleic acid is an ω-6 fatty acid, whereas linolenic acid is an ω-3 fatty acid. Notably, ω-6 fatty acids tend to be proinflammatory, whereas ω-3 fatty acids are lesser so. Fats/oils Fats and oils are the primary energy storage forms of animals and are also known as triacylglycerols and triglycerides, since they consist of a glycerol molecule linked via ester bonds to three fatty acids (Figure 2.196). Fats and oils have the same basic structure. We give the name fat to those compounds that are solid at room temperature and the name oil to those that are liquid at room temperature. Note that biological oils are not the same as petroleum oils. Increasing the number of unsaturated fatty acids (and the amount of unsaturation in a given fatty acid) in a fat decreases the melting temperature of it. Organisms like fish, which live in cool environments, have fats with more unsaturation and this is why fish oil contains polyunsaturated fatty acids. Adipocytes Fats are stored in the body in specialized cells known as adipocytes. Enzymes known as lipases release fatty acids from fats by hydrolysis reactions (Figure 2.197). Triacylglycercol lipase (pancreatic - Figure 2.198) is able to cleave the first two fatty acids from the fat. A second enzyme, monoacylglycerol lipase, cleaves the last fatty acid. Fats can be synthesized by replacing the phosphate on phosphatidic acid with a fatty acid. Glycerophospholipids Glycerophospholipids (phosphoglycerides) are important components of the lipid bilayer of cellular membranes. Phosphoglycerides are structurally related to fats, as both are derived from phosphatidic acid (Figure 2.199). Phosphatidic acid is a simple glycerophospholipid that is usually converted into phosphatidyl compounds. These are made by esterifying various groups, such as ethanolamine, serine, choline, inositol, and others (Figure 2.200) to the phosphate of phosphatidic acid. All of these compounds form lipid bilayers in aqueous solution , due to their amphiphilic nature. Phosphatidylethanolamines
Since all glycerolipids can have a variety of fatty acids at positions 1 and 2 on the glycerol, they all are families of compounds. The phosphatidylethanolamines are found in all living cells and are one of the most common phosphatides, making up about 25% of them. They are common constituents of brain tissue and in the spinal cord, making up as much as 45% of the total phospholipids. Phosphatidylethanolamines are asymmetrically distributed across membranes, being preferentially located on the inner leaflet (closest to the cytoplasm) of the plasma membrane. Metabolically, phosphatidylethanloamines are precursors of phosphatidylcholines. Phosphatidylserines Phosphatidylserines are another group of phosphatidyl compounds that are preferentially distributed across the lipid bilayer of the plasma membrane. Like the phosphatidylethanolamines, phosphatidylserines are preferentially located on the inner leaflet of the plasma membrane. When apoptosis (cell suicide) occurs, the preferential distribution is lost and the phosphatidylserines appear on the outer leaflet where they serve as a signal to macrophages to bind and destroy the cell. Phosphatidylcholines Phosphatidylcholines (Figure 2.201) are another group of important membrane components. They tend to be found more commonly on the outer leaflet of the plasma membrane. Nutritionally, the compounds are readily obtained from eggs and soybeans. Phosphatidylcholines are moved across membranes by Phosphatidylcholine transfer protein (PCTP). This protein, which is sensitive to the levels of phosphatidylcholines, acts to stimulate the activity of a thioesterase (breaks thioester bonds, such as acyl-CoAs) and activates PAX3 transcription factors. Cardiolipins Cardiolipins are an unusual set of glycerophospholipids in containing two diacylglycerol backbones joined in the middle by a diphosphoglycerol (Figure 2.202). It is an important membrane lipid, constituting about 20% of the inner mitochondrial membrane and is found in organisms from bacteria to humans. In both plants and animals, it is found almost totally in the inner mitochondrial membrane. The molecules appear to be required for both Complex IV and Complex III of the electron transport chain to maintain its structure. The ATP synthase enzyme (Complex V) of the oxidative phosphorylation system also binds four molecules of cardiolipin. It has been proposed that cardiolipin functions as a proton trap in the process of proton pumping by Complex IV. Cardiolipin also plays a role in apoptosis. As shown in Figure 2.203, oxidation of cardiolipin by a cardiolipin-specific oxygenase causes cardiolipin to move from the inner mitochondrial membrane to the outer one, helping to form a permeable pore and facilitating the transport of cytochrome c out of the intermembrane space and into the cytoplasm - a step in the process of apoptosis. Diacylglycerol Diacylglycerol (also called diglyceride and DAG - Figure 2.204) is an important intermediate in metabolic pathways. It is produced, for example, in the first step of the hydrolysis of fat and is also produced when membrane lipids, such as PIP2 (phosphatidylinositol-4,5-bisphosphate) are hydrolyzed by phospholipase C in a signaling cascade. DAG is itself a signaling compound, binding to protein kinase C to activate it to phosphorylate substrates. Synthesis of DAG begins with glycerol-3-phosphate, which gains two fatty acids from two acyl-CoAs to form phosphatidic acid. Dephosphorylation of phosphatidic acid produces DAG. DAG can also be rephosphorylated by DAG kinase to re-make phosphatidic acid or another fatty acid can be added to make fat. Inositol Though technically not a lipid itself, inositol is found in many lipids. Inositol is a derivative of cyclohexane containing six hydroxyl groups - one on each carbon (Figure 2.205. It has nine different stereoisomers of which one, cis-1,2,3,5-trans-4,6- cyclohexanehexol (called myo-inositol) is the most common. It has a sweet taste (half that of sucrose). Numerous phosphorylated forms of the compound exist, from a single phosphate to six (one on each carbon). Phytic acid, for example, in plants, has six phosphates (Figure 2.206) that it uses to store phosphate. Inositol is produced from glucose and was once considered vitamin B8, but is made by the body in adequate amounts, so it is not now considered a vitamin. Phosphorylated forms of inositol are found in phosphoinositides, such as PIP2 and PIP3, both of which are important in signaling processes. Some of these include insulin signaling, fat catabolism, calcium regulation, and assembly of the cytoskeleton. Phosphoinositides Compounds based on phosphatidylinositol (PI) are often called phosphoinositides. These compounds have important roles in signaling and membrane trafficking. Hydroxyls on carbons 3,4, and 5 of the inositol ring are targets for phosphorylation by a variety of kinases. Seven different combinations are used. Steric hindrance inhibits phosphorylation of carbons 2 or 6. Naming of these phosphorylated compounds follows generally as PI(#P)P, PI(#P, #P)P, or PI(#P, #P, #P)P where #P refers to the number of the carbon where a phosphate is located. For example, PI(3)P refers to a phosphatidyl compound with a phosphate added to carbons 3 of the inositol ring, whereas PI(3,4,5)P is a phosphatidyl compound with a phosphate added to carbons 3,4,and 5. Phosphatidylinositol-4,5- bisphosphate Phosphatidylinositol-4,5-bisphosphate (PIP2 - Figure 2.207) is a phospholipid of plasma membranes that functions in the phospholipase C signaling cascade. In this signaling pathway, hydrolysis catalyzed by phospholipase C releases inositol-1,4,5- trisphosphate (IP3) and diacylglycerol. Synthesis of PIP2 begins with phosphatidylinositol, which is phosphorylated at position 4 followed by phosphorylation at position 5 by specific kinases. PIP2 can be phosphorylated to form the signaling molecule known as phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Along with PIP3, PIP2 serves as a docking phospholipid for the recruitment of proteins that play roles in signaling cascades. Binding of PIP2 is also required by inwardly directed potassium channels. Phosphatidylinositol (3,4,5)- trisphosphate
Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is an important molecule for the activation of signaling proteins, such as AKT, which activates anabolic signaling pathways related to growth and survival. PIP3 can be dephosphorylated by phosphatase PTEN to yield PIP2 and can be synthesized from PIP2 by kinase action of Class I PI 3- kinases. Kinase activity to synthesize PIP3 results in movement of PIP3-binding proteins to the plasma membrane. They include Akt/ PKB, PDK1, Btk1, and ARNO and each is activated by binding to PIP3. Plasmalogens A special class of the glycerophospholipids are the plasmalogens (Figure 2.209). They differ in containing a vinyl ether linkage at position 1 of glycerol, in contrast to other glycerophopsholipids, which have an ester linkage at this position. Position 2 of each is an ester. The precursor for the ether linkage is typically a 16 or 18 carbon saturated alcohol or an 18 carbon unsaturated alcohol. At the phosphate tail, the most commonly attached groups are ethanolamine or choline. Plasmalogens are found abundantly in humans in heart (30-40% of choline phospholipids). 30% of the glycerophospholipids in brain are plasmalogens and 70% of the ethanolamine lipids of the myelin sheath of nerve cells are plasmalogens. Though their function is not understood, it is believed that plasmalogens may provide some protection against reactive oxygen species and have roles in signaling. Lecithin Lecithin is a generic term for a combination of lipid substances that includes phosphoric acid, glycerol, glycolipids, triglycerides, and phospholipids. Lecithin is a wetting agent helpful with emulsification and encapsulation and is even used as an anti-sludge additive in motor lubricants. Lecithin is used in candy bars to keep cocoa and cocoa butter from separating. Though considered safe as a food ingredient, lecithin can be converted by gut bacteria to trimethylamine-N-oxide which may contribute to cholesterol deposition and atherosclerosis. Sphingolipids Fatty acids are also components of a broad class of molecules called sphingolipids. Sphingolipids are structurally similar to glycerophospholipids, though they are synthesized completely independently of them starting with palmitic acid and the amino acid serine. Sphingolipids are named for the amino alcohol known as sphingosine (Figure 2.210), though they are not directly synthesized from it. Figure 2.211 shows the generalized structure of sphingolipids. If the R-group is a hydrogen, the molecule is called a ceramide. When the R-group is phosphoethanolamine the resulting molecule is sphingomyelin, an important component of the myelin sheath and lipid membranes. If a single, simple sugar is instead added, a cerebroside is created (Figure 2.212). Addition of a complex oligosaccharide creates a ganglioside. Complex sphingolipids may play roles in cellular recognition and signaling. Sphingolipids are found most abundantly in plasma membrane and are almost completely absent from mitochondrial and endoplasmic reticulum membranes. In animals, dietary sphingolipids have been linked to reduced colon cancer, reductions in LDLs, and increases in HDLs. Like the glycerophospholipids, sphingolipids are amphiphilic. Most sphingolipids except sphingomyelin do not contain phosphate. Eicosanoids Fatty acids made from omega-6 and omega-3 fatty acids include three important fatty acids containing 20 carbons. They include arachidonic acid (an ω-6 fatty acid with four double bonds (Δ-5,8,11,14) - Figure 2.213), eicosapentaenoic acid (an ω-3 fatty acid with five double bonds, and dihomo-γ-linolenic acid (an ω-6 fatty acid with three double bonds). The class of compounds known as eicosanoids is made by oxidation of these compounds. Subclasses include include prostaglandins, prostacyclins, thromboxanes, lipoxins, leukotrienes, and endocannabinoids (Figures 2.214-2.219). Eicosanoids play important roles affecting inflammation, immunity, mood, and behavior. Prostaglandins A collection of molecules acting like hormones, prostaglandins are derived from arachidonic acid and have many differing (even conflicting) physiological effects. These include constriction or dilation of vascular smooth muscle cells, induction of labor, regulation of inflammation, and action on the thermoregulatory center of the hypothalamus to induce fever, among others. Prostaglandins are grouped with the thromboxanes (below) and prostacyclins (below), as prostanoids. The prostanoids, which all contain 20 carbons are a subclass of the eicosanoids. Prostaglandins are found in most tissues of higher organisms. They are autocrine or paracrine compounds produced from essential fatty acids. The primary precursor of the prostaglandins is the fatty acid known as arachidonic acid and the prostaglandin made from it is known as PGH2 (Figure 2.214), which, in turn is a precursor of other prostaglandins, as well as the prostacyclins and thromboxanes. Interesting prostaglandins PGD2 - inhibits hair follicle growth, vasodilator, causes bronchial constriction, higher in lungs of asthmatics than others. PGE2 (Figure 2.215) - exerts effects in labor (soften cervix, uterine contraction), stimulates bone resorption by osteoclasts, induces fever, suppresses T-cell receptor signaling, vasodilator, inhibits release of noradrenalin from sympathetic nerve terminals. It is a potent activator of the Wnt signaling pathway. A prostaglandin can have opposite effects, depending on which receptor it binds to. Binding of PGE2 to the EP1 receptor causes bronchoconstriction and smooth muscle contraction, whereas binding of the same molecule to the EP2 receptor causes bronchodilation and smooth muscle relaxation. PGF (Figure 2.216)- uterine contractions, induces labor, bronchoconstriction. PGI2 - vasodilation, bronchodilation, inhibition of platelet aggregation. Thromboxanes Thromboxanes play roles in clot formation and named for their role in thrombosis. They are potent vasoconstrictors and facilitate platelet aggregation. They are synthesized in platelets, as well. The anti-clotting effects of aspirin have their roots in the inhibition of synthesis of PGH2, which is the precursor of the thromboxanes. The most common thromboxanes are A2 (Figure 2.217) and B2. Prostacyclin
Prostacyclin (also known as prostaglandin I2 or PGI2 - Figure 2.218) counters the effects of thromboxanes, inhibiting platelet activation and acting as vasodilators. It is produced from PGH2 by action of the enzyme prostacyclin synthase. Leukotrienes Another group of eicosanoid compounds are the leukotrienes (Figure 2.219). Like prostaglandins, leukotrienes are made from arachidonic acid. The enzyme catalyzing their formation is a dioxygenase known as arachidonate 5-lipoxygenase. Leukotrienes are involved in regulating immune responses. They are found in leukocytes and other immunocompetent cells, such as neutrophils, monocytes, mast cells, eosinophils, and basophils. Leukotrienes are associated with production of histamines and prostaglandins, which act as mediators of inflammation. Leukotrienes also trigger contractions in the smooth muscles of the bronchioles. When overproduced, they may pay a role in asthma and allergic reactions. Some treatments for asthma aim at inhibiting production or action of leukotrienes. Cholesterol Arguably, no single biomolecule has generated as much discussion and interest as has cholesterol (Figure 2.220). Certainly, from the perspective of the Nobel Prize committee, no small molecule even comes close, with 13 people having been awarded prizes for work on it. Evidence for cholesterol’s importance comes from the study of brain tissue where it comprises 10-15% of the dry mass. Membrane flexibility In animal cells, cholesterol provides for membrane flexibility that allows for cellular movement that is in contrast to plant and bacterial cells with fixed structures. Cholesterol is made in many cells of the body, with the liver making the greatest amount. The anabolic pathway leading to synthesis of cholesterol is known as the isoprenoid pathway and branches of it lead to other molecules including other fat-soluble vitamins. Cholesterol is only rarely found in prokaryotes (Mycoplasma, which requires it for growth, is an exception) and is found in only trace amounts in plants. Instead, plants synthesize similar compounds called phytosterols (Figure 2.221). On average, the body of a 150 pound adult male makes about 1 gram of cholesterol per day, with a total content of about 35 grams. Packaging Cholesterol’s (and other lipids’) hydrophobicity requires special packaging into lipoprotein complexes (called chylomicrons, VLDLs, IDLs, LDLs, and HDLs) for movement in the lymph system and bloodstream. Though cholesterol can be made by cells, they also take it up from the blood supply by absorbing cholesterol-containing LDLs directly in a process called receptor-mediated endocytosis. Oxidative damage to LDLs can lead to formation of atherosclerotic plaques and this is why cholesterol has gotten such a negative image in the public eye. The liver excretes cholesterol through the bile for elimination into the digestive system, but the compound is recycled there. Reducing cholesterol levels Strategies for reducing cholesterol in the body focus primarily on three areas - reducing consumption, reducing endogenous synthesis, and reducing the recycling. Dietary considerations, such as saturated fat versus unsaturated fat consumption are currently debated. Dietary trans fats, though, correlate with incidence of coronary heart disease. Consumption of vegetables may provide some assistance with reducing levels of cholesterol recycled in the digestive system, because plant phytosterols compete with cholesterol for reabsorption and when this happens, a greater percentage of cholesterol exits the body in the feces. Drugs related to penicillin are also used to inhibit cholesterol recycling. One of these is ezetimibe, shown in Figure 2.224. Genetic defects in the cholesterol movement system are a cause of the rare disease known as familial hypercholesterolemia in which the blood of afflicted individuals contains dangerously high levels of LDLs. Left untreated, the disease is often fatal in the first 10-20 years of life. While LDLs have received (and deserve) a bad rap, another group of lipoprotein complexes known as the HDLs (high density lipoprotein complexes) are known as “good cholesterol” because their levels correlate with removal of debris (including cholesterol) from arteries and reduce inflammation. Membrane function In membranes, cholesterol is important as an insulator for the transmission of signals in nerve tissue and it helps to manage fluidity of membranes over a wide range of temperatures. Stacked in the lipid bilayer, cholesterol decreases a membrane’s fluidity and its permeability to neutral compounds, as well as protons and sodium ions. Cholesterol may play a role in signaling by helping with construction of lipid rafts within the cell membrane. Vitamin A Vitamin A comes in three primary chemical forms, retinol (storage in liver - Figure 2.225), retinal (role in vision - Figure 2.226), and retinoic acid (roles in growth and development). All vitamin A forms are diterpenoids and differ only in the chemical form of the terminal group. Retinol is mostly used as the storage form of the vitamin. Retinol is commonly esterified to a fatty acid and kept in the liver. In high levels, the compound is toxic. Retinol is obtained in the body by hydrolysis of the ester or by reduction of retinal. Importantly, neither retinal nor retinol can be made from retinoic acid. Retinoic acid is important for healthy skin and teeth, as well as bone growth. It acts in differentiation of stem cells through a specific cellular retinoic acid receptor. Sources Good sources of vitamin A are liver and eggs, as well as many plants, including carrots, which have a precursor, β-carotene (Figure 2.227) from which retinol may be made by action of a dioxygenase. Light sensitivity The conjugated double bond system in the side chain of vitamin A is sensitive to light and can flip between cis and trans forms on exposure to it. It is this response to light that makes it possible for retinal to have a role in vision in the rods and cones of the eyes. Here, the aldehyde form (retinal) is bound to the protein rhodopsin in the membranes of rod and cone cells.
When exposed to light of a particular wavelength, the “tail” of the retinal molecule will flip back and forth from cis to trans at the double bond at position 11 of the molecule. When this happens, a nerve signal is generated that signals the brain of exposure to light. Slightly different forms of rhodopsin have different maximum absorption maxima allowing the brain to perceive red, green and blue specifically and to assemble those into the images we see (Figure 2.228). Cones are the cells responsible for color vision, whereas rods are mostly involved in light detection in low light circumstances. Deficiency and surplus Deficiency of vitamin A is common in developing countries and was inspiration for the design and synthesis of the geneticallymodified golden rice, which is used as a source of vitamin A to help prevent blindness in children. Overdose of vitamin A, called hypervitaminosis A is dangerous and can be fatal. Excess vitamin A is also suspected to be linked to osteoporosis. In smokers, excess vitamin A is linked to an increased rate of lung cancer, but non-smokers have a reduced rate. Vitamin D The active form of vitamin D plays important roles in the intestinal absorption of calcium and phosphate and thus in healthy bones. Technically, vitamin D isn’t even a vitamin, as it is a compound made by the body. Rather, it behaves more like a hormone. Derived from ultimately from cholesterol, vitamin D can be made in a reaction catalyzed by ultraviolet light. In the reaction, the intermediate 7-dehydrocholesterol is converted to cholecalciferol (vitamin D3) by the uv light (Figure 2.229). The reaction occurs most readily in the bottom two layers of the skin shown in Figure 2.230. Forms of vitamin D Five different compounds are referred to as vitamin D. They are Vitamin D1 - A mixture of ergocalciferol and lumisterol Vitamin D2 - Ergocalciferol Vitamin D3 - Cholecalciferol Vitamin D4 - 22-Dihydroergocalciferol Vitamin D5 - Sitocalciferol Vitamin D3 is the most common form used in vitamin supplements and it and vitamin D2 are commonly obtained in the diet, as well. The active form of vitamin D, calcitriol (Figure 2.231), is made in the body in controlled amounts. This proceeds through two steps from cholecalciferol. First, a hydroxylation in the liver produces calcidiol and a second hydroxylation in the kidney produces calcitriol. Monocyte macrophages can also synthesize vitamin D and they use is as a cytokine to stimulate the innate immune system. Mechanism of action Calcitriol moves in the body bound to a vitamin D binding protein, which delivers it to target organs. Calcitriol inside of cells acts by binding a vitamin D receptor (VDR), which results in most of the vitamin’s physiological effects. After binding calcitriol, the VDR migrates to the nucleus where it acts as a transcription factor to control levels of expression of calcium transport proteins (for example) in the intestine. Most tissues respond to VDR bound to calcitriol and the result is moderation of calcium and phosphate levels in cells. Deficiency/excess Deficiency of vitamin D is a cause of the disease known as rickets, which is characterized by soft, weak bones and most commonly is found in children. It is not common in the developed world, but elsewhere is of increasing concern. Excess of vitamin D is rare, but has toxic effects, including hypercalcemia, which results in painful calcium deposits in major organs. Indications of vitamin D toxicity are increased urination and thirst. Vitamin D toxicity can lead to mental retardation and many other serious health problems. Vitamin E Vitamin E comprises a group of two compounds (tocopherols and tocotrienols - Figure 2.232) and stereoisomers of each. It is commonly found in plant oils. The compounds act in cells as fat-soluble antioxidants. α-tocopherol (Figure 2.233), the most active form of the vitamin, works 1) through the glutathione peroxidase protective system and 2) in membranes to interrupt lipid peroxidation chain reactions. In both actions, vitamin E reduces levels of reactive oxygen species in cells. Action Vitamin E scavenges oxygen radicals (possessing unpaired electrons) by reacting with them to produce a tocopheryl radical. This vitamin E radical can be converted back to its original form by a hydrogen donor. Vitamin C is one such donor. Acting in this way, Vitamin E helps reduce oxidation of easily oxidized compounds in the lipid peroxidation reactions (Figure 2.234). Vitamin E also can affect enzyme activity. The compound can inhibit action of protein kinase C in smooth muscle and simultaneously activate catalysis of protein phosphatase 2A to remove phosphates, stopping smooth muscle growth. Deficiency/excess Deficiency of vitamin E can lead to poor conduction of nerve signals and other issues arising from nerve problems. Low levels of the vitamin may be a factor in low birth weights and premature deliveries. Deficiency, however, is rare, and not usually associated with diet. Excess Vitamin E reduces vitamin K levels, thus reducing the ability to clot blood. Hypervitaminosis of vitamin E in conjunction with aspirin can be life threatening. At lower levels, vitamin E may serve a preventative role with respect to atherosclerosis by reducing oxidation of LDLs, a step in plaque formation. Vitamin K Like the other fat-soluble vitamins, Vitamin K comes in multiple forms (Figure 2.235) and is stored in fat tissue in the body. There are two primary forms of the vitamin - K1 and K2 and the latter has multiple sub-forms . Vitamins K3, K4, and K5 are made synthetically, not biologically. Action Vitamin K is used as a co-factor for enzymes that add carboxyl groups to glutamate side chains of proteins to increase their affinity for calcium. Sixteen such proteins are known in humans. They include proteins involved in blood clotting (prothrombin (called Factor II), Factors VII, IX, and X), bone metabolism (osteocalcin, also called bone Gla protein (BGP), matrix Gla protein (MGP), and periostin) and others.
Modification of prothrombin is an important step in the process of blood clotting (see HERE). Reduced levels of vitamin K result in less blood clotting, a phenomenon sometimes referred to as blood thinning. Drugs that block recycling of vitamin K (Figure 2.236) by inhibiting the vitamin K epoxide reductase, produce lower levels of the vitamin and are employed in treatments for people prone to excessive clotting. Warfarin (coumadin) is one such compound that acts in this way and is used therapeutically. Individuals respond to the drug differentially, requiring them to periodically be tested for levels of clotting they possess, lest too much or too little occur. Sources Vitamin K1 is a stereoisomer of the plant photosystem I electron receptor known as phylloquinone and is found abundantly in green, leafy vegetables. Phylloquinone is one source of vitamin K, but the compound binds tightly to thylakoid membranes and tends to have low bioavailability. Vitamin K2 is produced by microbes in the gut and is a primary source of the vitamin. Infants in the first few days before they establish their gut flora and people taking broad spectrum antibiotics may have reduced levels, as a result. Dietary deficiency is rare in the absence of damage to the small bowel. Others at risk of deficiency include people with chronic kidney disease and anyone suffering from a vitamin D deficiency. Deficiencies produce symptoms of easy bruising, heavy menstrual bleeding, anemia, and nosebleeds. Steroids Steroids, such as cholesterol are found in membranes and act as signaling hormones in traveling through the body. Steroid hormones are all made from cholesterol and are grouped into five categories - mineralocorticoids (21 carbons), glucocorticoids (21 carbons), progestagens (21 carbons), androgens (19 carbons), and estrogens (18 carbons). Mineralocorticoids Mineralocorticoids are steroid hormones that influence water and electrolyte balances. Aldosterone (Figure 2.238) is the primary mineralocorticoid hormone, though other steroid hormones (including progesterone) have some functions like it. Aldosterone stimulates kidneys to reabsorb sodium, secrete potassium, and passively reabsorb water. These actions have the effect of increasing blood pressure and blood volume. Mineralocorticoids are produced by the zona glomerulosa of the cortex of the adrenal gland. Glucocorticoids Glucocorticoids (GCs) bind to glucocorticoid receptors found in almost every vertebrate animal cell and act in a feedback mechanism in the immune system to reduce its activity. GCs are used to treat diseases associated with overactive immune systems. These include allergies, asthma, and autoimmune dis- Figure 2.237 - Steroid numbering scheme Image by Pehr Jacobson eases. Cortisol (Figure 2.239) is an important glucocorticoid with cardiovascular, metabolic, and immunologic functions. The synthetic glucocorticoid known as dexamethasone has medical applications for treating rheumatoid arthritis, bronchospasms (in asthma), and inflammation due to its increased potency (25-fold) compared to cortisol. Glucocorticoids are produced primarily in the zona fasciculata of the adrenal cortex. Progestagens Progestagens (also called gestagens) are steroid hormones that work to activate the progesterone receptor upon binding to it. Synthetic progestagens are referred to as progestins. The most common progestagen is progesterone (also called P4 - Figure 2.240) and it has functions in maintaining pregnancy. Progesterone is produced primarily in the diestrus phase of the estrous cycle by the corpus luteum of mammalian ovaries. In pregnancy, the placenta takes over most progesterone production. Androgens Androgens are steroid hormones that act by binding androgen receptors to stimulate development and maintenance of male characteristics in vertebrates. Androgens are precursors of estrogens (see below). The primary androgen is testosterone (Figure 2.241). Other important androgens include dihydrotestosterone (stimulates differentiation of penis, scrotum, and prostate in embryo) and androstenedione (common precursor of male and female hormones). Estrogens The estrogen steroid hormones are a class of compounds with important roles in menstrual and estrous cycles. They are the most important female sex hormones. Estrogens act by activating estrogen receptors inside of cells. These receptors, in turn, affect expression of many genes. The major estrogens in women include estrone (E1), estradiol (E2 - Figure 2.242), and estriol (E3). In the reproductive years, estradiol predominates. During pregnancy, estriol predominates and during menopause, estrone is the major estrogen. Estrogens are made from the androgen hormones testosterone and androstenedione in a reaction catalyzed by the enzyme known as aromatase. Inhibition of this enzyme with aromatase inhibitors, such as exemestane, is a strategy for stopping estrogen production. This may be part of a chemotherapeutic treatment when estrogenresponsive tumors are present. Cannabinoids Cannabinoids are a group of chemicals that bind to and have effects on brain receptors (cannabinoid receptors), repressing neurotransmitter release. Classes of these compounds include endocannabinoids (made in the body), phytocannabinoids (made in plants, such as marijuana), and synthetic cannabinoids (man-made). Endocannabinoids are natural molecules derived from arachidonic acid. Cannabinoid receptors are very abundant, comprising the largest number of G-protein- 247 Figure 2.243 - Tetrahydrocannabinol - Active ingredient in marijuana coupled receptors found in brain. The best known phytocannabinoid is Δ-9- tetrahydrocannabinol (THC), the primary psychoactive ingredient (of the 85 cannabinoids) of marijuana (Figure 2.243). Anandamide Anandamide (N-arachidonoylethanolamine - Figure 2.244) is an endocannabinoid neurotransmitter derived from arachidonic acid. It exerts its actions primarily through the CB1 and CB2 cannabinoid receptors, the same ones bound by the active ingredient in marijuana, Δ9-tetrahydrocannabinol. Anandamide has roles in stimulating eating/appetite and affecting motivation and pleasure. It has been proposed to play a role in “runners high,” an analgesic effect experienced from exertion, especially among runners. Anandamide appears to impair memory function in rats.
Anandamide has been found in chocolate and two compounds that mimic its effects (N-oleoylethanolamine and Nlinoleoylethanolamine) are present as well. The enzyme fatty acid amide hydrolase (FAAH) breaks down anandamide into free arachidonic acid and ethanolamine. Lipoxins Lipoxins (Figure 2.245) are eicosanoid compounds involved in modulating immune responses and they have anti-inflammatory effects. When lipoxins appear in inflammation it begins the end of the process. Lipoxins act to attract macrophages to apoptotic cells at the site of inflammation and they are engulfed. Lipoxins further act to start the resolution phase of the inflammation process. At least one lipoxin (aspirin-triggered LX4) has its synthesis stimulated by aspirin. This occurs as a byproduct of aspirin’s acetylation of COX-2. When this occurs, the enzyme’s catalytic activity is re-directed to synthesis of 15R-hydroxyeicosatetraenoic acid (HETE) instead of prostaglandins. 15R-HETE is a procursor of 15-epimer lipoxins, including aspirin-triggered LX4. Heme Heme groups are a collection of protein/ enzyme cofactors containing a large heterocyclic aromatic ring known as a porphyrin ring with a ferrous (Fe++) ion in the middle. An example porphyrin ring with an iron (found in Heme B of hemoglobin), is shown in Figure 2.246. When contained in a protein, these are known collectively as hemoproteins (Figure 2.247). Heme, of course, is a primary component of hemoglobin, but it is also found in other proteins, such as myoglobin, cytochromes, and the enzymes catalase and succinate dehydrogenase. Hemoproteins function in oxygen transport, catalysis, and electron transport. Heme is synthesized in the liver and bone marrow in a pathway that is conserved across a wide range of biology. Porphobilinogen Porphobilinogen (Figure 2.248) is a pyrrole molecule involved in porphyrin metabolism. It is produced from aminolevulinate by action of the enzyme known as ALA dehydratase. Porphobilinogen is acted upon by the enzyme porphobilinogen deaminase. Deficiency of the latter enzyme (and others in porphyrin metabolism) can result in a condition known as porphyria, which results in accumulation of porphobilinogen in the cytoplasm of cells. The disease can manifest itself with acute abdominal pain and numerous psychiatric issues. Both Vincent van Gogh and King ` George III are suspected to have suffered from porphyria, perhaps causing the “madness of King George III.” Porphyria is also considered by some to be the impetus for the legend of vampires seeking blood from victims, since the color of the skin in non-acute forms of the disease can be miscolored, leading some to perceive that as a deficiency of hemoglobin and hence the “thirst” for blood imagined for vampires. Dolichols Dolichol is a name for a group of non-polar molecules made by combining isoprene units together. Phosphorylated forms of dolichols play central roles in the N-glycosylation of proteins. This process, which occurs in the endoplasmic reticulum of eukaryotic cells, begins with a membrane-embedded dolichol pyrophosphate (Figure 2.249) to which an oligosaccharide is attached (also see HERE). This oligosaccharide contains three molecules of glucose, nine molecules of mannose and two molecules of N-acetylglucosamine. Interestingly, the sugars are attached to the dolichol pyrophosphate with the pyrophosphate pointing outwards (away from) the endoplasmic reticulum, but after attachment, the dolichol complex flips so that the sugar portion is situated on the inside of the endoplasmic reticulum. There, the entire sugar complex is transferred to the amide of an asparagine side chain of a target protein. The only asparagine side chains to which the attachment can be made are in proteins where the sequences Asn-X-Ser or Asn-X-Thr occur. Sugars can be removed/added after the transfer to the protein. Levels of dolichol in the human brain increase with age, but in neurodegenerative diseases, they decrease. Terpenes Terpenes are members of a class of nonpolar molecules made from isoprene units. Terpenes are produced primarily by plants and by some insects. Terpenoids are a related group of molecules that contain functional groups lacking in terpenes. Terpenes have a variety of functions. In plants, they often play a defensive role protecting from insects. The name of terpene comes from turpentine, which has an odor like some of the terpenes. Terpenes are common components of plant resins (think pine) and they are widely used in medicines and as fragrances. Hops, for example, gain some of their distinctive aroma and flavor from terpenes. Not all terpenes, however have significant odor. Synthesis Terpenes, like steroids, are synthesized starting with simple building blocks known as isoprenes. There are two of them - dimethylallyl pyrophosphate and the related isopentenyl pyrophosphate and (Figures 2.252 and 2.253) which combine 1-2 units at a time to make higher order structures. Terpene synthesis overlaps and includes steroid synthesis. Terpenes and terpenoids are classified according to how many isoprene units they contain. They include hemiterpenes (one unit), monoterpenes (two units), sesquiterpenes (three units), diterpenes (four units), sesterterpenes (five units), triterpenes (six units), sesquarterpenes (seven units), tetraterpenes (eight units), polyterpenes (many units). Another class of terpene-containing molecules, the norisoterpenoids arise from peroxidase-catalyzed reactions on terpene molecules. Examples Common terpenes include monoterpenes of terpineol (lilacs), limonene (citrus), myrcene (hops), linalool (lavender), and pinene (pine). Higher order terpenes include taxadiene (diterpene precursor of taxol), lycopene (tetraterpenes), carotenes (tetraterpenes), and natural rubber (polyterpenes). Steroid precursors geranyl pyrophosphate (monoterpene derivative), farnesyl pyrophosphate (sesquiterpene derivative), and squalene (triterpene) are all terpenes or derivatives of them. Vitamin A and phytol are derived from diterpenes. Caffeine
Caffeine is the world’s most actively consumed psychoactive drug (Figure 2.255). A methylxanthine alkaloid, caffeine is closely related to adenine and guanine and this is responsible for many effects on the body. Caffeine blocks the binding of adenosine on its receptor and consequently prevents the onset of drowsiness induced by adenosine. Caffeine readily crosses the blood-brain barrier and stimulates release of neurotransmitters. Caffeine stimulates portions of the autonomic nervous system and inhibits the activity of phosphodiesterase. The latter has the result of raising cAMP levels in cells, which activates protein kinase A and activates glycogen breakdown, inhibits TNF-α and leukotriene synthesis, which results in reduction of inflammation and innate immunity. Caffeine also has effects on the cholinergic system (acetylcholinesterase inhibitor), is an inositol triphosphate receptor 1 antagonist, and is a voltage independent activator of ryanodin receptors (a group of calcium channels found in skeletal muscle, smooth muscle, and heart muscle cells). The half-life of caffeine in the body varies considerably. In healthy adults, it has a half-life of about 3-7 hours. Nicotine decreases the half-life and contraceptives and pregnancy can double it. The liver metabolizes caffeine, so the health of the liver is a factor in the halflife. CYP1A2 of the cytochrome P450 oxidase enzyme is primarily responsible. Caffeine is a natural pesticide in plants, paralyzing predator bugs. Lipoprotein complexes and lipid movement in the body Lipoprotein complexes are combinations of apolipoproteins and lipids bound to them that solubilize fats and other non-polar molecules, such as cholesterol, so they can travel in the bloodstream between various tissues of the body. The apolipoproteins provide the emulsification necessary for this. Lipoprotein complexes are formed in tiny “balls” with the water soluble apolipoproteins on the outside and non-polar lipids, such as fats, cholesteryl esters, and fat soluble vitamins on the inside. They are categorized by their densities. These include (from highest density to the lowest) high density lipoproteins (HDLs), Low Density Lipoproteins (LDLs), Intermediate Density Lipoproteins (IDLs), Very Low Density Lipoproteins (VLDLs) and the chylomicrons. These particles are synthesized in the liver and small intestines. Apolipoproteins Each lipoprotein complex contain a characteristic set of apolipoproteins, as shown in Figure 2.256. ApoC-II and ApoC-III are notable for their presence in all the lipoprotein complexes and the roles they play in activating (ApoC-II) or inactivating (ApoC-III) lipoprotein lipase. Lipoprotein lipase is a cellular enzyme that catalyzes the breakdown of fat from the complexes. ApoE (see below) is useful for helping the predict the likelihood of the occurrence of Alzheimer's disease in a patient. Gene editing ApoB-48 and ApoB-100 are interesting in being coded by the same gene, but a unique mRNA sequence editing event occurs that converts one into the other. ApoB-100 is made in the liver, but ApoB-48 is made in the small intestine. The small intestine contains an enzyme that deaminates the cytidine at nucleotide #2153 of the common mRNA. This changes it to a uridine and changes the codon it is in from CAA (codes for glutamine) to UAA (stop codon). The liver does not contain this enzyme and does not make the change in the mRNA. Consequently, a shorter protein is synthesized in the intestine (ApoB-48) than the one that is made in the liver (ApoB-100) using the same gene sequence in the DNA. Movement The movement of fats in the body is important because they are not stored in all cells. Only specialized cells called adipocytes store fat. There are three relevant pathways in the body for moving lipids. As described below, they are 1) the exogenous pathway; 2) the endogenous pathway, and 3) the reverse transport pathway. Exogenous pathway Dietary fat entering the body from the intestinal system must be transported, as appropriate, to places needing it or storing it. This is the function of the exogenous pathway of lipid movement in the body. All dietary lipids (fats, cholesterol, fat soluble vitamins, and other lipids) are moved by it. In the case of dietary fat, it begins its journey after ingestion first by being solubilized by bile acids in the intestinal tract. After passing through the stomach, pancreatic lipases clip two fatty acids from the fat, leaving a monoacyl glycerol. The fatty acids and monoacyl glycerol are absorbed by intestinal cells (enterocytes) and reassembled back into a fat, and then this is mixed with phospholipids, cholesterol esters, and apolipoprotein B-48 and processed to form chylomicrons (Figures 2.258 & 2.259) in the Golgi apparatus and smooth endoplasmic reticulum. Exocytosis These are exocytosed from the cell into lymph capillaries called lacteals. The chylomicrons pass through the lacteals and enter the bloodstream via the left subclavian vein. Within the bloodstream, lipoprotein lipase breaks down the fats causing the chylomicron to shrink and become what is known as a chylomicron remnant. It retains its cholesterol and other lipid molecules. The chylomicron remnants travel to the liver where they are absorbed (Figure 2.260). This is accomplished by receptors in the liver that recognize and bind to the ApoE of the chylomicrons. The bound complexes are then internalized by endocytosis, degraded in the lysosomes, and the cholesterol is disbursed in liver cells. Endogenous pathway
The liver plays a central role in managing the body’s needs for lipids. When lipids are needed by the body or when the capacity of the liver to contain more lipids than is supplied by the diet, the liver packages up fats and cholesteryl esters into Very Low Density Lipoprotein (VLDL) complexes and exports them via the endogenous pathway. VLDL complexes contain ApoB-100, ApoC-I, ApoC-II, ApoC-III, and ApoE apolipoproteins. VLDLs enter the blood and travel to muscles and adipose tissue where lipoprotein lipase is activated by ApoC-II. In the muscle cells, the released fatty acids are taken up and oxidized. By contrast, in the adipoctyes, the fatty acids are taken up and reassembled back into triacylglycerides (fats) and stored in fat droplets. Removal of fat from the VLDLs causes them to shrink, first to Intermediate Density Lipoprotein (IDL) complexes (also called VLDL remnants) and then to Low Density Lipoprotein (LDL) complexes. Shrinking of VLDLs is accompanied by loss of apolipoproteins so that LDLs are comprised primarily of ApoB-100. This lipoprotein complex is important because cells have receptors for it to bind and internalize it by receptor-mediated endocytosis (Figure 2.261). Up until this point, cholesterol and cholesteryl esters have traveled in chylomicrons, VLDLs, and IDLs as fat has been stripped stripped away. For cholesterol compounds to get into the cell from the lipoprotein complexes, they must be internalized by cells and that is the job of receptormediated endocytosis. Reverse transport pathway Another important consideration of the movement of lipids in the body is the reverse transport pathway (Figure 2.260). It is also called the reverse cholesterol transport pathway, since cholesterol is the primary molecule involved. This pathway involves the last class of lipoprotein complexes known as the High Density Lipoproteins (HDLs). In contrast to the LDLs, which are commonly referred to as “bad cholesterol” (see below also), the HDLs are known as “good cholesterol.” HDLs are synthesized in the liver and small intestine. They contain little or no lipid when made (called depleted HDLs), but serve the role of “scavenger” for cholesterol in the blood and from remnants of other (damaged) lipoprotein complexes in the blood. To perform its task, HDLs carry the enzyme known as lecithincholesterol acyl transferase (LCAT), which they use to form cholesteryl esters using fatty acids from lecithin (phosphatidylcholine) and then they internalize them. The cholesterol used for this purpose comes from the bloodstream, from macrophages, and from foam cells (macrophage-LDL complexes - Figure 2.262). Addition of cholesteryl esters causes the HDL to swell and Figure 2.261 - The process of receptor-mediated endocytosis Image by Aleia Kim when it is mature, it returns its load of cholesterol back to the liver or, alternatively, to LDL molecules for endocytosis. HDLs have the effect of lowering levels of cholesterol and it is for that reason they are described as “good cholesterol.” Regulation of lipid transfer It is important that cells get food when they need it so some control of the movement of nutrients is critical. The liver, which plays the central role in modulating blood glucose levels, is also important for performing the same role for lipids. It accomplishes this task the use of specialized LDL receptors on its surface. Liver LDL receptors bind LDLs that were not taken up by other cells in their path through the bloodstream. High levels of LDLs are a signal to the liver to reduce the creation of VLDLs for release. People with the genetic disease known as familial hypercholesterolemia, which manifests with dangerously high levels of LDLs, lack properly functioning LDL receptors on their liver cells.Figure 2.263 - Progression of atherosclerosis Wikipedia Figure 2.263 - Progression of atherosclerosis Wikipedia Figure 2.263 - Progression of atherosclerosis Wikipedia In sufferers of this disease, the liver never gets the signal that the LDL levels are high. In fact, to the liver, it appears that all VLDLs and LDLs are being taken up by peripheral tissues, so it creates more VLDLs to attempt to boost levels. Untreated, the disease used to be fatal early, but newer drugs like the statins have significantly increased life spans of patients. Cellular needs for the contents of LDLs are directly linked to the levels of synthesis of LDL receptors on their membranes. As cells are needing more cholesterol, their synthesis of components for receptors goes up and it decreases as need diminishes. Good cholesterol / bad cholesterol It is commonly accepted that “high cholesterol” levels are not healthy. This is due, at least indirectly, to the primary carriers of cholesterol, the LDLs. A primary function of the LDLs is to deliver cholesterol and other lipids directly into cells by receptor mediated endocytosis (Figure 2.237). High levels of LDLs, though, are correlated with formation of atherosclerotic plaques (Figure 2.263 & 2.264) and incidence of atherosclerosis, leading to the description of them as “bad cholesterol.” This is because when LDL levels are very high, plaque formation begins. It is thought that reactive oxygen species (higher in the blood of smokers) causes partial oxidation of fatty acid groups in the LDLs. When levels are high, they tend to accumulate in the extracellular matrix of the epithelial cells on the inside of the arteries. Macrophages of the immune system take up the damaged LDLs (including the cholesterol). Since macrophages can’t control the amount of cholesterol they take up, cholesterol begins to accumulate in them and they take on appearance that leads to their being described as “foam cells.” With too much cholesterol, the foam cells, however, are doomed to die by the process of programmed cell death (apoptosis). Accumulation of these, along with scar tissue from inflammation result in formation of a plaque. Plaques can grow and block the flow of blood or pieces of them can break loose and plug smaller openings in the blood supply, ultimately leading to heart attack or stroke. Good cholesterol
On the other hand, high levels of HDL are inversely correlated with atherosclerosis and arterial disease. Depleted HDLs are able to remove cholesterol from foam cells. This occurs as a result of contact between the ApoA-I protein of the HDL and a transport protein on the foam cell (ABC-G1). Another transport protein in the foam cell, ABCA-1 transports extra cholesterol from inside the cell to the plasma membrane where it is taken up into the HDL and returned to the liver or to LDLs by the reverse transport cholesterol pathway. Deficiency of the ABCA-1 gene leads to Tangier disease. In this condition, HDLs are almost totally absent because they remain empty as a result of not being able to take up cholesterol from foam cells, so they are destroyed by the body. ApoE and Alzheimer’s disease ApoE is a component of the chylomicrons and is also found in brain, macrophages, kidneys, and the spleen. In humans, it is found in three different alleles, E2, E3, and E4. The E4 allele (present at about 14% of the population) is associated with increased likelihood of contracting Alzheimer's disease. People heterozygous for the allele are 3 times as likely to contract the disease and those homozygous for it are 15 times as likely to do so. It is not known why this gene or allele is linked to the disease. The three alleles differ only slightly in amino acid sequence, but the changes do cause notable structural differences. The E4 allele is associated with increased calcium ion levels and apoptosis after injury. Alzheimer’s disease is associated with accumulation of aggregates of the β- amyloid peptide. ApoE does enhance the proteolytic breakdown of it and the E4 isoform is not as efficient in these reactions as the other isoforms.
Source: BiochemFFA_2_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy All of the proteins on the face of the earth are made up of the same 20 amino acids. Linked together in long chains called polypeptides, amino acids are the building blocks for the vast assortment of proteins found in all living cells. "It is one of the more striking generalizations of biochemistry ...that the twenty amino acids and the four bases, are, with minor reservations, the same throughout Nature." - Francis Crick All amino acids have the same basic structure, which is shown in Figure 2.1. At the “center” of each amino acid is a carbon called the α carbon and attached to it are four groups - a hydrogen, an α- carboxyl group, an α-amine group, and an R-group, sometimes referred to as a side chain. The α carbon, carboxyl, and amino groups are common to all amino acids, so the R-group is the only unique feature in each amino acid. (A minor exception to this structure is that of proline, in which the end of the R-group is attached to the α-amine.) With the exception of glycine, which has an R-group consisting of a hydrogen atom, all of the amino acids in proteins have four different groups attached to them and consequently can exist in two mirror image forms, L and D. With only very minor exceptions, every amino acid found in cells and in proteins is in the L configuration. There are 22 amino acids that are found in proteins and of these, only 20 are specified by the universal genetic code. The others, selenocysteine and pyrrolysine use tRNAs that are able to base pair with stop codons in the mRNA during translation. When this happens, these unusual amino acids can be incorporated into proteins. Enzymes containing selenocysteine, for example, include glutathione peroxidases, tetraiodothyronine 5' deiodinases, thioredoxin reductases, formate dehydrogenases, glycine reductases, and selenophosphate synthetase. Pyrrolysine-containing proteins are much rarer and are mostly confined to archaea. Essential and non-essential Nutritionists divide amino acids into two groups - essential amino acids (must be in the diet because cells can’t synthesize them) and non-essential amino acids (can be made by cells). This classification of amino acids has little to do with the structure of amino acids. Essential amino acids vary considerable from one organism to another and even differ in humans, depending on whether they are adults or children. Table 2.1 shows essential and non-essential amino acids in humans. Some amino acids that are normally nonessential, may need to be obtained from the diet in certain cases. Individuals who do not synthesize sufficient amounts of arginine, cysteine, glutamine, proline, selenocysteine, serine, and tyrosine, due to illness, for example, may need dietary supplements containing these amino acids. Table 2.1 - Essential and non-essential amino acids Non-protein amino acids There are also α-amino acids found in cells that are not incorporated into proteins. Common ones include ornithine and citrulline. Both of these compounds are intermediates in the urea cycle. Ornithine is a metabolic precursor of arginine and citrulline can be produced by the breakdown of arginine. The latter reaction produces nitric oxide, an important signaling molecule. Citrulline is the metabolic byproduct. It is sometimes used as a dietary supplement to reduce muscle fatigue. R-group chemistry Table 2.2 - Amino acid categories (based on R-group properties) We separate the amino acids into categories based on the chemistry of their R-groups. If you compare groupings of amino acids in different textbooks, you will see different names for the categories and (sometimes) the same amino acid being categorized differently by different authors. Indeed, we categorize tyrosine both as an aromatic amino acid and as a hydroxyl amino acid. It is useful to classify amino acids based on their R-groups, because it is these side chains that give each amino acid its characteristic properties. Thus, amino acids with (chemically) similar side groups can be expected to function in similar ways, for example, during protein folding. Non-polar amino acids
• Alanine (Ala/A) is one of the most abundant amino acids found in proteins, ranking second only to leucine in occurrence. A D-form of the amino acid is also found in bacterial cell walls. Alanine is non-essential, being readily synthesized from pyruvate. It is coded for by GCU, GCC, GCA, and GCG. • Glycine (Gly/G) is the amino acid with the shortest side chain, having an R-group consistent only of a single hydrogen. As a result, glycine is the only amino acid that is not chiral. Its small side chain allows it to readily fit into both hydrophobic and hydrophilic environments. • Glycine is specified in the genetic code by GGU, GGC, GGA, and GGG. It is nonessential to humans. • Isoleucine (Ile/I) is an essential amino acid encoded by AUU, AUC, and AUA. It has a hydrophobic side chain and is also chiral in its side chain. • Leucine (Leu/L) is a branched-chain amino acid that is hydrophobic and essential. Leucine is the only dietary amino acid reported to directly stimulate protein synthesis in muscle, but caution is in order, as 1) there are conflicting studies and 2) leucine toxicity is dangerous, resulting in "the four D's": diarrhea, dermatitis, dementia and death . Leucine is encoded by six codons: UUA,UUG, CUU, CUC, CUA, CUG. • Methionine (Met/M) is an essential amino acid that is one of two sulfurcontaining amino acids - cysteine is the other. Methionine is non-polar and encoded solely by the AUG codon. It is the “initiator” amino acid in protein synthesis, being the first one incorporated into protein chains. In prokaryotic cells, the first methionine in a protein is formylated. • Proline (Pro/P) is the only amino acid found in proteins with an R-group that joins with its own α-amino group, making a secondary amine and a ring. Proline is a non-essential amino acid and is coded by CCU, CCC, CCA, and CCG. It is the least flexible of the protein amino acids and thus gives conformational rigidity when present in a protein. Proline’s presence in a protein affects its secondary structure. It is a disrupter of α-helices and β-strands. Proline is often hydroxylated in collagen (the reaction requires Vitamin C - ascorbate) and this has the effect of increasing the protein’s conformational stability. Proline hydroxylation of hypoxia-inducible factor (HIF) serves as a sensor of oxygen levels and targets HIF for destruction when oxygen is plentiful. • Valine (Val/V) is an essential, non-polar amino acid synthesized in plants. It is noteworthy in hemoglobin, for when it replaces glutamic acid at position number six, it causes hemoglobin to aggregate abnormally under low oxygen conditions, resulting in sickle cell disease. Valine is coded in the genetic code by GUU, GUC, GUA, and GUG. Carboxyl Amino Acids • Aspartic acid (Asp/D) is a non-essential amino acid with a carboxyl group in its Rgroup. It is readily produced by transamination of oxaloacetate. With a pKa of 3.9, aspartic acid’s side chain is negatively charged at physiological pH. Aspartic acid is specified in the genetic code by the codons GAU and GAC. • Glutamic acid (Glu/E), which is coded by GAA and GAG, is a non-essential amino acid readily made by transamination of α- ketoglutarate. It is a neurotransmitter and has an R-group with a carboxyl group that readily ionizes (pKa = 4.1) at physiological pH. Amine amino acids • Arginine (Arg/R) is an amino acid that is, in some cases, essential, but non-essential in others. Premature infants cannot synthesize arginine. In addition, surgical trauma, sepsis, and burns increase demand for arginine. Most people, however, do not need arginine supplements. Arginine’s side chain contains a complex guanidinium group with a pKa of over 12, making it positively charged at cellular pH. It is coded for by six codons - CGU, CGC, CGA, CGG, AGA, and AGG. • Histidine (His/H) is the only one of the proteinaceous amino acids to contain an imidazole functional group. It is an essential amino acid in humans and other mammals. With a side chain pKa of 6, it can easily have its charge changed by a slight change in pH. Protonation of the ring results in two NH structures which can be drawn as two equally important resonant structures. • Lysine (Lys/K) is an essential amino acid encoded by AAA and AAG. It has an Rgroup that can readily ionize with a charge of +1 at physiological pH and can be posttranslationally modified to form acetyllysine, hydroxylysine, and methyllysine. It can also be ubiquitinated, sumoylated, neddylated, biotinylated, carboxylated, and pupylated, and. O-Glycosylation of hydroxylysine is used to flag proteins for export from the cell. Lysine is often added to animal feed because it is a limiting amino acid and is necessary for optimizing growth of pigs and chickens. Aromatic amino acids • Phenylalanine (Phe/ F) is a non-polar, essential amino acid coded by UUU and UUC. It is a metabolic precursor of tyrosine. Inability to metabolize phenylalanine arises from the genetic disorder known as phenylketonuria. Phenylalanine is a component of the aspartame artificial sweetener. • Tryptophan (Trp/W) is an essential amino acid containing an indole functional group. It is a metabolic precursor of serotonin, niacin, and (in plants) the auxin phytohormone. Though reputed to serve as a sleep aid, there are no clear research results indicating this. • Tyrosine (Tyr/Y) is a non-essential amino acid coded by UAC and UAU. It is a target for phosphorylation in proteins by tyrosine protein kinases and plays a role in signaling processes. In dopaminergic cells of the brain, tyrosine hydroxylase converts tyrosine to l-dopa, an immediate precursor of dopamine. Dopamine, in turn, is a precursor of norepinephrine and epinephrine. Tyrosine is also a precursor of thyroid hormones and melanin. Hydroxyl amino acids
• Serine (Ser/S) is one of three amino acids having an R-group with a hydroxyl in it (threonine and tyrosine are the others). It is coded by UCU, UCC, UCA, UGC, AGU, and AGC. Being able to hydrogen bond with water, it is classified as a polar amino acid. It is not essential for humans. Serine is precursor of many important cellular compounds, including purines, pyrimidines, sphingolipids, folate, and of the amino acids glycine, cysteine, and tryptophan. The hydroxyl group of serine in proteins is a target for phosphorylation by certain protein kinases. Serine is also a part of the catalytic triad of serine proteases. • Threonine (Thr/T) is a polar amino acid that is essential. It is one of three amino acids bearing a hydroxyl group (serine and tyrosine are the others) and, as such, is a target for phosphorylation in proteins. It is also a target for Oglycosylation of proteins. Threonine proteases use the hydroxyl group of the amino acid in their catalysis and it is a precursor in one biosynthetic pathway for making glycine. In some applications, it is used as a pro-drug to increase brain glycine levels. Threonine is encoded in the genetic code by ACU, ACC, ACA, and ACG. Tyrosine - see HERE. Other amino acids • Asparagine (Asn/N) is a non-essential amino acid coded by AAU and AAC. Its carboxyamide in the R-group gives it polarity. Asparagine is implicated in formation of acrylamide in foods cooked at high temperatures (deep frying) when it reacts with carbonyl groups. Asparagine can be made in the body from aspartate by an amidation reaction with an amine from glutamine. Breakdown of asparagine produces malate, which can be oxidized in the citric acid cycle. • Cysteine (Cys/C) is the only amino acid with a sulfhydryl group in its side chain. It is nonessential for most humans, but may be essential in infants, the elderly and individuals who suffer from certain metabolic diseases. Cysteine’s sulfhydryl group is readily oxidized to a disulfide when reacted with another one. In addition to being found in proteins, cysteine is also a component of the tripeptide, glutathione. Cysteine is specified by the codons UGU and UGC. • Glutamine (Gln/Q) is an amino acid that is not normally essential in humans, but may be in individuals undergoing intensive athletic training or with gastrointestinal disorders. It has a carboxyamide side chain which does not normally ionize under physiological pHs, but which gives polarity to the side chain. Glutamine is coded for by CAA and CAG and is readily made by amidation of glutamate. Glutamine is the most abundant amino acid in circulating blood and is one of only a few amino acids that can cross the blood-brain barrier. • Selenocysteine (Sec/U) is a component of selenoproteins found in all kingdoms of life. It is a component in several enzymes, including glutathione peroxidases and thioredoxin reductases. Selenocysteine is incorporated into proteins in an unusual scheme involving the stop codon UGA. Cells grown in the absence of selenium terminate protein synthesis at UGAs. However, when selenium is present, certain mRNAs which contain a selenocysteine insertion sequence (SECIS), insert selenocysteine when UGA is encountered. The SECIS element has characteristic nucleotide sequences and secondary structure base-pairing patterns. Twenty five human proteins contain selenocysteine. • Pyrrolysine (Pyl/O) is a twenty second amino acid, but is rarely found in proteins. Like selenocysteine, it is not coded for in the genetic code and must be incorporated by unusual means. This occurs at UAG stop codons. Pyrrolysine is found in methanogenic archaean organisms and at least one methane-producing bacterium. Pyrrolysine is a component of methane-producing enzymes. Ionizing groups pKa values for amino acid side chains are very dependent upon the chemical environment in which they are present. For example, the R-group carboxyl found in aspartic acid has a pKa value of 3.9 when free in solution, but can be as high as 14 when in certain environments inside of proteins, though that is unusual and extreme. Each amino acid has at least one ionizable amine group (α- amine) and one ionizable carboxyl group (α- carboxyl). When these are bound in a peptide bond, they no longer ionize. Some, but not all amino acids have R-groups that can ionize. The charge of a protein then arises from the charges of the α-amine group, the α- carboxyl group. and the sum of the charges of the ionized R-groups. Titration/ionization of aspartic acid is depicted in Figure 2.10. Ionization (or deionization) within a protein’s structure can have significant effect on the overall conformation of the protein and, since structure is related to function, a major impact on the activity of a protein. Most proteins have relatively narrow ranges of optimal activity that typically correspond to the environments in which they are found (Figure 2.11). It is worth noting that formation of peptide bonds between amino acids removes ionizable hydrogens from both the α- amine and α- carboxyl groups of amino acids. Thus, ionization/ deionization in a protein arises only from 1) the amino terminus; 2) carboxyl terminus; 3) R-groups; or 4) other functional groups (such as sulfates or phosphates) added to amino acids post-translationally - see below. Carnitine Not all amino acids in a cell are found in proteins. The most common examples include ornithine (arginine metabolism), citrulline (urea cycle), and carnitine (Figure 2.12). When fatty acids destined for oxidation are moved into the mitochondrion for that purpose, they travel across the inner membrane attached to carnitine. Of the two stereoisomeric forms, the L form is the active one. The molecule is synthesized in the liver from lysine and methionine. From exogenous sources, fatty acids must be activated upon entry into the cytoplasm by being joined to coenzyme A. The CoA portion of the molecule is replaced by carnitine in the intermembrane space of the mitochondrion in a reaction catalyzed by carnitine acyltransferase I. The resulting acylcarnitine molecule is transferred across the inner mitochondrial membrane by the carnitineacylcarnitine translocase and then in the matrix of the mitochondrion, carnitine acyltransferase II replaces the carnitine with coenzyme A (Figure 6.88). Catabolism of amino acids
We categorize amino acids as essential or non-essential based on whether or not an organism can synthesize them. All of the amino acids, however, can be broken down by all organisms. They are, in fact, a source of energy for cells, particularly during times of starvation or for people on diets containing very low amounts of carbohydrate. From a perspective of breakdown (catabolism), amino acids are categorized as glucogenic if they produce intermediates that can be made into glucose or ketogenic if their intermediates are made into acetyl-CoA. Figure 2.13 shows the metabolic fates of catabolism of each of the amino acids. Note that some amino acids are both glucogenic and ketogenic. Post-translational modifications After a protein is synthesized, amino acid side chains within it can be chemically modified, giving rise to more diversity of structure and function (Figure 2.14). Common alterations include phosphorylation of hydroxyl groups of serine, threonine, or tyrosine. Lysine, proline, and histidine can have hydroxyls added to amines in their R-groups. Other modifications to amino acids in proteins include addition of fatty acids (myristic acid or palmitic acid), isoprenoid groups, acetyl groups, methyl groups, iodine, carboxyl groups, or sulfates. These can have the effects of ionization (addition of phosphates/sulfates), deionization (addition of acetyl group to the R-group amine of lysine), or have no effect on charge at all. In addition, N-linked- and O-linkedglycoproteins have carbohydrates covalently attached to side chains of asparagine and threonine or serine, respectively. Some amino acids are precursors of important compounds in the body. Examples include epinephrine, thyroid hormones, Ldopa, and dopamine (all from tyrosine), serotonin (from tryptophan), and histamine (from histidine). Building Polypeptides Although amino acids serve other functions in cells, their most important role is as constituents of proteins. Proteins, as we noted earlier, are polymers of amino acids. Amino acids are linked to each other by peptide bonds, in which the carboxyl group of one amino acid is joined to the amino group of the next, with the loss of a molecule of water. Additional amino acids are added in the same way, by formation of peptide bonds between the free carboxyl on the end of the growing chain and the amino group of the next amino acid in the sequence. A chain made up of just a few amino acids linked together is called an oligopeptide (oligo=few) while a typical protein, which is made up of many amino acids is called a polypeptide (poly=many). The end of the peptide that has a free amino group is called the N-terminus (for NH2), while the end with the free carboxyl is termed the C-terminus (for carboxyl). As we’ve noted before, function is dependent on structure, and the string of amino acids must fold into a specific 3-D shape, or conformation, in order to make a functional protein. The folding of polypeptides into their functional forms is the topic of the next section.
Source: BiochemFFA_2_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Proteins are the workhorses of the cell. Virtually everything that goes on inside of cells happens as a result of the actions of proteins. Among other things, protein enzymes catalyze the vast majority of cellular reactions, mediate signaling, give structure both to cells and to multicellular organisms, and exert control over the expression of genes. Life, as we know it, would not exist if there were no proteins. The versatility of proteins arises because of their varied structures. Proteins are made by linking together amino acids, with each protein having a characteristic and unique amino acid sequence. To get a sense for the diversity of proteins that can be made using 20 different amino acids, consider that the number of different combinations possible with 20 amino acids is 20n, where n=the number of amino acids in the chain. It becomes apparent that even a dipeptide made of just two amino acids joined together gives us 202 = 400 different combinations. If we do the calculation for a short peptide of 10 amino acids, we arrive at an enormous 10,240,000,000,000 combinations. Most proteins are much larger than this, making the possible number of proteins with unique amino acid sequences unimaginably huge. Levels of Structure The significance of the unique sequence, or order, of amino acids, known as the protein’s primary structure, is that it dictates the 3-D conformation the folded protein will have. This conformation, in turn, will determine the function of the protein. We shall examine protein structure at four distinct levels (Figure 2.17) - 1) how sequence of the amino acids in a protein (primary structure) gives identity and characteristics to a protein (Figure 2.18); 2) how local interactions between one part of the polypeptide backbone and another affect protein shape (secondary structure); 3) how the polypeptide chain of a protein can fold to allow amino acids to interact with each other that are not close in primary structure (tertiary structure); and 4) how different polypeptide chains interact with each other within a multi-subunit protein (quaternary structure). At this point, we should provide a couple of definitions. We use the term polypeptide to refer to a single polymer of amino acids. It may or may not have folded into its final, functional form. The term protein is sometimes used interchangeably with polypeptide, as in “protein synthesis”. It is generally used, however, to refer to a folded, functional molecule that may have one or more subunits (made up of individual polypeptides). Thus, when we use the term protein, we are usually referring to a functional, folded polypeptide or peptides. Structure is essential for function. If you alter the structure, you alter the function - usually, but not always, this means you lose all function. For many proteins, it is not difficult to alter the structure. Proteins are flexible, not rigidly fixed in structure. As we shall see, it is the flexibility of proteins that allows them to be amazing catalysts and allows them to adapt to, respond to, and pass on signals upon binding of other molecules or proteins. However, proteins are not infinitely flexible. There are constraints on the conformations that proteins can adopt and these constraints govern the conformations that proteins display. Subtle changes Even very tiny, subtle changes in protein structure can give rise to big changes in the behavior of proteins. Hemoglobin, for example, undergoes an incredibly small structural change upon binding of one oxygen molecule, and that simple change causes the remainder of the protein to gain a considerably greater affinity for oxygen that the protein didn’t have before the structural change. Sequence, structure and function As discussed earlier, the number of different amino acid sequences possible, even for short peptides, is very large. No two proteins with different amino acid sequences (primary structure) have identical overall structure. The unique amino acid sequence of a protein is reflected in its unique folded structure. This structure, in turn, determines the protein’s function. This is why mutations that alter amino acid sequence can affect the function of a protein. Protein Synthesis Synthesis of proteins occurs in the ribosomes and proceeds by joining the carboxyl terminus of the first amino acid to the amino terminus of the next one (Figure 2.19). The end of the protein that has the free α-amino group is referred to as the amino terminus or N-terminus. The other end is called the carboxyl terminus or C-terminus , since it contains the only free α-carboxyl group. All of the other α-amino groups and α-carboxyl groups are tied up in forming peptide Figure 2.19 Linking of amino acids through peptide bond formation bonds that join adjacent amino acids together. Proteins are synthesized starting with the amino terminus and ending at the carboxyl terminus. Schematically, in Figure 2.18, we can see how sequential R-groups of a protein are arranged in an alternating orientation on either side of the polypeptide chain. Organization of R-groups in this fashion is not random. Steric hindrance can occur when consecutive R-groups are oriented on the same side of a peptide backbone (Figure 2.20) Primary Structure Primary structure is the ultimate determinant of the overall conformation of a protein. The primary structure of any protein arrived at its current state as a result of mutation and selection over evolutionary time. Primary structure of proteins is mandated by the sequence of DNA coding for it in the genome. Regions of DNA specifying proteins are known as coding regions (or genes). The base sequences of these regions directly specify the sequence of amino acids in proteins, with a one-to-one correspondence between the codons (groups of three consecutive bases) in the DNA and the amino acids in the encoded protein. The sequence of codons in DNA, copied into messenger RNA, specifies a sequence of amino acids in a protein. (Figure 2.21).
The order in which the amino acids are joined together in protein synthesis starts defining a set of interactions between amino acids even as the synthesis is occurring. That is, a polypeptide can fold even as it is being made. The order of the R-group structures and resulting interactions are very important because early interactions affect later interactions. This is because interactions start establishing structures - secondary and tertiary. If a helical structure (secondary structure), for example, starts to form, the possibilities for interaction of a particular amino acid Rgroup may be different than if the helix had not formed (Figure 2.22). R-group interactions can also cause bends in a polypeptide sequence (tertiary structure) and these bends can create (in some cases) opportunities for interactions that wouldn’t have been possible without the bend or prevent (in other cases) similar interaction possibilities. Secondary Structure As protein synthesis progresses, interactions between amino acids close to each other begin to occur, giving rise to local patterns called secondary structure. These secondary structures include the well known α- helix and β-strands. Both were predicted by Linus Pauling, Robert Corey, and Herman Branson in 1951. Each structure has unique features. α-helix The α-helix has a coiled structure, with 3.6 amino acids per turn of the helix (5 helical turns = 18 amino acids). Helices are predominantly right handed - only in rare cases, such as in sequences with many glycines can left handed α- helices form. In the α-helix, hydrogen bonds form between C=O groups and N-H groups in the polypeptide backbone that are four amino acids distant. These hydrogen bonds are the primary forces stabilizing the α-helix. We use the terms rise, repeat, and pitch to describe the parameters of any helix. The repeat is the number of residues in a helix before it begins to repeat itself. For an α-helix, the repeat is 3.6 amino acids per turn of the helix. The rise is the distance the helix elevates with addition of each residue. For an α-helix, this is 0.15 nm per amino acid. The pitch is the distance between complete turns of the helix. For an α-helix, this is 0.54 nm. The stability of an α-helix is enhanced by the presence of the amino acid aspartate. β strand/sheet A helix is, of course, a three-dimensional object. A flattened form of helix in two dimensions is a common description for a β- strand. Rather than coils, β-strands have bends and these are sometimes referred to as pleats, like the pleats in a curtain. β-strands can be organized to form elaborately organized structures, such as sheets, barrels, and other arrangements. Higher order β-strand structures are sometimes called supersecondary structures), since they involve interactions between amino acids not close in primary sequence. These structures, too, are stabilized by hydrogen bonds between carbonyl oxygen atoms and hydrogens of amine groups in the polypeptide backbone (Figure 2.28). In a higher order structure, strands can be arranged parallel (amino to carboxyl orientations the same) or anti-parallel (amino to carboxyl orientations opposite of each other (in Figure 2.27, the direction of the strand is shown by the arrowhead in the ribbon diagrams). Turns Turns (sometimes called reverse turns) are a type of secondary structure that, as the name suggests, causes a turn in the structure of a polypeptide chain. Turns give rise to tertiary structure ultimately, causing interruptions in the secondary structures (α- helices and β-strands) and often serve as connecting regions between two regions of secondary structure in a protein. Proline and glycine play common roles in turns, providing less flexibility (starting the turn) and greater flexibility (facilitating the turn), respectively. There are at least five types of turns, with numerous variations of each giving rise to many different turns. The five types of turns are • δ-turns - end amino acids are separated by one peptide bond • γ-turns - separation by two peptide bonds •β-turns - separation by three peptide bonds •α-turns - separation by four peptide bonds •π-turns - separation by five bonds Of these, the β-turns are the most common form and the δ-turns are theoretical, but unlikely, due to steric limitations. Figure 2.29 depicts a β- turn. 310 helices In addition to the α-helix, β-strands, and various turns, other regular, repeating structures are seen in proteins, but occur much less commonly. The 310 helix is the fourth most abundant secondary structure in proteins, constituting about 10-15% of all helices. The helix derives its name from the fact that it contains 10 amino acids in 3 turns. It is right-handed. Hydrogen bonds form between amino acids that are three residues apart. Most commonly, the 310 helix appears at the amine or carboxyl end of an α-helix. Like the α-helix, the 310 helix is stabilized by the presence of aspartate in its sequence. π-helices A π-helix may be thought of as a special type of α- helix. Some sources describe it as an α-helix with an extra amino acid stuck in the middle of it (Figure 2.32). π-helices are not exactly rare, occurring at least once in as many as 15% of all proteins. Like the α- helix, the π-helix is right-handed, but where the α-helix has 18 amino acids in 5 turns, the π-helix has 22 amino acids in 5 turns. π-helices typically do not stretch for very long distances. Most are only about 7 amino acids long and the sequence almost always occurs in the middle of an α-helical region. Ramachandran plots In 1963, G.N. Ramachandran, C. Ramakrishnan, and V. Sasisekharan described a novel way to describe protein structure. If one considers the backbone of a polypeptide chain, it consists of a repeating set of three bonds. Sequentially (in the amino to carboxyl direction) they are 1) a rotatable bond (ψ) between α-carbon and α-carboxyl preceding the peptide bond (see HERE), 2) a non-rotatable peptide bond (ω) between the α-carboxyl and α-amine groups), and 3) a rotatable bond (φ) between the α-amine and α-carbon following the peptide bond (see HERE). Note in Figures 2.33 and 2.34 that the amino to carboxyl direction is right to left.
The presence of the carbonyl oxygen on the α-carboxyl group allows the peptide bond to exist as a resonant structure, meaning that it behaves some of the time as a double bond. Double bonds cannot, of course, rotate, but the bonds on either side of it have some freedom of rotation. The φ and ψ angles are restricted to certain values, because some angles will result in steric hindrance. In addition, each type of secondary structure has a characteristic range of values for φ and ψ. Ramachandran and colleagues made theoretical calculations of the energetic stability of all possible angles from 0° to 360° for each of the φ and ψ angles and plotted the results on a Ramachandran Plot (also called a φ-ψ plot), delineating regions of angles that were theoretically the most stable (Figure 2.35). Three primary regions of stability were identified, corresponding to φ-ψ angles of β-strands (top left), right handed α- helices (bottom left), and lefthanded α-helices (upper right). The plots of predicted stability are remarkably accurate when compared to φ-ψ angles of actual proteins. Secondary structure prediction Table 2.3 - Relative tendencies of each amino acid to be in a secondary structure. Higher values indicate greater tendency Image by Penelope Irving By comparing primary structure (amino acid sequences) to known 3D protein structures, one can tally each time an amino acid is found in an α-helix, β-strand/sheet, or a turn. Computer analysis of thousands of these sequences allows one to assign a likelihood of any given amino acid appearing in each of these structures. Using these tendencies, one can, with up to 80% accuracy, predict regions of secondary structure in a protein based solely on amino acid sequence. This is seen in Table 2.3. Occurrence in primary sequence of three consecutive amino acids with relative tendencies higher than one is an indicator that that region of the polypeptide is in the corresponding secondary structure. An online resource for predicting secondary structures called PSIPRED is available HERE. Hydrophobicity Table 2.4 - Hydropathy Scores The chemistry of amino acid Rgroups affects the structures they are most commonly found in. Subsets of their chemical properties can give clues to structure and, sometimes, cellular location. A prime example is the hydrophobicity (wateravoiding tendencies) of some Rgroups. Given the aqueous environment of the cell, such R-groups are not likely to be on the outside surface of a folded protein. However, this rule does not hold for regions of protein that may be embedded within the lipid bilayers of cellular/ organelle membranes. This is because the region of such proteins that form the transmembrane domains are are buried in the hydrophobic environment in the middle of the lipid bilayer. Not surprisingly, scanning primary sequences for specifically sized/spaced stretches of hydrophobic amino acids can help to identify proteins found in membranes. Table 2.4 shows hydrophobicity values for R-groups of the amino acids. In this set, the scale runs from positive values (hydrophobic) to negative values (hydrophilic). A KyteDoolittle Hydropathy plot for the RET protooncogene membrane protein is shown in Figure 2.36. Two regions of the protein are very hydrophobic as can be seen from the peaks near amino acids 5-10 and 630-640. Such regions might be reasonably expected to be situated either within the interior of the folded protein or to be part of transmembrane domains. Random coils Some sections of a protein assume no regular, discernible structure and are sometimes said to lack secondary structure, though they may have hydrogen bonds. Such segments are described as being in random coils and may have fluidity to their structure that results in them having multiple stable forms. Random coils are identifiable with spectroscopic methods, such as circular dichroism Wikipedia and nuclear magnetic resonance (NMR) in which distinctive signals are observed. See also metamorphic proteins (HERE) and intrinsically disordered proteins (HERE). Supersecondary structure Another element of protein structure is harder to categorize because it incorporates elements of secondary and tertiary structure. Dubbed supersecondary structure (or structural motifs), these structures contain multiple nearby secondary structure components arranged in a specific way and that appear in multiple proteins. Since there are many ways of making secondary structures from different primary structures, so too can similar motifs arise from different primary sequences. An example of a structural motif is shown in Figure 2.37. Tertiary structure Proteins are distinguished from each other by the sequence of amino acids comprising them. The sequence of amino acids of a protein determines protein shape, since the chemical properties of each amino acid are forces that give rise to intermolecular interactions to begin to create secondary structures, such as α-helices and β-strands. The sequence also defines turns and random coils that play important roles in the process of protein folding. Since shape is essential for protein function, the sequence of amino acids gives rise to all of the properties a protein has. As protein synthesis proceeds, individual components of secondary structure start to interact with each other, giving rise to folds that bring amino acids close together that are not near each other in primary structure (Figure 2.38). At the tertiary level of structure, interactions among the R-groups of the amino acids in the protein, as well as between the polypeptide backbone and amino acid side groups play a role in folding. Globular proteins
Folding gives rise to distinct 3-D shapes in proteins that are non-fibrous. These proteins are called globular. A globular protein is stabilized by the same forces that drive its formation. These include ionic interactions, hydrogen bonding, hydrophobic forces, ionic bonds, disulfide bonds and metallic bonds. Treatments such as heat, pH changes, detergents, urea and mercaptoethanol overpower the stabilizing forces and cause a protein to unfold, losing its structure and (usually) its function (Figure 2.39). The ability of heat and detergents to denature proteins is why we cook our food and wash our hands before eating - such treatments denature the proteins in the microorganisms on our hands. Organisms that live in environments of high temperature (over 50°C) have proteins with changes in stabilizing forces - additional hydrogen bonds, additional salt bridges (ionic interactions), and compactness may all play roles in keeping these proteins from unfolding. Protein stabilizing forces Before considering the folding process, let us consider some of the forces that help to stabilize proteins. Hydrogen bonds Hydrogen bonds arise as a result of partially charged hydrogens found in covalent bonds. This occurs when the atom the hydrogen is bonded to has a greater electronegativity than hydrogen itself does, resulting in hydrogen having a partial positive charge because it is not able to hold electrons close to itself (Figure 2.40). Hydrogen partially charged in this way is attracted to atoms, such as oxygen and nitrogen that have partial negative charges, due to having greater electronegativities and thus holding electrons closer to themselves. The partially positively charged hydrogens are called donors, whereas the partially negative atoms they are attracted to are called acceptors. (See Figure 1.30). Individual hydrogen bonds are much weaker than a covalent bond, but collectively, they can exert strong forces. Consider liquid water, which contains enormous numbers of hydrogen bonds (Figure 2.41). These forces help water to remain liquid at room temperature. Other molecules lacking hydrogen bonds of equal or greater molecular weight than water, such as methane or carbon dioxide, are gases at the same temperature. Thus, the intermolecular interactions between water molecules help to “hold” water together and remain a liquid. Notably, only by raising the temperature of water to boiling are the forces of hydrogen bonding overcome, allowing water to become fully gaseous. Hydrogen bonds are important forces in biopolymers that include DNA, proteins, and cellulose. All of these polymers lose their native structures upon boiling. Hydrogen bonds between amino acids that are close to each other in primary structure can give rise to regular repeating structures, such as helices or pleats, in proteins (secondary structure). Ionic interactions Ionic interactions are important forces stabilizing protein structure that arise from ionization of R-groups in the amino acids comprising a protein. These include the carboxyl amino acids (HERE), the amine amino acids as well as the sulfhydryl of cysteine and sometimes the hydroxyl of tyrosine. Hydrophobic forces Hydrophobic forces stabilize protein structure as a result of interactions that favor the exclusion of water. Non-polar amino acids (commonly found in the interior of proteins) favor associating with each other and this has the effect of excluding water. The excluded water has a higher entropy than water interacting with the hydrophobic side chains. This is because water aligns itself very regularly and in a distinct pattern when interacting with hydrophobic molecules. When water is prevented from having these kinds of interactions, it is much more disordered that it would be if it could associate with the hydrophobic regions. It is partly for this reason that hydrophobic amino acids are found in protein interiors - so they can exclude water and increase entropy. Disulfide bonds Disulfide bonds, which are made when two sulfhydryl side-chains of cysteine are brought into close proximity, covalently join together different protein regions and can give great strength to the overall structure (Figures 2.42 & 2.43). An Ode to Protein Structure by Kevin Ahern The twenty wee amino A's Define a protein many ways Their order in a peptide chain Determines forms that proteins gain And when they coil, it leaves me merry Cuz that makes structures secondary It's tertiary, I am told That happens when a protein folds But folded chains are downright scary When put together quaternary They're nature's wonders, that's for sure Creating problems, making cures A fool can fashion peptide poems But proteins come from ribosoems These joined residues of cysteine are sometimes referred to as cystine. Disulfide bonds are the strongest of the forces stabilizing protein structure. van der Waals forces van der Waals forces is a term used to describe various weak interactions, including those caused by attraction between a polar molecule and a transient dipole, or between two temporary dipoles. van der Waals forces are dynamic because of the fluctuating nature of the attraction, and are generally weak in comparison to covalent bonds, but can, over very short distances, be significant. Post-translational modifications Post-translational modifications can result in formation of covalent bonds stabilizing proteins as well. Hydroxylation of lysine and proline in strands of collagen can result in cross-linking of these groups and the resulting covalent bonds help to strengthen and stabilize the collagen. Folding models Two popular models of protein folding are currently under investigation. In the first (diffusion collision model), a nucleation event begins the process, followed by secondary structure formation. Collisions between the secondary structures (as in the β-hairpin in Figure 2.37) allow for folding to begin. By contrast, in the nucleation-condensation model, the secondary and tertiary structures form together. Folding in proteins occurs fairly rapidly (0.1 to 1000 seconds) and can occur during synthesis - the amino terminus of a protein can start to fold before the carboxyl terminus is even made, though that is not always the case. Folding process
Protein folding is hypothesized to occur in a “folding funnel” energy landscape in which a folded protein’s native state corresponds to the minimal free energy possible in conditions of the medium (usually aqueous solvent) in which the protein is dissolved. As seen in the diagram (Figure 2.44), the energy funnel has numerous local minima (dips) in which a folding protein can become trapped as it moves down the energy plot. Other factors, such as temperature, electric/magnetic fields, and spacial considerations likely play roles. If external forces affect local energy minima during folding, the process and end-product can be influenced. As the speed of a car going down a road will affect the safety of the journey, so too do energy considerations influence and guide the folding process, resulting in fully functional, properly folded proteins in some cases and misfolded “mistakes” in others. Getting stuck As the folding process proceeds towards an energy minimum (bottom of the funnel in Figure 2.44), a protein can get “stuck” in any of the local minima and not reach the final folded state. Though the folded state is, in general, more organized and therefore has reduced entropy than the unfolded state, there are two forces that overcome the entropy decrease and drive the process forward. The first is the magnitude of the decrease in energy as shown in the graph. Since ΔG = ΔH -TΔS, a decrease in ΔH can overcome a negative ΔS to make ΔG negative and push the folding process forward. Favorable (decreased) energy conditions arise with formation of ionic bonds, hydrogen bonds, disulfide bonds, and metallic bonds during the folding process. In addition, the hydrophobic effect increases entropy by allowing hydrophobic amino acids in the interior of a folded protein to exclude water, thus countering the impact of the ordering of the protein structure by making the ΔS less negative. Structure prediction Computer programs are very good at predicting secondary structure solely based on amino acid sequence, but struggle with determining tertiary structure using the same information. This is partly due to the fact that secondary structures have repeating points of stabilization based on geometry and any regular secondary structure (e.g., α-helix) varies very little from one to another. Folded structures, though, have an enormous number of possible structures as shown by Levinthal’s Paradox. Spectroscopy Because of our inability to accurately predict tertiary structure based on amino acid sequence, proteins structures are actually determined using techniques of spectroscopy. In these approaches, proteins are subjected to varied forms of electromagnetic radiation and the ways they interact with the radiation allows researchers to determine atomic coordinates at Angstrom resolution from electron densities (see X-ray crystallography) and how nuclei spins interact (see NMR). Levinthal’s paradox In the late 1960s, Cyrus Levinthal outlined the magnitude of the complexity of the protein folding problem. He pointed out that for a protein with 100 amino acids, it would have 99 peptide bonds and 198 considerations for φ and ψ angles. If each of these had only three conformations, that would result in 3198 different possible foldings or 2.95x1094. Even allowing a reasonable amount of time (one nanosecond) for each possible fold to occur, it would take longer than the age of the universe to sample all of them, meaning clearly that the process of folding is not occurring by a sequential random sampling and that attempts to determine protein structure by random sampling were doomed to fail. Levinthal, therefore, proposed that folding occurs by a sequential process that begins with a nucleation event that guides the process rapidly and is not unlike the funnel process depicted in Figure 2.44. Diseases of protein misfolding The proper folding of proteins is essential to their function. It follows then that misfolding of proteins (also called proteopathy) might have consequences. In some cases, this might simply result in an inactive protein. Protein misfolding also plays a role in numerous diseases, such as Mad Cow Disease, Alzheimers, Parkinson’s Disease, and CreutzfeldJakob disease. Many, but not all, misfolding diseases affect brain tissue. Insoluble deposits Misfolded proteins will commonly form aggregates called amyloids that are harmful to tissues containing them because they change from being soluble to insoluble in water and form deposits. The process by which misfolding (Figure 2.45) occurs is not completely clear, but in many cases, it has been demonstrated that a “seed” protein which is misfolded can induce the same misfolding in other copies of the same protein. These seed proteins are known as prions and they act as infectious agents, resulting in the spread of disease. The list of human diseases linked to protein misfolding is long and continues to grow. A Wikipedia link is HERE. Prions Prions are infectious protein particles that cause transmissible spongiform encephalopathies (TSEs), the best known of which is Mad Cow disease. Other manifestations include the disease, scrapie, in sheep, and human diseases, such as CreutzfeldtJakob disease (CJD), Fatal Familial Insomnia, and kuru. The protein involved in these diseases is a membrane protein called PrP. PrP is encoded in the genome of many organisms and is found in most cells of the body. PrPc is the name given to the structure of PrP that is normal and not associated with disease. PrPSc is the name given to a misfolded form of the same protein, that is associated with the development of disease symptoms (Figure 2.45). Misfolded The misfolded PrPSc is associated with the TSE diseases and acts as an infectious particle. A third form of PrP, called PrPres can be found in TSEs, but is not infectious. The ‘res’ of PrPres indicates it is protease resistant. It is worth noting that all three forms of PrP have the same amino acid sequence and differ from each other only in the ways in which the polypeptide chains are folded. The most dangerously misfolded form of PrP is PrPSc, because of its ability to act like an infectious agent - a seed protein that can induce misfolding of PrPc , thus converting it into PrPSc. Function
The function of PrPc is unknown. Mice lacking the PrP gene do not have major abnormalities. They do appear to exhibit problems with long term memory, suggesting a function for PrPc . Stanley Prusiner, who discovered prions and coined the term, received the Nobel Prize in Medicine in 1997 for his work. I think that if I chanced to be on A protein making up a prion I’d twist it and for goodness sakes Stop it from making fold mistakes Amyloids Amyloids are a collection of improperly folded protein aggregates that are found in the human body. As a consequence of their misfolding, they are insoluble and contribute to some twenty human diseases including important neurological ones involving prions. Diseases include (affected protein in parentheses) - Alzheimer’s disease (Amyloid β), Parkinson’s disease (α-synuclein), Huntington’s disease (huntingtin), rheumatoid arthritis (serum amyloid A), fatal familial insomnia (PrPSc), and others. Amino acid sequence plays a role in amyloidogenesis. Glutamine-rich polypeptides are common in yeast and human prions. Trinucleotide repeats are important in Huntington’s disease. Where sequence is not a factor, hydrophobic association between β-sheets can play a role. Amyloid β Amyloid β refers to collections of small proteins (36-43 amino acids) that appear to play a role in Alzheimer’s disease. (Tau protein is the other factor.) They are, in fact, the main components of amyloid plaques found in the brains of patients suffering from the disease and arise from proteolytic cleavage of a larger amyloid precursor glycoprotein called Amyloid Precursor Protein, an integral membrane protein of nerve cells whose function is not known. Two proteases, β-secretase and γ- secretase perform this function. Amyloid β proteins are improperly folded and appear to induce other proteins to misfold and thus precipitate and form the amyloid characteristic of the disease. The plaques are toxic to nerve cells and give rise to the dementia characteristic of the disease. It is thought that aggregation of amyloid β proteins during misfolding leads to generation of reactive oxygen species and that this is the means by which neurons are damaged. It is not known what the actual function of amyloid β is. Autosomal dominant mutations in the protein lead to early onset of the disease, but this occurs in no more than 10% of the cases. Strategies for treating the disease include inhibition of the secretases that generate the peptide fragments from the amyloid precursor protein. Huntingtin Huntingtin is the central gene in Huntington’s disease. The protein made from it is glutamine rich, with 6-35 such residues in its wild-type form. In Huntington’s disease, this gene is mutated, increasing the number of glutamines in the mutant protein to between 36 and 250. The size of the protein varies with the number of glutamines in the mutant protein, but the wild-type protein has over 3100 amino acids and a molecular weight of about 350,000 Da. Its precise function is not known, but huntingtin is found in nerve cells, with the highest level in the brain. It is thought to possibly play roles in transport, signaling, and protection against apoptosis. Huntingtin is also required for early embryonic development. Within the cell, huntingtin is found localized primarily with microtubules and vesicles. Trinucleotide repeat The huntingtin gene contains many copies of the sequence CAG (called trinucleotide repeats), which code for the many glutamines in the protein. Huntington’s disease arises when extra copies of the CAG sequence are generated when the DNA of the gene is being copied. Expansion of repeated sequences can occur due to slipping of the polymerase relative to the DNA template during replication. As a result, multiple additional copies of the trinucleotide repeat may be made, resulting in proteins with variable numbers of glutamine residues. Up to 35 repeats can be tolerated without problem. The number of repeats can expand over the course of a person’s lifetime, however, by the same mechanism. Individuals with 36-40 repeats begin to show signs of the disease and if there are over 40, the disease will be present. Molecular chaperones The importance of the proper folding of proteins is highlighted by the diseases associated with misfolded proteins, so it is no surprise, then, that cells expend energy to facilitate the proper folding of proteins. Cells use two classes of proteins known as molecular chaperones, to facilitate such folding in cells. Molecular chaperones are of two kinds, the chaperones, and the chaperonins. An example of the first category is the Hsp70 class of proteins. Hsp stands for “heat shock protein”, based on the fact that these proteins were first observed in large amounts in cells that had been briefly subjected to high temperatures. Hsps function to assist cells in stresses arising from heat shock and exposure to oxidizing conditions or toxic heavy metals, such as cadmium and mercury. However, they also play an important role in normal conditions, where they assist in the proper folding of polypeptides by preventing aberrant interactions that could lead to misfolding or aggregation. The Hsp70 proteins are found in almost all cells and use ATP hydrolysis to stimulate structural changes in the shape of the chaperone to accommodate binding of substrate proteins. The binding domain of Hsp70s contains a β-barrel structure which wraps around the polypeptide chain of the substrate and has affinity for hydrophobic side chains of amino acids. As shown in Figure 2.50, Hsp70 binds to polypeptides as they emerge from ribosomes during protein synthesis. Binding of substrate stimulates ATP hydrolysis and this is facilitated by another heat shock protein known as Hsp40. The hydrolysis of ATP causes the Hsp70 to taken on a closed conformation that helps shield exposed hydrophobic residues and prevent aggregation or local misfolding. After protein synthesis is complete, ADP is released and replaced by ATP and this results in release of the substrate protein, which then allows the full length polypeptide to fold correctly. In heat shock
In times of heat shock or oxidative stress, Hsp70 proteins bind to unfolded hydrophobic regions of proteins to similarly prevent them from aggregating and allowing them to properly refold. When proteins are damaged, Hsp70 recruits enzymes that ubiquitinate the damaged protein to target them for destruction in proteasomes. Thus, the Hsp70 proteins play an important role in ensuring not only that proteins are properly folded, but that damaged or nonfunctional proteins are removed by degradation in the proteasome. Chaperonins A second class of proteins involved in assisting other proteins to fold properly are known as chaperonins. There are two primary categories of chaperonins - Class I (found in bacteria, chloroplasts, and mitochondria) and Class II (found in the cytosol of eukaryotes and archaebacteria). The best studied chaperonins are the GroEL/GroES complex proteins found in bacteria (Figure 2.51). GroEL/GroES may not be able to undo aggregated proteins, but by facilitating proper folding, it provides competition for misfolding as a process and can reduce or eliminate problems arising from improper folding. GroEL is a double-ring 14mer with a hydrophobic region that can facilitate folding of substrates 15-60 kDa in size. GroES is a singlering heptamer that binds to GroEL in the presence of ATP and functions as a cover over GroEL. Hydrolysis of ATP by chaperonins induce large conformational changes that affect binding of substrate proteins and their folding. It is not known exactly how chaperonins fold proteins. Passive models postulate the chaperonin complex functioning inertly by preventing unfavorable intermolecular interactions or placing restrictions on spaces available for folding to occur. Active models propose that structural changes in the chaperonin complex induce structural changes in the substrate protein. Protein breakdown Another protein complex that has an important function in the lifetime dynamics of proteins is the proteasome (Figure 2.52). Proteasomes, which are found in all eukaryotes and archaeans, as well as some bacteria, function to break down unneeded or damaged proteins by proteolytic degradation. Proteasomes help to regulate the concentration of some proteins and degrade ones that are misfolded. The proteasomal degradation pathway plays an important role in cellular processes that include progression through the cell cycle, modulation of gene expression, and response to oxidative stresses. Degradation in the proteasome yields short peptides seven to eight amino acids in length. Threonine proteases play important roles. Breakdown of these peptides yields individual amino acids, thus facilitating their recycling in cells. Proteins are targeted for degradation in eukaryotic proteasomes by attachment to multiple copies of a small protein called ubiquitin (8.5 kDa - 76 amino acids). The enzyme catalyzing the reaction is known as ubiquitin ligase. The resulting polyubiquitin chain is bound by the proteasome and degradation begins. Ubiquitin was named due to it ubiquitously being found in eukaryotic cells. Ubiquitin Ubiquitin (Figure 2.53) is a small (8.5 kDa) multi-functional protein found in eukaryotic cells. It is commonly added to target proteins by action of ubiquitin ligase enzymes (E3 in Figure 2.54). One (ubiquitination) or many (polyubiquitination) ubiquitin molecules may be added. Attachment of the ubiquitin is through the side chain of one of seven different lysine residues in ubiquitin. The addition of ubiquitin to proteins has many effects, the best known of which is targeting the protein for degradation in the proteasome. Proteasomal targeting is seen when polyubiquitination occurs at lysines #29 and 48. Polyubiquitination or monoubiquitination at other lysines can result in altered cellular location and changed protein-protein interactions. The latter may alter affect inflammation, endocytic trafficking, translation and DNA repair. Ubiquitin ligase malfunction Parkin is a Parkinson’s disease-related protein that, when mutated, is linked to an inherited form of the disease called autosomal recessive juvenile Parkinson’s disease. The function of the protein is not known, but it is a component of the E3 ubiquitin ligase system responsible for transferring ubiquitin from the E2 protein to a lysine side chain on the target protein. It is thought that mutations in parkin lead to proteasomal dysfunction and a consequent inability to break down proteins harmful to dopaminergic neurons. This results in the death or malfunction of these neurons, resulting in Parkinson’s disease. Intrinsically disordered proteins Movie 2.1 - Dynamic movement of cytochrome C in solution Wikipedia As is evident from the many examples described elsewhere in the book, the 3-D structure of proteins is important for their function. But, increasingly, it is becoming evident that not all proteins fold into a stable structure. Studies on the so-called intrinsically disordered proteins (IDPs) in the past cou- ple of decades has shown that many proteins are biologically active, even thought they fail to fold into stable structures. Yet other proteins exhibit regions that remain unfolded (IDP regions) even as the rest of the polypeptide folds into a structured form. Intrinsically disordered proteins and disordered regions within proteins have, in fact, been known for many years, but were regarded as an anomaly. It is only recently, with the realization that IDPs and IDP regions are widespread among eukaryotic proteins, that it has been recognized that the observed disorder is a "feature, not a bug". Movie 2.2 SUMO-1, a protein with intrinsically disordered sections Wikipedia
Comparison of IDPs shows that they share sequence characteristics that appear to favor their disordered state. That is, just as some amino acid sequences may favor the folding of a polypeptide into a particular structure, the amino acid sequences of IDPs favor their remaining unfolded. IDP regions are seen to be low in hydrophobic residues and unusually rich in polar residues and proline. The presence of a large number of charged amino acids in the IDPs can inhibit folding through charge repulsion, while the lack of hydrophobic residues makes it difficult to form a stable hydrophobic core, and proline discourages the formation of helical structures. The observed differences between amino acid sequences in IDPs and structured proteins have been used to design algorithms to predict whether a given amino acid sequence will be disordered. What is the significance of intrinsically disordered proteins or regions? The fact that this property is encoded in their amino acid sequences suggests that their disorder may be linked to their function. The flexible, mobile nature of some IDP regions may play a crucial role in their function, permitting a transition to a folded structure upon binding a protein partner or undergoing post-translational modification. Studies on several wellknown proteins with IDP regions suggest some answers. IDP regions may enhance the ability of proteins like the lac repressor to translocate along the DNA to search for specific binding sites. The flexibility of IDPs can also be an asset in protein-protein interactions, especially for proteins that are known to interact with many different protein partners. For example, p53 has IDP regions that may allow the protein to interact with a variety of functional partners. Comparison of the known functions of proteins with predictions of disorder in these proteins suggests that IDPs and IDP regions may disproportionately function in signaling and regulation, while more structured proteins skew towards roles in catalysis and transport. Interestingly, many of the proteins found in both ribosomes and spliceosomes are predicted to have IDP regions that may play a part in correct assembly of these complexes. Even though IDPs have not been studied intensively for very long, what little is known of them suggests that they play an important and underestimated role in cells. Metamorphic proteins Another group of proteins that have recently changed our thinking about protein structure and function are the so-called metamorphic proteins. These proteins are capable of forming more than one stable, folded state starting with a single amino acid sequence. Although it is true that multiple folded conformations are not ruled out by the laws of physics and chemistry, metamorphic proteins are a relatively new discovery. It was known, of course, that prion proteins were capable of folding into alternative structures, but metamorphic proteins appear to be able to toggle back and forth between two stable structures. While in some cases, the metamorphic protein undergoes this switch in response to binding another molecule, some proteins that can accomplish this transition on their own. An interesting example is the signaling molecule, lymphotactin. Lymphotactin has two biological functions that are carried out by its two conformers- a monomeric form that binds the lymphotactin receptor and a dimeric form that binds heparin. It is possible that this sort of switching is more widespread than has been thought. Refolding denatured proteins All information for protein folding is contained in the amino acid sequence of the protein. It may seem curious then that most proteins do not fold into their proper, fully active form after they have been+++ denatured and the denaturant is removed. A few do, in fact. One good example is bovine ribonuclease (Figure 2.55). Its catalytic activity is very resistant to heat and urea and attempts to denature it don’t work very well. However, if one treats the enzyme with β-mercaptoethanol (which breaks disulfide bonds) prior to urea treatment and/or heating, activity is lost, indicating that the covalent disulfide bonds help stabilize the overall enzyme structure and when they are broken, denaturation can readily occur. When the mixture cools back down to room temperature, over time some enzyme activity reappears, indicating that ribonuclease re-folded under the new conditions. Interestingly, renaturation will occur maximally if a tiny amount of β-mercaptoethanol is left in the solution during the process. The reason for this is because β- mercaptoethanol permits reduction (and breaking) of accidental, incorrect disulfide bonds during the folding process. Without it, these disulfide bonds will prevent proper folds from forming. Irreversible denaturation Most enzymes, however, do not behave like bovine ribonuclease. Once denatured, their activity cannot be recovered to any significant There are not very many ways Inactivating RNase It’s stable when it’s hot or cold Because disulfides tightly hold If you desire to make it stall Use hot mercaptoethanol extent. This may seem to contradict the idea of folding information being inherent to the sequence of amino acids in the protein. It does not. Most enzymes don’t refold properly after denaturation for two reasons. First, normal folding may occur as proteins are being made. Interactions among amino acids early in the synthesis are not “confused” by interactions with amino acids later in the synthesis because those amino acids aren’t present as the process starts. Chaperonins’ role In other cases, the folding process of some proteins in the cell relied upon action of chaperonin proteins (see HERE). In the absence of chaperonins, interactions that might result in misfolding occur, thus preventing proper folding. Thus, early folding and the assistance of chaperonins eliminate some potential “wrong-folding” interactions that can occur if the entire sequence was present when folding started. Quaternary structure A fourth level of protein structure is that of quaternary structure. It refers to structures that arise as a result of interactions between multiple polypeptides. The units can be identical multiple copies or can be different polypeptide chains. Adult hemoglobin is a good example of a protein with quaternary structure, being composed of two identical chains called α and two identical chains called β.
Thumbnail: The cell membrane, also called the plasma membrane or plasmalemma, is a semipermeable lipid bilayer common to all living cells. It contains a variety of biological molecules, primarily proteins and lipids, which are involved in a vast array of cellular processes. It also serves as the attachment point for both the intracellular cytoskeleton and, if present, the cell wall. Image used with permission (CC BU-SA 3.0; Dhatfield and LadyofHats). 03: Membranes Source: BiochemFFA_3_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Lipid bilayers The protective membrane around cells contains many components, including cholesterol, proteins, glycolipids, glycerophospholipids, and sphingolipids. The last two of these will, when mixed vigorously with water, spontaneously form what is called a lipid bilayer (Figure 3.1), which serves as a protective boundary for the cell that is largely impermeable to the movement of most materials across it. With the notable exceptions of water, carbon dioxide, carbon monoxide, and oxygen, most polar/ionic require transport proteins to help them to efficiently navigate across the bilayer. The orderly movement of these compounds is critical for the cell to be able to 1) get food for energy; 2) export materials; 3) maintain osmotic balance; 4) create gradients for secondary transport; 5) provide electromotive force for nerve signaling; and 6) store energy in electrochemical gradients for ATP production (oxidative phosphorylation or photosynthesis). In some cases, energy is required to move the substances (active transport). Facilitated Diffusion In other cases, no external energy is required and they move by diffusion through specific cellular channels. This is referred to as facilitated diffusion. Before we discuss movement of materials across membranes, it is appropriate we discuss the composition of cellular membranes. Plasma membranes differ from cell walls both in the materials comprising them and in their flexibility. Cell walls will be covered near the end of this chapter. Though some cells do not have cell walls (animal cells) and others do (bacteria, fungi, and plants), there is commonality among cells in that they all possess plasma membranes. There is also commonality in the components of the membranes, though the relative amount of constituents varies. Figures 3.1 and 3.2 illustrate the structure and environments of plasma membranes. All plasma membranes contain a significant amount of amphiphilic substances linked to fatty acids. These include the glycerophospholipids and the sphingolipids. The fatty acid(s) are labeled as hydrophobic tails in the figures. Hydrophilic heads The composition of the hydrophilic heads varies considerably. In glycerophospholipids, a phosphate is always present, of course, and it is often esterified to another substance to make a phosphatide (Figure 3.3). Common compounds linked to the phosphate (at the X position) include serine, ethanolamine, and choline. These vary in the their charges so in this way, the charge on the external or internal surface can be controlled. Cells tend to have more negative charges on the exterior half of the lipid bilayer (called the outer leaflet) and more positive charges on the interior half (inner leaflet). Sphingolipids In sphingolipids (Figure 3.4), the hydrophilic head can contain a phosphate linked to ethanolamine or choline and this describes the structure of sphingomyelin, an important component of neural membranes. Most sphingolipids lack the phosphate and have instead a hydrophilic head of a single sugar (cerebrosides) or a complex oligosaccharide (gangliosides). Water exclusion In each case, the glycerophospholipid or sphingolipid has one end that is polar and one end that is non-polar. As we saw in the organization of amino acids with hydrophobic side chains occurring preferentially on the inside of a folded protein to exclude water, so too do the non-polar portions of these amphiphilic molecules arrange themselves so as to exclude water. Remember that the cytoplasm of a cell is mostly water and the exterior of the cell is usually bathed in an aqueous layer. It therefore makes perfect sense that the polar portions of the membrane molecules arrange themselves as they do - polar parts outside interacting with water and non-polar parts in the middle of the bilayer avoiding/excluding water. Composition Bias The plasma membrane has distinct biases of composition relative to its inside and the outside (Figure 3.7). First, glycosylation (of lipids and proteins) has the sugar groups located almost exclusively on the outside of the cell, away from the cytoplasm (Figure 3.8). Among the membrane lipids, sphingolipids are much more commonly glycosylated than glycerophospholipids. In addition, some of the glyerophospholipids are found preferentially on one side or the other (Figure 3.7). Phosphatidylserine and phosphatidylethanolamine are found preferentially within the inner leaflet of the plasma membrane, whereas phosphatidylcholine tends to be located on the outer leaflet. In the process of apoptosis, the phosphatidylserines appear on the outer leaflet where they serve as a signal to macrophages to bind and destroy the cell. Sphingolipids are found preferentially in the plasma membrane and are almost completely absent from mitochondrial and endoplasmic reticulum membranes (Figure 3.9). Organelle membranes Bias of lipid composition also exists with respect to organelle membranes. The unusual diphosphodiglycerolipid known as cardiolipin, for example, is almost only found in mitochondrial membranes (see HERE) and like phosphatidylserine, its movement is an important step in apoptosis. In signaling, phosphatidylinositols play important roles providing second messengers upon being cleaved (see HERE). Lateral Diffusion
Movement of lipids within each leaflet of the lipid bilayer occurs readily and rapidly due to membrane fluidity. This type of movement is called lateral diffusion and can be measured by the technique called FRAP (Figure 3.10, see HERE also). In this method, a laser strikes and stains a section of the lipid bilayer of a cell, leaving a spot as shown in B. Over time, the stain diffuses out ultimately across the entire lipid bilayer, much like a drop of ink will diffuse throughout when added to a glass of water. A measurement of the rate of diffusion gives an indication of the fluidity of a membrane. Transverse Diffusion While the movement in lateral diffusion occurs rapidly, movement of molecules from one leaflet over to the other leaflet occurs much more slowly. This type of molecular movement is called transverse diffusion and is almost nonexistent in the absence of enzyme action. Remember that there is a bias of distribution of molecules between leaflets of the membrane, which means that something must be moving them. There are three enzymes that catalyze movement of compounds in transverse diffusion. Flippases move membrane glycerophospholipids/ sphingolipids from outer leaflet to inner leaflet (cytoplasmic side) of cell. Floppases move membrane lipids in the opposite direction. Scramblases move in either direction. Other components of lipid bilayer Besides glycerophospholipids and sphingolipids, there are other materials commonly found in lipid bilayers of cellular membranes. Two important prominent ones are cholesterol (Figure 3.13) and proteins. Besides serving as a metabolic precursor of steroid hormones and the bile acids, cholesterol’s main role in cells is in the membranes. The flatness and hydrophobicity of the sterol rings allow cholesterol to interact with the nonpolar portions of the lipid bilayer while the hydroxyl group on the end can interact with the hydrophilic part. Membrane fluidity Cholesterol’s function in the lipid bilayer is complex (Figure 3.13). It influences membrane fluidity. Figure 3.14 shows the phase transition for a membrane as it is heated, moving from a more “frozen” character to that of a more “fluid” one as the temperature rises. The mid-point of this transition, referred to as the Tm, is influenced by the fatty acid composition of the lipid bilayer compounds. Longer and more saturated fatty acids will favor higher Tm values, whereas unsaturation and short fatty acids will favor lower Tm values. It is for this reason that fish, which live in cool environments, have a higher level of unsaturated fatty acids in them - to use to make membrane lipids that will remain fluid at ocean temperatures. Interestingly, cholesterol does not change the Tm value, but instead widens the transition range between frozen and fluid forms of the membrane, allowing it to have a wider range of fluidity. Lipid Rafts Cholesterol is also abundantly found in membrane structures called lipid rafts. Depicted in Figure 3.15, lipid rafts are organized structures within the membrane typically containing signaling molecules and other integral membrane proteins. Lipid rafts affect membrane fluidity, neurotransmission, and trafficking of receptors and membrane proteins. Features Distinguishing features of the rafts is that they are more ordered than the bilayers surrounding them, containing more saturated fatty acids (tighter packing and less disorganization, as a result) and up to 5 times as much cholesterol. They also are relatively rich in sphingolipids, with as much as 50% greater quantities of sphingomyelin than surrounding areas of the bilayer. The higher concentration of cholesterol in the rafts may be due to its greater ability to associate with sphingolipids (Figure 3.16). Some groups, such as prenylated proteins, like RAS, may be excluded from lipid rafts. Lipid rafts may provide concentrating platforms after individual protein receptors bind to ligands in signaling. After receptor activation takes place at a lipid raft, the signaling complex would provide protection from nonraft enzymes that could inactivate the signal. For example, a common feature of signaling systems is phosphorylation, so lipid rafts might provide protection against dephosphorylation by enzymes called phosphatases. Lipid rafts appear to be involved in many signal transduction processes, such as T cell antigen receptor signaling, B cell antigen receptor signaling, EGF receptor signaling, immunoglobulin E signaling, insulin receptor signaling and others. For more on signaling, see HERE. Barrier Transport of materials across membranes is essential for a cell to exist. The lipid bilayer is an effective barrier to the entry of most molecules and without a means of allowing food molecules to enter a cell, it would die. The primary molecules that move freely across the lipid bilayer are small, uncharged ones, such as H2O, CO2, CO, and O2, so larger molecules, like glucose, that the cell needs for energy, would be effectively excluded if there were not proteins to facilitate its movement across the membrane. Figure 3.17 depicts the barrier that the lipid bilayer provides to movement across it and the pressures (ionic attraction, in this case) that can affect movement. Potential energy from charge and concentration differences are harvested by cells for purposes that include synthesis of ATP, and moving materials against a concentration gradient in a process called active transport. Membrane proteins Proteins in a lipid bilayer can vary in quantity enormously, depending on the membrane. Protein content by weight of various membranes typically ranges between 30 and 75% by weight. Some mitochondrial membranes can have up to 90% protein. Proteins linked to and associated with membranes come in several types. Transmembrane proteins Transmembrane proteins are integral membrane proteins that completely span from one side of a biological membrane to the other and are firmly embedded in the membrane (Figure 3.18). Transmembrane proteins can function as docking sites for attachment (to the extracellular matrix, for example), as receptors in the cellular signaling system, or facilitate the specific transport of molecules into or out of the cell. Example of integrated/ transmembrane proteins include those involved in transport (e.g., Na+/K+ ATPase), ion channels (e.g., potassium channel of nerve cells) and signal transduction across the lipid bilayer (e.g., GProtein Coupled Receptors).
Peripheral membrane proteins interact with part of the bilayer (usually does not involve hydrophobic interactions), but do not project through it. A good example is phospholipase A2, which cleaves fatty acids from glycerophospholipids in membranes. Associated membrane proteins typically do not have external hydrophobic regions, so they cannot embed in a portion of the lipid bilayer, but are found near them. Such association may arise as a result of interaction with other proteins or molecules in the lipid bilayer. A good example is ribonuclease. Anchored membrane proteins Anchored membrane proteins are not themselves embedded in the lipid bilayer, but instead are attached to a molecule (typically a fatty acid) that is embedded in the membrane (Figure 3.19). The oncogene family of proteins known as ras are good examples. These proteins are anchored to the lipid bilayer by attachment to non-polar farnesyl groups catalyzed by the enzyme farnesyltransferase. Finer classification A more detailed classification scheme further categorizes the integral and anchored proteins into six different types (Figure 3.20). Type I and Type II have only one portion of the protein pass through the membrane. They differ in the orientation of the amine and carboxyl end with respect to inside/outside. Type I transmembrane proteins have the amino terminus on the outside and carboxy terminus on the inside, whereas Type II proteins have this reversed. Type III proteins are a single polypeptide chain that has multiple regions of it cross back and forth across the membrane, often to form a channel. Type IV is a multi-polypeptide protein which has multiple crossings of the membrane. Type V transmembrane proteins do not have a part of them that crosses the membrane, but they are anchored to the membrane by a lipid (such as a fatty acid) embedded in the lipid bilayer. Type VI transmembrane proteins both have a portion of them that crosses the membrane and they are attached to a lipid embedded in the lipid bilayer. Blood Types Cells have hundreds-thousands of membrane proteins and the protein composition of a membrane varies with its function and location. Glycoproteins embedded in membranes play important roles in cellular identification. Blood types, for example, differ from each other in the structure of the carbohydrate chains projecting out from the surface of the glycoprotein in their membranes (Figure 3.21). Osmotic Pressure Membranes provide barriers/boundaries for most molecules, but the permeability of water across a lipid bilayer creates a variable that must be considered. The variable here is osmotic pressure. Osmotic pressure (loosely) refers to the tendency of a solution to take in water by the process of osmosis. In Figure 3.22, one can see a visual representation of the concept of the pressure. A U-shaped tube has at its bottom a semipermeable membrane. Water can pass through the membrane, but sugar molecules (C6H12O6) cannot. On the left side, sugar is added creating a concentration difference between the right and left chambers. Water diffuses across the membrane from right to left in an attempt to equalize the concentrations, causing the level of the right side to decrease and the left side to increase. The pressure resulting from the differences in height is felt at the membrane. Equalizing concentrations The liquid on the right does not completely move to the left, though, as might be expected if the only force involved is equalizing the concentration of sugar across the membrane (no sugar on right = no water). Instead, an equilibrium of sorts of water levels is reached even though the concentrations don’t equal out. The pressure existing at the membrane then from the differences in level corresponds to the osmotic pressure of the mixture. The osmotic pressure of a solution is the pressure difference needed to halt the flow of solvent across a semipermeable membrane. Osmotic pressure can also be thought of as the pressure required to counter osmosis. The osmotic pres- Figure 3.21 - Blood types arise from cell surface glycoproteins Figure 3.22 - Osmotic pressure. Water diffuses leftwards to try to equalize the solute concentration. The pressure realized at the membrane in the right figure is the osmotic pressure sure of a dilute solution mathematically behaves like the ideal gas law \[P_{osmotic} = \dfrac{nRT}{V}\] where n is the number of moles, R is the gas constant, T is the temperature in Kelvin, and V is the volume. It is more convenient in solutions to work with molarity, so \[P_{osmotic}= MR^* T\] where M is the molarity of the dissolved molecules, R* is the gas constant expressed in (L atm)/(K mol), and T is the temperature. The Greek letter Π is used to refer to the Posmoticterm, so \[Π = MR^* T\] Remember when calculating the molarity to include the molarity of each particle. For example, when one dissolves sucrose in solution, it does not split into smaller particles, so \[Molarity_{Particles} = Molarity_{Sucrose}\] However, for salts, like KOH, which forms two ions in solution (K+ and OH- ), MolarityParticles= 2* MolarityKOH. Significant consideration Osmotic pressure is a significant consideration for cells. Consider the fact that water can move freely across cellular membranes, but most of the contents of the cell, such as proteins, DNA, ions, sugars, etc., cannot. Second, the concentration of these compounds inside the cell is different than the concentration of them outside of the cell. Third, since water can move through the lipid bilayer, it will tend to move in the direction that will tend to equalize solute. Hypotonic, hypertonic, isotonic We consider three situations (Figure 3.23). First, if the concentration of solutes is greater inside the cell than outside, water will tend to move into the cell, causing the cell to swell. This circumstance is called hypotonic. Conversely, if the solute concentration is greater outside the cell than inside of it, water will exit the cell and the cell with shrink. This is a hypertonic situation. Last, if the concentrations of solutes into and outside of the cell is equal, this is called an isotonic solution. Here, no movement of water occurs across the cell membrane and the cell retains its size.
Source: BiochemFFA_3_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Movement of materials across membranes As noted earlier, it is essential for cells to be able to uptake nutrients. This function along with movement of ions and other substances is provided by proteins/protein complexes that are highly specific for the compounds they move. Selective movement of ions by membrane proteins and the ions’ extremely low permeability across the lipid bilayer are important for helping to maintain the osmotic balance of the cell and also for providing for the most important mechanism for it to make ATP - the process of oxidative phosphorylation. Terminology A protein involved in moving only one molecule across a membrane is called a uniport (Figure 3.25). Proteins that move two molecules in the same direction across the membrane are called symports (also called synporters, synports, or symporters). If two molecules are moved in opposite directions across the bilayer, the protein is called an antiport. Proteins involved in moving ions are called ionophores. If the action of a protein in moving ions across a membrane results in a net change in charge, the protein is described as electrogenic and if there is no change in charge the protein is described as electroneutral (Figure 3.26). When the driving force for movement through the membrane protein is simply diffusion, the process is called facilitated diffusion or passive transport and when the process requires other energy input, the process is called active transport. Channels and transporters With respect to movement of materials through membrane proteins, there is a difference between channels (sometimes called pores) and transporters. Channels largely provide openings with some specificity and molecules pass through them at close to the rate of diffusion. They usually involve movement of water or ions. Examples would be the sodium or potassium channels of nerve cells. Transporters have high specificity and transfer rates that are orders of magnitude slower. Transport proteins include the sodium-potassium pump, the sodium-calcium exchanger, and lactose permease, amongst many others). Facilitated diffusion As noted, the driving force for facilitated diffusion is concentration, meaning that in facilitated diffusion, materials will only move from a higher concentration to a lower concentration and that at the end of the process, the concentration of materials on each side of a bilayer will be equal (Figure 3.28). This may work well in many cases. For example, the blood concentration of glucose is sufficiently high that red blood cells can use facilitated diffusion as a means of acquiring glucose. Other cells, further removed from the blood supply where the glucose concentration is lower, must use active transport mechanisms because there is not a sufficient concentration of glucose to provide cells with the glucose they need. Ion channels Ion channels are pore-forming membrane proteins in the membranes of all cells that regulate movement of selected ions across a membrane (Figures 3.29 & 3.30). They help to establish the resting membrane potential and to affect action potentials and other electrical signals. They are very important in the process of nerve transmission. Ion channels control the flow of ions across secretory and epithelial cells, and consequently help to regulate cell volume by affecting osmotic pressure. Ion channels are essential features of almost all cells, functioning as selective “tunnels” that restrict movement through them to ions with specific characteristics (typically size). The size of the opening is very narrow (usually one or two atoms wide) and is able to select even against ions that are too small. Control mechanisms Ion channels are controlled by mechanisms that include voltage, ligands, light, temperature, and mechanical deformation (stretch activated). Ligand-gated ion channels (LGICs) are transmembrane proteins which open to selectively allow ions such as Na+, K+, Ca++, or Cl− to pass through the membrane in response to the binding of a ligand messenger. Sound waves cause mechanical deformation of hair cells in the ear. This results in the opening of ion channels and initiation of a nerve signal to the brain. Sodium ion channels in the tongue for sugar receptors open in response to binding of sucrose, allowing sodium concentration in the nerve cell to increase and initiate a nerve signal to the brain. In this case, the default for the gate is to be closed and it opens in response to binding of a ligand (sucrose). In light sensing cells of the eye, calcium gates are open by default, but stimulation by light causes them to close, triggering a series of events that result in a signal being sent the brain about the perception of light. Thus, in this case, the stimulus (light) causes an open channel to close. Moving the other direction, nerve signals originating in the brain travel to muscle tissue and through a complicated set of exchanges, result in the opening of calcium gates of muscle cells, increasing the concentration of calcium and stimulating muscular contraction (see HERE). Voltage gated channels are essential for transmission of nerve signals, a process discussed in more depth HERE. Ion movement through channels The ability of ion channels to select against ions too large is intuitive - the size of the opening in the ion channel simply isn’t big enough for a larger ion to fit through the opening. Potassium, for example, passes through sodium channels rarely because the opening is too small. Potassium channels that are selective for potassium ions must be big enough to allow potassium to enter, but if size were the only selection means, then sodium ions would also readily pass through potassium channels, since sodium ions (0.95 Å) are smaller than potassium ions (1.33 Å). In order for potassium channels to select against sodium ions and favor potassium ions, other considerations come into play. Hydration shell
To understand this unique selectivity, it is important to understand how ions move through channels. Before an ion can pass through a channel, it must first be dissociated from (stripped of) the water molecules in its hydration shell - water molecules surrounding ions in aqueous solutions (Figure 3.32). This process requires an input of energy. The initial energy required to strip the water molecules from the hydration shell has been compared to the activation energy of an enzymatic reaction. Comparable to enzymes Just as enzymes lower the activation energy of enzymatic reactions and thus allow them to more readily occur, so too do channel proteins lower the energy requirements for a molecule to traverse a lipid bilayer. In the absence of the channel protein, the dehydration energy is mostly prohibitive for most polar molecules to occur, so very few make it across the lipid bilayer without the channel protein. This is why ion channel/transport proteins are so important to the cell. After the water has been stripped, the ion can pass through the channel and when it arrives at the other side of the channel, the diffusing ion becomes rehydrated, thus regaining the energy that was required initially to strip away the water molecules from the ion. Selectivity of the potassium channel The potassium channel (Figure 3.33) uses the dimensions of the potassium ion precisely to shepherd it through the channel. The sodium ion, which has different dimensions has a more difficult time making it through the channel despite its smaller size. The reason this is rooted in the energy required for dehydration. For potassium ions, after the water has been stripped off, precisely positioned carbonyl groups along the channel help to stabilize the ion as it moves. The sodium ion, on the other hand is too small and does not make efficient connections with carbonyl groups and thus has a more difficult path. Because of this, the energy difference between dehydration and rehydration of a sodium ion in a potassium channel is energetically unfavorable (requires net input of energy) but the same process for a potassium ion is energetically favorable (results in a net gain of energy). Movie 3.1 - Gramicidin A Wikipedia (animated gif, download to view) Energy factor Thus the selection in favor of potassium and against sodium ions in a potassium channel is based on energy, not physical size, whereas in the selection of sodium ions over potassium ions in a sodium channel, size is the primary consideration. Ion balance The movement of ions across a lipid bilayer is tightly regulated, and with good reason. Maintaining a proper balance of ions inside and outside of cells is important for maintaining osmotic balance. It is also important inside and outside of organelles like the mitochondria and chloroplasts for energy generation. If the ionic balance of a cell is sufficiently disturbed by an uncontrolled ionophore, a cell may die. Gramicidin Gramicidins (Movie 3.1) are antibiotic polypeptides synthesized by the soil bacterium known as Bacillus brevis. These small pentadecapeptides (15 amino acids) are synthesized by the bacterium to kill other bacteria. When released by the Bacillus brevis, the gramicidins insert themselves in the membranes of Gram positive bacteria and allow the movement of sodium ions into the target cells, ultimately killing them. Gramicidins can also cause hemolysis in humans so they cannot be used internally, but instead are used topically. Aquaporins Aquaporins are pore-containing integral membrane proteins that selectively permit passage of water molecules in and out of the cell, while preventing ions and other solutes from moving (Figures 3.34 & 3.35). Some aquaporins called aquaglyceroporins, also transport other small uncharged entities, such as glycerol, ammonia, urea, and CO2, across the membrane,. The water pores are completely impermeable to charged molecules, such as protons, which is important for the preserving the membrane's electrochemical potential difference. Porins Porins are proteins containing a β-barrel structure that crosses the cell membrane/wall and acts as a pore/channel through which specific molecules diffuse. Porins are found in the outer membrane of Gram-negative bacteria and some Gram-positive bacteria, mitochondria, and chloroplasts. Porins typically transport only one group of molecules or, in some cases, one specific molecule. Antibiotics, such as β-lactam and fluoroquinolone pass through porins to reach the cytosol of Gram negative bacteria. Bacteria may develop resistance to these antibiotics when a mutation occurs to the porin involved that results in exclusion of the antibiotics that would otherwise pass through. Transporter proteins Not all facilitated transport occurs through ion channel proteins. Transporter proteins, as noted earlier (HERE and Figure 3.27) facilitate movement of materials across a lipid bilayer, but are slower than ion channels. Figure 3.36 illustrates a transporter protein in action. As can be seen, transporter proteins rely on a specific receptor site for proper recognition of the molecule to be moved. Binding of the proper molecule causes a conformational change in the shape of the protein (an eversion) which results in a flipping of the open side of the protein to the other side of the lipid bilayer. In this way, the molecule is moved. Like ion channels, transporter proteins facilitate movement of materials in either direction, driven only by the concentration difference between one side and the other. Active transport All of the transport mechanisms described so far are driven solely by a concentration gradient - moving from higher concentrations in the direction of lower concentrations. These movements can occur in either direction and, as noted, result in equal concentrations on either side of the bilayer, if allowed to go to completion. Many times, however, cells must move materials against a concentration gradient and when this occurs, another source of energy is required. This process is known as active transport.
A good definition of active transport is that in active transport, at least one molecule is being moved against a concentration gradient. A common, but not exclusive, energy source is ATP (see Na+/K+ ATPase), but other energy sources are also employed. For example, the sodium-glucose transporter uses a sodium gradient as a force for actively transporting glucose into a cell. Thus, it is important to know that not all active transport uses ATP energy. Na+/K+ ATPase An important integral membrane transport protein is the Na+/K+ ATPase antiport (Figures 3.37 and 3.38), which moves three sodium ions out of the cell and two potassium ions into the cell with each cycle of action. In each case, the movement of ions is against the concentration gradient. Since three positive charges are moved out for each two positive charges moved in, the system is electrogenic. The protein uses the energy of ATP to create ion gradients that are important both in maintaining cellular osmotic pressure and (in nerve cells) for creating the sodium and potassium gradients necessary for signal transmission. Failure of the system to function results in swelling of the cell due to movement of water into the cell through osmotic pressure. The transporter expends about one fifth of the ATP energy of animal cells. The cycle of action occurs as follows: 1. Pump binds ATP followed by binding of 3 Na+ ions from cytoplasm of cell 2. ATP hydrolysis results in phosphorylation of aspartate residue of pump. ADP is released 3. Phosphorylated pump undergoes conformational change to expose Na+ ions to exterior of cell. Na+ ions are released. 4. Pump binds 2 extracellular K+ ions. 5. Pump dephosphorylates causing it to expose K+ ions to cytoplasm as pump returns to original shape. 6. Pump binds 3 Na+ ions, binds ATP and releases 2 K+ ions to restart process The Na+/K+ ATPase is classified as a P-type ATPase. This category of pump is notable for having a phosphorylated aspartate intermediate and is present across the biological kingdoms - bacteria, archaeans, and eukaryotes. ATPase types ATPases have roles in either the synthesis or hydrolysis of ATP and come in several different forms. • F-ATPases (F1FO-ATPases) are present in mitochondria, chloroplasts and bacterial plasma membranes and are the prime ATP synthesizers for these systems. Each uses a proton gradient as its energy source for ATP production. Complex V of the mitochondrion is an F-type ATPase. • V-ATPases (V1VO-ATPases) are mostly found in vacuoles of eukaryotes . They utilize energy from ATP hydrolysis to transport solutes and protons into vacuoles and lysosomes, thus lowering their pH values. The V-type and F-type ATPases are very similar in structure. The V-type (Figure 3.39) uses ATP to pump protons into vacuoles and lysosomes, whereas F-types use proton gradients of the mitochondria and chloroplasts to make ATP. • A-ATPases (A1AO-ATPases) are found in archaeans and are similar to F-ATPases in function. • P-ATPases (E1E2-ATPases) are in bacteria, fungi and in eukaryotic plasma membranes and organelles. They transport a diversity of ions across membranes. Each has a common mechanism of action which include autophosphorylation of a conserved aspartic acid side chain within it. Examples of P-type ATPases include the Na+/K+ ATPase and the calcium pump. • E-ATPases are enzymes found on the cell surface. They hydrolyze a range of extracellular nucleoside triphosphates, including ATP. Nerve transmission Now that you have seen how the Na+/K+ ATPase functions, it is appropriate to discuss how nerve cells use ion gradients created with it to generate and transmit nerve signals. Neurons are cells of the nervous system that use chemical and electrical signals to rapidly transmit information across the body (Figure 3.40). The sensory nerve system links receptors for vision, hearing, touch, taste, and smell to the brain for perception. Motor neurons run from the spinal cord to muscle cells. These neurons have a cell body and a very long, thin extension called an axon, that stretches from the cell body in the spinal cord all the way to the muscles they control. Nerve impulses travel down the axon to stimulate muscle contraction. Signals travel through neurons, ultimately arriving at junctions with other nerve cells or target cells such as muscle cells. Note that neurons do not make physical contact with each other or with muscle cells. The tiny space between two neurons or between a neuron and a muscle cell is called the synaptic cleft. At the synaptic cleft, the neuron releases neurotransmitters that exit the nerve cell and travel across the junction to a recipient cell where a response is generated. That response may be creating another nerve signal, if the adjacent cell is a nerve cell or it may be a muscular contraction if the recipient is a muscle cell (Figure 3.41). In considering information movement via nerve cells, then, we will discuss two steps - 1) creation and propagation of a signal in a nerve cell and 2) action of neurotransmitters exiting a nerve cell and transiting a synaptic junction. Signal source Creation of a nerve signal begins with a stimulus to the nerve cell. In the case of muscle contraction, the motor cortex of the brain sends signals to the appropriate motor neurons, stimulating them to generate a nerve impulse. How is such an impulse generated? Resting potential In the unstimulated state, all cells, including nerve cells, have a small voltage difference (called the resting potential) across the plasma membrane, arising from unequal pumping of ions across the membrane. The Na+/K+ ATPase, for example, pumps sodium ions out of the cell and potassium ions into cells. Since three sodium ions get pumped out for every two potassium ions pumped in, a charge and chemical gradient is created. It is the charge gradient that gives rise to the resting potential. Altering the gradients of ions across membranes provide the driving force for nerve signals. This happens as a result of opening and closing of gated ion channels. Opening of gates to allow ions to pass through the membrane swiftly changes the ionic balance across the membrane resulting in a new voltage difference called the action potential. It is the action potential that is the impetus of nerve transmission. Initiation of signal
The signal generated by a motor neuron begins with opening of sodium channels in the membrane of the nerve cell body causing a rapid influx of sodium ions into the nerve cell. This step, called depolarization (Figure 3.42), triggers an electrochemical signal - the action potential. Remember that the Na+/K+ ATPase has created a large sodium gradient, so sodium ions rush into the cell when sodium channels open. After the initial depolarization, potassium channel gates, responding to the depolarization, open, allowing potassium ions to rapidly diffuse out of the cell (remember K+ ions are more abundant inside of the cell). This phase is called the repolarization phase and during it, the sodium gates close. The rapid exit of potassium ions causes the voltage difference to “overshoot” the resting potential and potassium gates close. This followed by the so-called refractory period, when the Na+/K+ ATPase begins its work to re-establish the original conditions by pumping sodium ions out and potassium ions into the nerve cell. Eventually, the system recovers and the resting potential is re-established. The initiating end of the nerve cell is then ready for another signal. Propagation of action potential What we have described here is only the initiation of the nerve signal in one part of the nerve cell. For the signal to be received, the action potential must travel the entirety of the length of the nerve cell (the axon) and cause a chemical signal to be released into the synaptic cleft to get to its target. Propagating the nerve signal (action potential) in the original nerve cell is the function of all of the rest of the gated ion channels (Figure 3.43) positioned on the sides of the nerve cell. The sodium and potassium gates involved in propagation of the signal all act in response to voltage changes created by the electrochemical gradient moving down the nerve cell (Figure 3.44). Remember that opening of the initial gates at initiation of the signal created an influx of sodium ions and an efflux of potassium ions. Moving signal This chemical and electrical change that creates the action potential leaves the end of the nerve cell where it started and travels down the axon towards the other end of the nerve cell. Along the way, it encounters more sodium and potassium gated channels. In each case, these respond simply to the voltage change of the action potential and open and close, exactly in the same way the gates opened to start the signal. Thus, a rapid wave of increasing sodium ions and decreasing potassium ions moves along the nerve cell, propagated (and amplified) by gates opening and closing as the ions and charges move down the nerve cell. Eventually, the ionic tidal wave reaches the end of the nerve cell (axon terminal) facing the synaptic cleft. Crossing the synaptic cleft For the signal to be received by the intended target (postsynaptic cell) from the originating neuron (presynaptic neuron), it must cross the synaptic cleft and stimulate the neighboring cell (Figure 3.45). Communicating information across a synaptic cleft is the job of neurotransmitters. These are small molecules synthesized in nerve cells that are packaged in membrane vesicles called synaptic vesicles in the nerve cell. Neurotransmitters come in all shapes and chemical forms, from small chemicals like acetylcholine to peptides like neuropeptide Y. The most abundant neurotransmitter is glutamate, which acts at over 90% of the synapses in the human brain. Movie 3.2 - Movement of an action potential down a nerve cell - Wikipedia Into the cleft As the action potential in the presynaptic neuron approaches the axon terminus, synaptic vesicles begin to fuse with the membrane and their neurotransmitter contents spill into the synaptic cleft. Once in the cleft, the neurotransmitters diffuse, some of them reaching receptors on the postsynaptic cell. Binding of the neurotransmitter to the receptors on the membrane of the postsynaptic cell stimulates a response. For motor neurons, the postsynaptic cell will be a muscle cell, and the response will be muscle contraction/relaxation. At this point, the originating nerve cell has done its job and communicated its information to its immediate target. If the postsynaptic cell is a nerve cell, the process repeats in that cell until it gets to its destination. Na+/glucose transporter Absorbing nutrients from the digestive system is necessary for animal life. The sodium/glucose transport protein is an electrogenic symporter that moves glucose into intestinal cells. It is found in the intestinal mucosa and the proximal tubule of the nephron of the kidney. The sodium/glucose transport system functions in the latter to promote reabsorption of glucose. The pump works in conjunction with the Na+/K+ transport system. The gradient of sodium ions built up by the Na+/K+ pump is used as an energy source to drive movement of glucose into cells (see Figure 3.38). Use of an ion gradient established by a separate pump is known as secondary active transport. For intestinal mucosa, the pump transports glucose out of the gut and into gut cells. Later, the glucose is exported out the other side of the gut cells to the interstitial space for use in the body. Calcium pumps Calcium ions are necessary for muscular contraction and play important roles as signaling molecules within cells. In addition, when calcium concentrations rise too high, DNA in chromosomes can precipitate. Calcium concentration in cells is therefore managed carefully. It is kept very low in the cytoplasm as a result of action of pumps, both in the plasma membrane, which pump calcium outwards from the cytoplasm and in organelles, such as the endoplasmic reticulum (sarcoplasmic reticulum of muscle cells), which pump calcium out of the cytoplasm and into these organelles. Opening of calcium channels, then, increases calcium concentration quickly in the cytoplasm resulting in a quick response, whether the intention is signaling or contraction of a muscle. After the response is generated, the calcium is pumped back out of the cytoplasm by the respective calcium pumps. Some calcium pumps use ATP as an energy source to move calcium and others use ion gradients, such as sodium for the same purpose. Na+/Ca++ transporter
One calcium pump of interest uses the sodium gradient as an energy source. It is the sodium/calcium pump. This electrogenic antiport system uses sodium’s movement into the cell as a driving force to move calcium out of the cell, although its direction can reverse in some circumstances. The pump is a high capacity system to move a lot of calcium quickly, moving up to 5000 calcium ions per second and is found in many tissues with many functions. Digitalis One important function of the Na+/Ca++ pump occurs in heart cells. Ca++ is important for contraction of heart muscle. Calcium efflux from the cells is the normal operation of the pump, however, during the upstroke of the cycle, there is a large movement of sodium ions into the heart cell. When this occurs, the pump reverses and pumps Na+ out and Ca++ in briefly. Since calcium helps stimulate contraction of cardiac muscle, this can help make the heart beat stronger and is the focus of the use of digitalis to treat congestive heart failure. Digitalis blocks the sodium-potassium ATPase and interferes with the sodium ion gradient. As noted above, when the Na+ gradient is oriented in the wrong direction, calcium is pumped in. Digitalis is therefore used to treat congestive heart failure because it increases the concentration of calcium in the heart cells, favoring more forceful beats. ABC transporters ABC transporters are another class of transmembrane proteins that use ATP energy to transport things against concentration gradients (Figures 3.47 & 3.48). This protein superfamily includes hundreds of proteins (48 in humans alone) and spans all extant phyla from prokaryotes to humans. These proteins function not only in membrane transport, but also in processes that include DNA repair and the process of translation. Transport Substances that ABC transporters move across membranes include metabolic products, lipids, sterols, and drugs. ABC transporters function in multi-drug resistance of many cells, and provide resistance to antibiotics in bacteria as well as resistance to chemotherapy in higher cells by exporting drugs used to treat both of these types of cells. ABC transporters are divided into three main groups - 1) importers (prokaryotes only); 2) exporters (prokaryotes and eukaryotes), and 3) non-transporters with roles in DNA repair and translation. All ABC transport proteins have four protein domains - two that are cytoplasmic and two that are membrane bound. They are alternately open to the cytoplasmic or extracellular (or periplasmic) regions and this is controlled by hydrolysis of ATP. Disease ABC transporters have roles in cystic fibrosis and other inherited human diseases. They are very involved in development of resistance to multiple drugs by a diverse group of cells. ABC transporters provide multi-drug resistance by expelling drug(s) from cells. ABCB1 protein, for example, pumps tumor suppression drugs out of the cell. Another ABC transporter known as Pgp transports organic cationic or neutral compounds. Cystic fibrosis Cystic fibrosis (CF) is a an autosomal recessive genetic disorder arising from mutations in both copies of the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. This ABC transporter system, which moves chloride and thiocyanate ions across epithelial tissue membranes exerts its effect mostly in the lungs but the pancreas, liver, kidneys, and intestine are all also affected by it. Function CFTR has roles in the production of sweat, mucus, and digestive fluids. Manifestations of the disease include breathing difficulty and overproduction of mucus in the lungs. When CFTR is functional, these fluids are normally thin, but when the gene is non-functional, they become much thicker and are points of infection. CFTR contains two ATP-hydrolyzing domains and two cell membrane-crossing domains with 6 α-helices each. It can be activated by phosphorylation by a cAMP-dependent protein kinase. The carboxyl end of CFTR is linked to the cytoskeleton by a PDZ domain. Lactose permease Another integral membrane protein performing active transport is lactose permease. It facilitates the movement of the sugar lactose across the lipid bilayer of the cell membrane (Figures 3.49- 3.51). The transport mechanism is classified as a secondary active transport since it exploits the inwardly directed H+ electrochemical gradient as an energy source. When lactose is transported into cells, it is broken down into its substituent monosaccharide sugars - glucose and galactose - for energy creation. The enzyme catalyzing this reaction is known as lactase and deficiency of it in humans leads to lactose intolerance (see HERE). GLUTs GLUTs (GLUcose Transport proteins) are uniport, type III integral membrane proteins that participate in the transport of glucose across membranes into cells. GLUTs are found in all phyla and are abundant in humans, with 12 GLUT genes. GLUT1, in erythrocytes is well-studied. Through GLUT 1, glucose enters and passes through it via facilitated diffusion at a rate that is 50,000 higher than in its absence. GLUTs of various types are found in different cells of the body. The one in red blood cells is known as GLUT 1 and has 12 membrane-spanning hydrophobic helices. Though the structure of GLUT 1 is not known, it is speculated that the 12 helices form a chamber able to form hydrophilic bonds with glucose to facilitate its passage. GLUT 1 levels in erythrocytes go up as glucose levels decrease and decrease when glucose levels go down. GLUT 1 can also transport ascorbate (vitamin C) in addition to glucose in mammals (such as humans) that do not produce their own vitamin C. Glut 4 GLUT 4 is regulated by insulin and is found primarily in adipose and striated muscle tissue. Insulin alters intracellular trafficking pathways in response to increases in blood sugar to favor movement of various GLUT proteins (including GLUT 4) from intracellular vesicles to the cell membrane, thus stimulating uptake of the glucose. GLUT 4 is also found in the hippocampus where, if trafficking is disrupted, the result can be depressive behavior and cognitive dysfunction. For all of the GLUT proteins, a key to keeping the glucose in the cell is phosphorylation of it by the glycolysis enzyme, hexokinase, in the cytoplasm. Phosphorylated molecules cannot enter GLUTs and don’t have an easy means of exiting the cell. Distance Ed To the tune of “Mister Ed” Metabolic Melodies Website HERE
A course is a source, of course, of course Of all of the knowledge that we endorse A major force for better/worse is the campus Distance Ed It’s true to outsource a college course There are a few standards to be enforced The long and short’s we reinforce the campus Distance Ed Bridge A classroom class meets every week the same time every day But Distance Ed is most unique - its flexible schedule’s okay E-course is a source, of course, of course Of online assistance for lab reports You’re not enrolled in an online course? Then sign up for this! “You’ll love Distance Ed” Recording by David Simmons Lyrics by Kevin Ahern Recording by David Simmons Lyrics by Kevin Ahern 313 It's one o'clock and Ahern's talkin' Henderson and Hasselbalch and pKa's and Buffers I should know This song's for BB three five oh I hope that maybe He'll think the way we Wrote our answers Wasn't crazy I really need the Partial credit - so This song's for BB three five oh It's really groovy That it improves me Watching lectures In Quicktime movies I really need to Go and download those Podcasts for BB three five oh This Song's For BB 3-5-0 To the tune of "This Land is My Land" Metabolic Melodies Website HERE I'm feeling manic I'm in a panic I'd better study My old organic It has reactions That I need to know This song's for BB three five oh I know he said it That's why I dread it 'cause I skipped Friday's Extra credit 'twil pro'bly haunt me That lowly ze-ro Grade in BB three five oh It could be steric Or esoteric That carbons get so Anomeric I'm too hysteric Better let it go This song's for BB three five oh Recording by Tim Karplus Lyrics by Kevin Ahern Recording by Tim Karplus Lyrics by Kevin Ahern
Source: BiochemFFA_3_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy There are many functions and factors relating to cell membranes that don’t fit into broad categories. Those items will be the focus of this section. Endocytosis Besides transporter proteins and ion channels, another common way for materials to get into cells is by the process of endocytosis. Endocytosis is an alternate form of active transport for getting materials into cells. Some of these processes, such as phagocytosis, are able to import much larger particles than would be possible via a transporter protein. Like transporter proteins, endocytosis uses energy for the purpose (though it is not as visible as with protein transporters), but unlike protein transporters, the process is not nearly as specific for individual molecules. As a result, the process usually involves the importation of many different molecules each time it occurs. The list of compounds entering cells in this way includes LDLs and their lipid contents, but it also include things like iron (packaged in transferrin), vitamins, hormones, proteins, and even some viruses sneak in by this means. There are three types of endocytosis we will consider (Figure 3.53). Receptor mediated endocytosis The process of receptor mediated endocytosis is a relatively specific means of bringing molecules into cells because it requires the incoming material to be somehow associated with a specific cell surface receptor. In the example of Figure 3.53, the receptor is the cellular LDL receptor. Clathrin-coated invaginations, as shown in the figure are known as “coated pits.” The mechanism proceeds with an inward budding of the plasma membrane receptor (coated vesicles). Binding of the ligand (ApoB-100 of the LDL, for example, in Figure 3.54) to the LDL receptor leads to formation of a membrane invagination. The absorbed LDL particle fuses to form an early endosome (Figure 3.55) and contents are subsequently sorted and processed for use by the cell. The components from the coated vesicle are recycled to the plasma membrane for additional actions. Receptor mediated endocytosis can also play a role in internalization of cellular receptors that function in the process of signaling. Here, a receptor bound to a ligand is brought into the cell and may ultimately generate a response in the nucleus. While receptor mediated endocytosis of receptors can have the effect of communicating a signal inwards to the cell, it can also reduce the total amount of signaling occurring, since the number of receptors on the cell surface is decreased by the process. Non-clathrin endocytosis There are three types of endocytosis occurring in cells that do not involve clathrin. They are 1) caveolae-based endocytosis, 2) macropinocytosis, and 3) phagocytosis. Caveolae-based endocytosis is based on a receptor molecule known as caveolin. Caveolins are a class of integral membrane proteins that compartmentalize and concentrate signaling molecules in the process of endocytosis. They are named for the cave-like caveolae structures of the plasma membrane where they are found. Caveolins Caveolins have affinity for cholesterol and associate with it in the membrane of cells, causing the formation of membrane invaginations of about 50 nm. The caveolin proteins can oligomerize and this is important for the coating and formation of the cave-like structures. There are three caveolin genes found in vertebrate cells, CAV1, CAV2, and CAV3. Down-regulation of caveolin-1 results in less efficient cellular migration in vitro. Caveolins are implicated in both formation and suppression of tumors. High expression of them inhibits cancer-related growth factor signaling pathways, but some caveolin-expressing cancer cells are more aggressive and metastatic, possible due to an enhanced capacity for anchorage-independent growth. Macropinocytosis A phenomenon known as “cell drinking,” macropinocytosis literally involves a cell “taking a gulp” of the extracellular fluid. It does this, as shown in Figure 3.56, by a simple invagination of ruffled surface features of the plasma membrane. A pocket results, which pinches off internally to create a vesicle containing extracellular fluid and dissolved molecules. Within the cytosol, this internalized vesicle will fuse with endosomes and lysosomes. The process is non-specific for materials internalized. Phagocytosis Phagocytosis is a process whereby relatively large particles (0.75 µm in diameter) are intenalized. Cells of the immune system, such as neutrophils, macrophages, and others, use phagocytosis to internalize cell debris, apoptotic cells, and microorganisms. The process operates through specific receptors on the surface of the cell and phagocytosing cell engulfs its target by growing around it. The internalized structure is known as a phagosome, which quickly merges with a lysosome to create a phagolysosome (Figure 3.58), which subjects the engulfed particle to toxic conditions to kill it, if it is a cell, and/or to digest it into smaller pieces. In some cases, as shown in the figure, soluble debris may be released by the phagocytosing cell. Endosomes Internalized material from endocytosis that doesn’t involve phagocytosis passes through an internalized structure called an endosome. Endosomes are membrane bounded structures inside of eukaryotic cells that play a role in endocytosis (Figure 3.59). They have a sorting function for material internalized into the cell, providing for retrieval of materials not destined for destruction in the lysosomes. LDLs, for example, are targeted after endocytosis to the endosomes for processing before part of them is delivered to the lysosome. The endosomes can also receive molecules from the trans-Golgi network. These can be delivered to the lysosomes, as well, or redirected back to the Golgi. Endosomes come in three forms - 1) early, 2) late, and 3) recycling. Figure 3.59 - Internalization of the epidermal growth factor receptor (EGFR) into endosomes. Early (E) and late (M) endosomes and lysosomes (L) are labeled. - Wikipedia Exocytosis
The process of exocytosis is used by cells to export molecules out of cells that would not otherwise pass easily through the plasma membrane. In the process, secretory vesicles fuse with the plasma membrane and release their contents extracellularly. Materials, such as proteins and lipids embedded in the membranes of the vesicles become a part of the plasma membrane when fusion between it and the vesicles occurs. Membrane fusion Fusion is a membrane process where two distinct lipid bilayers merge their hydrophobic cores, producing one interconnected structure. Membrane fusion involving vesicles is the mechanism by which the processes of endocytosis and exocytosis occur. When the fusion proceeds through both leaflets of both bilayers, an aqueous bridge results and the contents of the two structures mix. Common processes involving membrane fusion (Figure 3.60) include fertilization of an egg by a sperm, separation of membranes in cell division, transport of waste products, and neurotransmitter release (Figure 3.61). Artificial membranes such as liposomes can also fuse with cellular membranes. Fusion is also important for transporting lipids from the point of synthesis inside the cell to the membrane where they are used. Entry of pathogens can also be governed by fusion, as many bilayer-coated viruses use fusion proteins in entering host cells. SNARE proteins Mediation of fusion of vesicles in exocytosis is carried out by proteins known as SNAREs (Soluble NSF Attachment Protein REceptor). This large superfamily of proteins spans a wide biological range, from yeast to mammals. Common vesicle fusions occur when synaptic vesicles dock with neurons (Figure 3.61) and release neurotransmitters. These are well-studied. The SNAREs involved in this process can be proteolytically cleaved by bacterial neurotoxins that give rise to the conditions of botulism and tetanus. SNAREs are found in two locations. v-SNAREs are found in the membranes of transport vesicles during the budding process, whereas t-SNARES can be found in the membranes of targeted compartments. The act of vesicle fusion coincides with increases of intracellular calcium. Fusion of synaptic vesicles in neurotransmission results in activation of voltage-dependent calcium channels in the targeted cell. Influx of calcium helps to stimulate vesicle fusion. In the endocrine system, binding of hormones to G protein coupled receptors activate the IP3/DAG system to increase levels of calcium. In the process of membrane fusion (Figure 3.62), the v-SNAREs of a secretory vesicle (upper left) interact with the t-SNAREs of a target membrane (bottom). The v- and t-SNAREs “zipper” themselves together to bring the membrane vesicle and the target membrane closer together. Zippering also causes flattening and lateral tension of the curved membrane surfaces, favoring hemifusion of the outer layers of each membrane. Continued tension results in subsequent fusion of the inner membranes as well, yielding opening of the contents of the vesicle chamber to its target (usually outside the cell). Shuttles Another way to transport items across a membrane for which there is no specific transport system available is the use of shuttles. Shuttles are important when there is no transport mechanism for moving material across a membrane for which no transport system exists. A great example is NADH. NADH is an important electron carrier that is produced in the cytoplasm and mitochondria of the cell. NADH produced in the mitochondrion goes directly to the electron transport system and delivers electrons to Complex I. NADH produced in the cytoplasm (such as from glycolysis) does not have this option, since the inner membrane of the mitochondrion is impermeable to the molecule and no transporter exists to move it across. The important part of the NADH is its electron cargo, so cells have evolved two ways to move the electrons into the mitochondrial matrix apart from NADH. Both methods involve shuttles. In each case, an acceptor molecule receives electrons from NADH and the reduced form of the acceptor molecule is transported. It gets transported into the matrix where it is oxidized (electrons are lost) and then donated to the electron transport system. Glycerol phosphate shuttle The first of these methods is the least efficient, but it is rapid. It found commonly in muscles which have needs for rapid energy and brain tissue. This shuttle is referred to as the glycerol phosphate shuttle and is shown in Figure 3.63. It operates in the intermembrane space between the inner and outer mitochondrial membranes. The outer mitochondrial membrane is very porous, allowing many materials to pass freely through it. In the intermembrane space, the cytoplasmic enzyme, glyceraldehyde-3-phosphate dehydrogenase (cGPD) catalyzes transfer of electrons from NADH to dihdydroxyacetone phosphate (#2 in the figure), yielding NAD+ and glyceraldehyde-3-phosphate (#1 in the figure). The glyceraldehyde-3-phosphate then binds to a glyceraldehyde-3-phosphate dehydrogenase (mGPD) embedded in the outer portion of the inner mitochondrial membrane. mGPD catalyzes the transfer of electrons from glyceraldehyde-3-phosphate to FAD, producing dihycroxyacetone phosphate and FADH2. FADH2 then transfers its electrons to the electron transport system through CoQ (Q above), forming CoQH2 (QH2 above). As will be discussed in the section on electron transport, this is not an efficient shuttle system because it does not result in production of as much ATP as occurs when electrons are transferred to NAD+ instead of FAD. Malate-aspartate shuttle A more efficient system of transferring electrons is the malate-aspartate shuttle and it is shown in Figure 3.64. As is apparent in the figure, this shuttle involves more steps than the glycerol phosphate shuttle, but the advantage of the malate-aspartate shuttle is that it is more efficient. NADH outside of the mitochondrion transfers its electrons to the shuttle and then NADH is re-made on the inside of the shuttle. No energy is expended in the process. When NADH accumulates in the cytoplasm, it moves to the intermembrane space where the enzyme malate dehydrogenase catalyzes the transfer of electrons to oxaloacetate to yield NAD+ and malate. A transport system for malate moves malate into the mitochondrial matrix in exchange for α-ketoglutarate.
Inside the mitochondrion, malate is reoxidized to oxaloacetate and electrons are given to NAD+ to recreate NADH. NADH then donates electrons to Complex I of the electron transport system. That’s really all there is to the shuttle. The remaining steps are simply to balance the equation of the process. Oxaloacetate accepts an amine group from glutamic acid to yield aspartic acid and α-ketoglutarate. Aspartate then moves out of the mitochondrion through an antiport transport protein that swaps it for glutamate. A series of reactions in the intermembrane space balance the equation. It is easy to get lost in the mess of balancing equations. The most important thing to understand here is that oxaloacetate accepts electrons on the outside to become malate which is the carrier of electrons across the membrane. Once inside the matrix, malate is converted back to oxaloacetate and its electrons are given to NAD+, forming NADH. Everything else is simple equation balancing. Acetyl-CoA shuttle Another kind of shuttle also involves the mitochondrion and in this case, the item being moved is a molecule, not a pair of electrons. The molecule of interest here is acetyl-CoA, for which no transport system operates, but which is needed in the cytoplasm for fatty acid synthesis when the cell has abundant energy. Acetyl-CoA is mostly in the mitochondrion and so long as the citric acid cycle is operating efficiently, its concentration is relatively stable. However, when the citric acid cycle slows, acetyl-CoA and the citrate made from it in the cycle begin to accumulate. A membrane transport system for citrate exists, so it gets moved out to the cytoplasm. In the cytoplasm, an enzyme known as citrate lyase cleaves citrate to acetyl-CoA and oxaloacetate. Oxaloacetate can be reduced to malate and moved back into the mitochondrion. As for acetyl-CoA, the more of that cells have in the cytoplasm, the more likely they will begin making fatty acids and fat, since acetyl-CoA is the starting material for fatty acid synthesis, which occurs in the cytoplasm. When does this process occur? As noted above, it occurs when the citric acid cycle stops and this occurs when levels of NADH and FADH2 increase. These, of course, increase when one is not burning off as many calories as one is consuming as a byproduct of respiratory control. Lack of exercise leads to higher levels of reduced electron carriers. Cell junctions Cells in multicellular organisms are in close contact with each other and links between them are called junctions. In vertebrate organisms, there are three main types of cell junctions and one of them (gap junctions) is important for movement of materials between cells. The three types are 1. Gap junctions 2. Adherens junctions, (Anchoring Junctions, desmosomes and hemidesmosomes) 3. Tight junctions Cell junctions in multicellular plants are structured differently from those in vertebrates and are called plasmodesmata. They too function in exchange of materials between individual cells. Gap junctions Gap junctions are specialized structures made up of two sets of structures called connexons (one from each cell - see Figure 3.65) directly link the cytoplasms of the connected cells. Gap junctions are regulated to control the flow of molecules, ions, and electrical impulses between cells. In plants, similar structures known as plasmodesmata traverse the cell wall (Figure 3.66) and perform similar functions. Adherens junctions Adherens junctions (Figure 3.67) are protein complexes on the cytoplasmic side of the cell membranes of epithelial and endothelial tissues that link cells to each other or to the extracellular matrix. They correspond to the fascia adherens found in non-epithelial/non-endothelial cells. Adherens junctions contain the following proteins - 1) α-catenin (binds cadherin through β-catenin); 2) β-catenin (attachment for α-catenin to cadherin; 3) γ-catenin (binds to cadherin); 4) cadherins (group of transmembrane proteins that dimerize with cadherins on adjacent cells; 5) p120 (also called Δ-catenin - binds to cadherin); 6) plakoglobin (catenin family protein homologous to and acting like β-catenin); 7) actin; 8) actinin; and 9) vinculin. Adherens junctions may help to maintain the actin contractile ring which forms in the process of cytokinesis. Tight junctions Tight junctions (Figure 3.68) are a network of protein strands that seal cells together and restrict the flow of ions in the spaces between them. The effect of their structure is to restrict the movement of materials through tissues by requiring them to pass through cells instead of around them. Tight junctions join together the cytoskeletons of cells and through their structure maintain their apical and basolateral polarity. GPI anchors Membrane proteins attached to glycosylphosphatidylinositol (also known as a GPI anchor) are referred to as being glypiated. The proteins, which play important roles in many biochemical processes, are attached to the GPI anchor at their carboxyl terminus. Phospholipases, such as phospholipase C can cut the bond between the protein and the GPI, freeing the protein from the outer cell membrane. Proteins destined to be glypiated have two signal sequences. They are 1) An N-terminal signal sequence and 2) A C-terminal signal sequence that is recognized by a GPI transamidase (GPIT). The N-terminal signal sequences is responsible for directing co-translational transport into the endoplasmic reticulum. The C-terminal sequence is recognized by GPI transamidase, which links the carboxy terminus of a protein to the GPI anchor. Liposomes The spontaneous ability of phosphoglycerolipid and sphingolipid compounds to form lipid bilayers is exploited in the formation of artificial membranous structures called liposomes (Figure 3.69). Liposomes are useful for delivering their contents into cells via membrane fusion. In the figure, items targeted for delivery to cells would be encased in the middle circular region of the liposome and when the liposome fuses with the cell membrane, it will deliver the contents directly into the cytoplasm. Hydropathy index
The interior portion of the lipid bilayer is very hydrophobic, which makes it very restrictive to movement of ions and polar substances across it. This property also places limitations on the types of amino acids that will interact with it as well. Because of this, transmembrane protein domains found in integral membrane proteins are biased in the amino acids that interact with either the lipid bilayer or the aqueous material on either side of it. Hydrophobic amino acids are found within the bilayer, whereas hydrophilic amino acids are found predominantly on the surfaces. An additional clue to identifying membrane crossing regions of a protein is that tryptophan or tyrosine is commonly positioned at non-polar/polar interface(s) of the lipid bilayer for integral proteins. Such an organization of amino acids can be recognized by computer analysis of amino acid sequences using what is called a hydropathy index/score (Figure 3.71). Though the names and the scorings vary, the idea is to assign a number (usually positive) to amino acid side chains with higher hydrophobicity and negative to those that are ionic. With these scores, a computer program can easily find the average scores of short amino acid segments (say 3 amino acids long) and then plot those values on a graph of hydrophobicity index versus position in polypeptide chain. Doing that for a transmembrane protein such as glycophorin results in the plot shown in Figure 3.72. It is apparent in the analysis that this is a transmembrane protein that has seven domains crossing the lipid bilayer, as labeled. Cell walls Cells walls are found in many cells, including plants, fungi, and bacteria, but are not found in animal cells. They are designed to provide strength and integrity and at least some protection against bursting from osmotic pressure (Figures 3.73-3.75). Gram positive bacteria (Figure 3.75) have the simplest cell wall design. Moving from outside the cell towards the cytoplasm there is an outer peptidoglycan layer for the cell wall followed by a periplasmic space, a plasma membrane, and then the cytoplasm. Gram negative bacteria add a second protective layer external to all of this, so for them, starting at the outside and moving inwards, one encounters an outer lipopolysaccharide layer, a periplasmic space, the peptidoglycan cell wall, a second periplasmic space, a plasma membrane and then the cytoplasm. Herbaceous plants have a rigid outer cell wall (primarily composed of cellulose, hemicellulose, and pectin) and an inner plasma membrane. Woody plants add a second level of wall with lignin between the cellulosic wall and the plasma membrane of herbaceous plants. BB Wonderland To the tune of “Winter Wonderland” Metabolic Melodies Website HERE Milam Hall - It’s 12:30 And Ahern’s gettin’ wordy He walks to and fro’ While not talkin’ slow Givin’ it to B-B-4-5-0 I was happy when the term got started Lecture notes and videos galore MP3s got added to my iPod But recitations sometimes were a bore And exams bit me roughly When the curve turned out ugly I don’t think it’s so My scores are too low Slidin’ by in B-B-4-5-0 Final-LY there’s an examination On December 9th at 6:00 pm I’ll have my card packed with information So I don’t have to memorize it then And I’ll feel like a smarty With my jam-packed note-cardy Just one more to go And then ho-ho-ho I’ll be done with B-B-4-5-0 Recording by David Simmons Lyrics by Kevin Ahern Recording by David Simmons Lyrics by Kevin Ahern 334 Thank God There's a Video To the tune of "Thank God I'm a Country Boy" Metabolic Melodies Website HERE There's a bundle of things a student oughta know And Ahern's talk isn't really very slow Learnin' ain't easy / the lectures kinda blow Thank God there's a video Well we’ve gone through the cycles and their enzymes too Studying the regulation everything is new I gotta admit that I haven’t got a clue What am I gonna do? So I got me a note card and bought me a Stryer Got the enzymes down and the names he requires I hope that I can muster up a little more desire Thank God there's a video Just got up to speed about the N-A-D Protons moving through Complex Vee Electrons dance in the cytochrome C Gotta hear the MP3 Fatty acid oxidation makes acetyl-CoA Inside the inner matrix of the mitochondri-ay It's very complicated, I guess I gotta say Thank God there's a video So I got me a note card and bought me a Stryer Got the enzymes down and the names he requires I hope that I can muster up a little more desire Thank God there's a video Replication's kind of easy in a simple kind of way Copyin' the bases in the plasmid DNAs Gs goes with Cs and Ts go with As Thanks to polymerase And the DNA's a template for the RNA Helices unwinding at T-A-T-A Termination happens, then the enzyme goes away Don't forget the poly-A So I got me a note card and bought me a Stryer Got the enzymes down and the names he requires I think that I can muster up a little more desire Thank God there's a video Recording by David Simmons Lyrics by Kevin Ahern Recording by David Simmons Lyrics by Kevin Ahern
Thumbnail: Enzyme changes shape by induced fit upon substrate binding to form enzyme-substrate complex. Hexokinase has a large induced fit motion that closes over the substrates adenosine triphosphate and xylose. Binding sites in blue, substrates in black and Mg2+ cofactor in yellow. (PDB: 2E2N, 2E2Q). Image used with permission (CC BY 4.0l Thomas Shafee) 04: Catalysis A printable version of this section is here: BiochemFFA_4_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy If there is a magical component to life, an argument can surely be made for it being catalysis. Thanks to catalysis, reactions that can take hundreds of years to complete in the uncatalyzed “real world,” occur in seconds in the presence of a catalyst. Chemical catalysts, such as platinum, can speed reactions, but enzymes (which are simply super-catalysts with a “twist,” as we shall see) put chemical catalysts to shame (Figure 4.1). To understand enzymatic catalysis, it is necessary first to understand energy. Chemical reactions follow the universal trend of moving towards lower energy, but they often have a barrier in place that must be overcome. The secret to catalytic action is reducing the magnitude of that barrier. Before discussing enzymes, it is appropriate to pause and discuss an important concept relating to chemical/biochemical reactions. That concept is equilibrium and it is very often misunderstood. The “equi" part of the word relates to equal, as one might expect, but it does not relate to absolute concentrations. What happens when a biochemical reaction is at equilibrium is that the concentrations of reactants and products do not change over time. This does not mean that the reactions have stopped. Remember that reactions are reversible, so there is a forward reaction and a reverse reaction: if you had 8 molecules of A, and 4 of B at the beginning, and 2 molecules of A were converted to B, while 2 molecules of B were simultaneously converted back to A, the number of molecules of A and B remain unchanged, i.e., the reaction is at equilibrium. However, you will notice that this does not mean that there are equal numbers of A and B molecules. Concentration Matters So, contrary to the perceptions of many students, the concentrations of products and reactants are not equal at equilibrium, unless the ΔG°’ for a reaction is zero, because when this is the case, $ΔG = \ln \left(\dfrac{[\rm{Products}]}{[\rm{Reactants}]} \right)$ since the ΔG°’ is zero. Because ΔG itself is zero at equilibrium, then $[Products] = [Reactants].$ This is the only circumstance where $[Products] = [Reactants]$ at equilibrium. Reiterating, at equilibrium, the concentrations of reactant and product do not change over time. That is, for a reaction $A \rightleftharpoons B [A]$ at time zero when equilibrium is reached, $[A]_{T_0}$, will be the same 5 minutes later (assuming A and B are chemically stable). Thus, $[A]_{T_0} = [A]_{T+5}$ Similarly, $[B]_{T_0} = [B]_{T+5}$ For that matter, at any amount of time X after equilibrium has been reached, $[A]T0 = [A]T+5 = [A]TX$ and $[B]T0 = [B] T+5 = [B]TX$ However, unless ΔG°’ = 0, it is wrong to say [A]T0 = [B]T0 As we study biochemical reactions and reaction rates, it is important to remember that 1) reactions do not generally start at equilibrium; 2) all reactions move in the direction of equilibrium; and 3) reactions in cells behave just like those in test tubes - they do not begin at equilibrium, but they move towards it. Dynamic reactions The reactions occurring in cells, though, are very dynamic and complex. In a test tube, they can be studied one at a time. In cells, the product of one reaction is often the substrate for another one. Reactions in cells are interconnected in this way, giving rise to what are called metabolic pathways. There are, in fact, thousands of different interconnected reactions going on continuously in cells. Attempts to study a single reaction in the chaos of a cell is daunting to say the least. For this reason, biochemists isolate enzymes from cells and study reactions individually. It is with this in mind that we begin our consideration of the phenomenon of catalysis by describing, first, the way in which enzymes work. Activation energy Figure 4.2 schematically depicts the energy changes that occur during the progression of a simple reaction. In order for the reaction to proceed, an activation energy must be overcome in order for the reaction to occur. In Figure 4.3, the activation energy for a catalyzed reaction is overlaid. As you can see, the reactants start at the same energy level for both catalyzed and uncatalyzed reactions and that the products end at the same energy for both as well. The catalyzed reaction, however, has a lower energy of activation (dotted line) than the uncatalyzed reaction. This is the secret to catalysis - overall ΔG for a reaction does NOT change with catalysis, but the activation energy is lowered. Figure 4.3 - Energy changes during the course of an uncatalyzed reaction (solid green line) and a catalyzed reaction (dotted green line). Image by Aleia Kim Reversibility The extent to which reactions will proceed forward is a function of the size of the energy difference between the product and reactant states. The lower the energy of the products compared to the reactants, the larger the percentage of molecules that will be present as products at equilibrium. It is worth noting that since an enzyme lowers the activation energy for a reaction that it can speed the reversal of a reaction just as it speeds a reaction in the forward direction. At equilibrium, of course, no change in concentration of reactants and products occurs. Thus, enzymes speed the time required to reach equilibrium, but do not affect the balance of products and reactants at equilibrium. Exceptions The reversibility of enzymatic reactions is an important consideration for equilibrium, the measurement of enzyme kinetics, for Gibbs free energy, for metabolic pathways, and for physiology. There are some minor exceptions to the reversibility of reactions, though. They are related to the disappearance of a substrate or product of a reaction. Consider the first reaction below which is catalyzed by the enzyme carbonic anhydrase: $CO_2 + H_2O \rightleftharpoons H_2CO_3 \rightleftharpoons HCO_3^- + H^+$
In the forward direction, carbonic acid is produced from water and carbon dioxide. It can either remain intact in the solution or ionize to produce bicarbonate ion and a proton. In the reverse direction, water and carbon dioxide are produced. Carbon dioxide, of course, is a gas and can leave the solution and escape. When reaction molecules are removed, as they would be if carbon dioxide escaped, the reaction is pulled in the direction of the molecule being lost and reversal cannot occur unless the missing molecule is replaced. In the second reaction occurring on the right, carbonic acid (H2CO3) is “removed” by ionization. This too would limit the reaction going back to carbon dioxide in water. This last type of “removal” is what occurs in metabolic pathways. In this case, the product of one reaction (carbonic acid) is the substrate for the next (formation of bicarbonate and a proton). In the metabolic pathway of glycolysis, ten reactions are connected in this manner and reversing the process is much more complicated than if just one reaction was being considered. General mechanisms of action As noted above, enzymatically catalyzed reactions are orders of magnitude faster than uncatalyzed and chemical-catalyzed reactions. The secret of their success lies in a fundamental difference in their mechanisms of action. Every chemistry student has been taught that a catalyst speeds a reaction without being consumed by it. In other words, the catalyst ends up after a reaction just the way it started so it can catalyze other reactions, as well. Enzymes share this property, but in the middle, during the catalytic action, an enzyme is transiently changed. In fact, it is the ability of an enzyme to change that leads to its incredible efficiency as a catalyst. Changes These changes may be subtle electronic ones, more significant covalent modifications, or structural changes arising from the flexibility inherent in enzymes, but not present in chemical catalysts. Flexibility allows movement and movement facilitates alteration of electronic environments necessary for catalysis. Enzymes are, thus, much more efficient than rigid chemical catalysts as a result of their abilities to facilitate the changes necessary to optimize the catalytic process. Substrate binding Another important difference between the mechanism of action of an enzyme and a chemical catalyst is that an enzyme has binding sites that not only ‘grab’ the substrate (molecule involved in the reaction being catalyzed), but also place it in a position to be electronically induced to react, either within itself or with another substrate. The enzyme itself may play a role in the electronic induction or the induction may occur as a result of substrates being placed in very close proximity to each other. Chemical catalysts have no such ability to bind substrates and are dependent upon them colliding in the right orientation at or near their surfaces. Active site Reactions in an enzyme are catalyzed at a specific location within it known as the ‘active site’. Substrates bind at the active site and are oriented to provide access for the relevant portion of the molecule to the electronic environment of the enzyme where catalysis occurs. Enzyme flexibility As mentioned earlier, a difference between an enzyme and a chemical catalyst is that an enzyme is flexible. Its slight changes in shape (often arising from the binding of the substrate itself) help to optimally position substrates for reaction after they bind. Figure 4.5 - Lysozyme with substrate binding site (blue), active site (red) and bound substrate (black). Wikipedia Induced fit These changes in shape are explained, in part, by Koshland’s Induced Fit Model of Catalysis (Figure 4.6), which illustrates that not only do enzymes change substrates, but that substrates also transiently change enzyme structure. At the end of the catalysis, the enzyme is returned to its original state. Koshland’s model is in contrast to the Fischer Lock and Key model, which says simply that an enzyme has a fixed shape that is perfectly matched for binding its substrate(s). Enzyme flexibility also is important for control of enzyme activity. Enzymes alternate between the T (tight) state, which is a lower activity state and the R (relaxed) state, which has greater activity. Induced Fit The Koshland Induced Fit model of catalysis postulates that enzymes are flexible and change shape on binding substrate. Changes in shape help to 1) aid binding of additional substrates in reactions involving more than one substrate and/or 2) facilitate formation of an electronic environment in the enzyme that favors catalysis. This model is in contrast to the Fischer Lock and Key Model of catalysis which considers enzymes as having pre-formed substrate binding sites. Ordered binding The Koshland model is consistent with multi-substrate binding enzymes that exhibit ordered binding of substrates. For these systems, binding of the first substrate induces structural changes in the enzyme necessary for binding the second substrate. There is considerable experimental evidence supporting the Koshland model. Hexokinase, for example, is one of many enzymes known to undergo significant structural alteration after binding of substrate. In this case, the two substrates are brought into very close proximity by the induced fit and catalysis is made possible as a result. Reaction types Enzymes that catalyze reactions involving more than one substrate, such as $A + B \rightleftharpoons C + D$ can act in two different ways. Enzymatic reactions can be of several types, as shown in Figure 4.7. In one mechanism, called sequential reactions, at some point in the reaction, both substrates will be bound to the enzyme. There are, in turn, two different ways in which this can occur - random and ordered. Figure 4.7 - Categories of enzymatic reactions Types of Reactions Single Substrate - Single Product A ⇄ B Single Substrate - Multiple Products A ⇄ B + C Multiple Substrates - Single Products A + B ⇄ C Multiple Substrates - Multiple Products A + B ⇄ C + D Consider lactate dehydrogenase, which catalyzes the reaction below: $NADH + Pyruvate → Lactate + NAD^+$
This enzyme requires that NADH must bind prior to the binding of pyruvate. As noted earlier, this is consistent with an induced fit model of catalysis. In this case, binding of the NADH changes the enzyme shape/environment so that pyruvate can bind and without binding of NADH, the substrate cannot access the pyruvate binding site. This type of multiple substrate reaction is called sequential ordered binding, because the binding of substrates must occur in the right order for the reaction to proceed. Random binding A second mechanism of binding/catalysis is exhibited by creatine kinase which catalyzes the following reaction: $Creatine + ATP → \text{Creatine phosphate} + ADP$ For this enzyme, substrates can bind to it in any order. Creatine kinase displays sequential random binding. It is worth mentioning that random binding is not inconsistent with Koshland’s induced fit model. Rather, random binding simply means that the enzyme’s induced fit doesn’t affect substrate binding sites and involves other parts of the enzyme. In summary, sequential binding can occur as ordered binding or as random binding. Double displacement reactions Not all enzymes that catalyze multi-substrate reactions, though, bind A and B by the sequential mechanisms above. This other category of enzyme includes those that exhibit what are called “ping-pong” (or double displacement) mechanisms. In these enzymes, the enzyme functions as both a catalyst and a carrier of a group between individually bound substrates. Examples of this type of enzyme include the class of enzymes known as transaminases. A general form of the reactions catalyzed by these enzymes is shown in Figure 4.8. In reversible transaminase reactions, an oxygen and an amine are swapping between the molecules. It can be represented as follows (where N is the amine and O is the oxygen). A=O + C=N ⇄ B-N + D=O This reaction occurs not as one transfer reaction swapping the N and the O, but rather as a set of two half-reactions. In this case, the enzyme is a both donor and a carrier of the group being swapped. The first half-reaction goes as follows A=O + Enz-N ⇄ B-N + Enz=O Next a second half-reaction goes as C-N + Enz=O ⇄ D=O + Enz=N The sum of these half-reactions then is A=O + C=N ⇄ B-N + D=O Note that at no time did the enzyme bind both A and C simultaneously. It is also important to recognize that the enzyme existed in two states - Enz=O and Enz-N. The shuffling of the enzyme between these two states is what gives rise to the ping-pong name of this mechanism - it literally goes back and forth like a ping-pong ball in a table tennis match. Enzyme kinetics To understand how an enzyme enhances the rate of a reaction, we must understand enzyme kinetics. We present a model here proposed by Leonor Michaelis and Maud Menten. In order to understand the model, it is necessary to understand a few parameters. First, we describe a reaction in simple terms proceeding as follows E + S ⇄ ES -> E + P where E is enzyme, S is substrate, and P is product. In this scheme, ES is the Enzyme-Substrate complex, which is simply the enzyme bound to its substrate. We could define the ES state a bit further with E + S ⇄ ES -> ES* -> EP -> E + P where ES* is the activated state and EP is the enzyme-product complex before release of the product. The first consideration we have is velocity. The velocity of a reaction is the rate of creation of product over time, measured as the concentration of product per time. The time is a critical consideration when measuring velocity. In a closed system (in which an enzyme operates), all reactions will advance towards equilibrium. Enzymatically catalyzed reactions are no different in the end result from non-enzymatic reactions, except that they get to equilibrium faster. Equilibrium At equilibrium, the ratio of product to reactant does not change. That is a property of equilibrium. Since the system is closed, the concentration of product over time will not change. The velocity will thus be zero under these conditions and we will have learned nothing about the reaction if we wait too long to study it. Velocity Consequently, in Michaelis-Menten kinetics, velocity is measured as initial velocity (V0). This is accomplished by measuring the rate of formation of product early in the reaction before equilibrium is established and under these conditions, there is very little if any of the reverse reaction occurring. The other two assumptions are related. First, we use conditions where there is much more substrate than enzyme. This makes sense. If the substrate is not in great excess, then the enzyme’s conversion of substrate to product will occur much faster than the enzyme can bind substrate. Waiting for substrate Thus, the enzyme would “wait” for substrate to bind if there were not sufficient amounts of it to bind to the enzyme in a timely fashion (when substrate concentration is low). This would not give an accurate measure of velocity, since the enzyme would be inactive a good deal of the time. Because of this, we assume saturation of the enzyme with substrate will give a maximal velocity of the reaction. Steady state Figure 4.17 - Steady state versus non-steady state conditions Last, the high concentration of substrate combined with measuring initial conditions results in studying reactions that are under so-called steady state conditions (Figure 4.17). When steady state occurs, the concentration of the ES complex over time is not significantly changing during the period of analysis. Reiterating, the three assumptions for Michaelis-Menten kinetics are 1. Measurement of initial velocity of a reaction 2. Substrate in great excess compared to enzyme 3. Reaction conditions occurring under steady state Experimental considerations Now we turn our attention to how studies of the kinetic properties of an enzyme are conducted. To perform an analysis, one would do the following experiment - 20 different tubes would be set up with enzyme buffer (to keep the enzyme stable), the same amount of enzyme, and then a different amount of substrate in each tube, ranging from tiny amounts in the first tubes to very large amounts in the last tubes. The reaction would be allowed to proceed for a fixed, short amount of time and then the reaction would be stopped and the amount of product contained in each tube would be determined.
The initial velocity (V0) of the reaction then would be the concentration of product found in each tube divided by the time that the reaction was allowed to run. Data from the experiment would be plotted on a graph using initial velocity (V0) on the Y-axis and the concentration of substrate on the X-axis, each tube, of course having a unique reaction velocity corresponding to a unique substrate concentration. For an enzyme following Michaelis-Menten kinetics, a curve like that shown in Figure 4.18 or 4.19 would result. At low concentration of substrate, it is limiting and the enzyme converts it into product as soon as it can bind it. Consequently, at low concentrations of substrate, the rate of increase of [P] is almost linear with [S] (Figure 4.19). Figure 4.19 - Linear relationship between [P] and [S] at low [S] Non-linear increase As the substrate concentration increases, however, the velocity of the reaction in tubes with higher substrate concentration ceases to increase linearly and instead begins to flatten out, indicating that as the substrate concentration gets higher and higher, the enzyme has a harder time keeping up to convert the substrate to product. Saturation Not surprisingly, when the enzyme becomes completely saturated with substrate, it will not have to wait for substrate to diffuse to it and will therefore be operating at maximum velocity. For an enzyme following Michaelis-Menten kinetics will have its velocity (v) at any given substrate concentration given by the following equation: Vmax Two terms in the equation above require explanation. The first is Vmax. It refers to the maximum velocity of an enzymatic reaction. Maximum velocity for a reaction occurs when an enzyme is saturated with substrate. Saturation is important because it means (per the assumption above) that none of the enzyme molecules are “waiting” for substrate after a product is released. Saturation ensures that another substrate is always instantly available. The unit of Vmax is concentration of product per time = [P]/time. On a plot of initial velocity versus substrate concentration (V0 vs. [S]), Vmax is the value on the Y axis that the curve asymptotically approaches (dotted line in Figure 4.20). It should be noted that the value of Vmax depends on the amount of enzyme used in a reaction. If you double the amount of enzyme used, you will double the Vmax. If one wanted to compare the velocities of two different enzymes, it would be necessary to use the same amounts of enzyme in the reaction each one catalyzes. Km The second term is Km (also known as Ks). Referred to as the Michaelis constant, Km is the substrate concentration that causes the enzyme to work at half of maximum velocity (Vmax/2). What it measures, in simple terms, is the affinity an enzyme has for its substrate. The value of Km is inversely related to the affinity of the enzyme for its substrate. Enzymes with a high Km value will have a lower affinity for their substrate (will take more substrate to get to Vmax/2) whereas those with a low Km will have high affinity and take less substrate to get to Vmax/2. The unit of Km is concentration. Affinities of enzymes for substrates vary considerably, so knowing Km helps us to understand how well an enzyme is suited to the substrate being used. Measurement of Km depends on the measurement of Vmax. Common mistake A common mistake students make in describing Vmax is saying that Km = Vmax /2. This is, of course, not true. Km is a substrate concentration and is the amount of substrate it takes for an enzyme to reach Vmax /2. On the other hand Vmax /2 is a velocity and a velocity certainly cannot equal a concentration. Kcat It is desirable to have a measure of velocity that is independent of enzyme concentration. Remember that Vmax depended on the amount of enzyme used. For this, we use the Kcat, also known as the turnover number. Kcat is a number that requires one to first determine Vmax for an enzyme and then divide the Vmax by the concentration of enzyme used to determine Vmax. Thus, Kcat = Vmax /[Enzyme] Since Vmax has units of concentration per time and [Enzyme] has units of concentration, the units on Kcat are time-1. While that might seem unintuitive, it means that the value of Kcat is the number of molecules of product made by each molecule of enzyme in the time given. So, a Kcat value of 1000/sec means each enzyme molecule in the reaction at Vmax is producing 1000 molecules of product per second. Note that since Kcat is a calculated value, it cannot be read from a V vs [S] graph as Vmax and Km can. Amazing Kcat values A Kcat value of 1000 molecules of product per enzyme per second might seem like a high value, but there are enzymes known (carbonic anhydrase, for example) that have a Kcat value of over 600,000/second (Figure 4.21). This astonishing value illustrates clearly why enzymes seem almost magical in their action. In contrast to $V_{max}$, which varies with the amount of enzyme used, Kcat is a constant for an enzyme under given conditions. As seen earlier, enzymes that follow Michaelis-Menten kinetics produce hyperbolic plots of Velocity (V0) versus Substrate Concentration [S] (Figure 4.18). Not all enzymes, though, follow Michaelis-Menten kinetics. Many enzymes have multiple protein subunits and these sometimes interact differently upon binding of a substrate or an external molecule. See ATCase (HERE) for an example. Perfect enzymes Now, if we think about what an ideal enzyme might be, it would be one that has a very high velocity and a very high affinity for its substrate. That is, it wouldn’t take much substrate to get to Vmax/2 and the Kcat would be very high. Such enzymes would have values of Kcat / Km that are maximum. Interestingly, there are several enzymes that have this property and their maximal Kcat / Km values are all approximately the same. Such enzymes are referred to as being “perfect” because they have reached the maximum possible value. Figure 4.22 - Kcat/Km values for perfect enzymes. Image by Aleia Kim Diffusion limitation
Why should there be a maximum possible value of Kcat / Km? The answer is that movement of substrate to the enzyme becomes the limiting factor for perfect enzymes. Movement of substrate by diffusion in water has a fixed rate at any temperature and that limitation ultimately determines the maximum speed an enzyme can catalyze at. In a macroscopic world analogy, factories can’t make products faster than suppliers can deliver materials. It is safe to say for a perfect enzyme that the only speed limit it has is the rate of substrate diffusion in water. Given the “magic” of enzymes alluded to earlier, it might seem that all enzymes should have evolved to be “perfect.” There are very good reasons why most of them have not. Speed Speed is a dangerous thing. The faster a reaction proceeds in catalysis by an enzyme, the harder it is to control. As we all know from learning to drive, speeding causes accidents. Just as drivers need to have speed limits for operating automobiles, so too must cells exert some control on the ‘throttle’ of their enzymes. In view of this, one might wonder then why any cells have evolved any enzymes to perfection. There is no single answer to the question, but a common one is illustrated by triose phosphate isomerase, which catalyzes a reaction in glycolysis shown in Figure 4.24. The enzyme appears to have evolved this ability because at lower velocities, there is breakdown of an unstable enediol intermediate that then readily forms methyl glyoxal, a cytotoxic compound (Figure 4.25). Speeding up the reaction provides less opportunity for the unstable intermediate to accumulate and fewer undesirable byproducts to be made. Dissociation constant In studying proteins and ligands, it is important to understand the “tightness” with which a protein (P) “holds onto” a ligand (L). This is measured with the dissociation constant ($K_d$). The formation of a ligand-protein complex $LP$ occurs as $L + P \rightleftharpoons LP$ The dissociation of the complex, therefore, is the reverse of this reaction, or $LP \rightleftharpoons L + P$ so the corresponding dissociation constant is defined as $K_d = \dfrac{[L][P]}{[LP]}$ where $[L]$, $[P]$, and $[LP]$ are the molar concentrations of the protein, ligand and the complex when they are joined together. Smaller values of $K_d$ indicate tight binding, whereas larger values indicate loose binding. The dissociation constant is the inverse of the association constant. $K_a = \dfrac{1}{K_d}$ Where multiple molecules bond together, such as $J_xK_y \rightleftharpoons xJ + yK$ The complex $J_xK_y$ is breaking down into $x$ subunits of $J$ and $y$ subunits of $K$. The dissociation constant is then defined as $K_d = \dfrac{[J]^x[K]^y}{[J_xK_y]}$ where $[J]$, $[K]$, and $[J_xK_y]$ are the concentrations of J, K, and the complex $J_xK_y$, respectively. Lineweaver-Burk plots The study of enzyme kinetics is typically the most math intensive component of biochemistry and one of the most daunting aspects of the subject for many students. Although attempts are made to simplify the mathematical considerations, sometimes they only serve to confuse or frustrate students. Such is the case with modified enzyme plots, such as Lineweaver-Burk (Figure 4.26). Indeed, when presented by professors as simply another thing to memorize, who can blame students? In reality, both of these plots are aimed at simplifying the determination of parameters, such as $K_m$ and $V_{max}$. In making either of these modified plots, it is important to recognize that the same data is used as in making a V0 vs. [S] plot. The data are simply manipulated to make the plotting easier. Figure 4.26 - A Lineweaver-Burk plot of $1/V_0$ vs $1/[S]$. Image by Aleia Kim Double reciprocal For a LineWeaver-Burk plot, the manipulation is using the reciprocal of the values of both the velocity and the substrate concentration. The inverted values are then plotted on a graph as 1/V0 vs. 1/[S]. Because of these inversions, Lineweaver-Burk plots are commonly referred to as ‘double-reciprocal’ plots. As can be seen in Figure 4.26, the value of Km on a Lineweaver Burk plot is easily determined as the negative reciprocal of the x-intercept , whereas the Vmax is the inverse of the y-intercept. Other related manipulation of kinetic data include Eadie-Hofstee diagrams, which plots V0 vs V0/[S] and gives Vmax as the Y-axis intercept with the slope of the line being -Km. Molecularity of reactions The term molecularity refers to the number of molecules that must come together in order for a reaction to take place. Reactions of the sort of A -> B (where ‘A’ is the reactant and ‘B’ is the product) are unimolecular, since A is directly changed into B. The rate of the reaction is related only to the concentration of reactant A. For a bimolecular reaction where A + B ⇄ C the reaction depends on the concentration of both A and B and its rate will be related to the product of the concentration of A and of B. Coenzymes Organic molecules that assist enzymes and facilitate catalysis are co-factors called coenzymes. The term co-factor is a broad category usually subdivided into inorganic ions and coenzymes. If the coenzyme is very tightly or covalently bound to the enzyme, it is referred to as a prosthetic group. Enzymes without their co-factors are inactive and referred to as apoenzymes. Enzymes containing all of their co-factors are called holoenzymes. Pre-steady state kinetic studies In the study of kinetic rates of enzymatic reactions, time zero is a very critical point. It establishes when the mixing of substrate with enzyme and measurement of formation of product begins. At time zero, there is no product. As shown in Figure 4.29, the appearance of product (on a short time scale) goes through an early burst phase with a steep slope for [product]/time and then changes. Figure 4.29 - Burst phase of product formation This change occurs during a critical period in an enzymatic reaction and gives information about the rate of reaction cycles. The duration of the burst phase tells how long a single reaction turnover occurs, whereas the slow of the line post-burst phase tells the amount of “functional” enzyme performing the reaction.
After the burst phase, the slope of the line of the amount of product versus time decreases. This is due to the reaction entering conditions of steady state, used to study Michaelis-Menten kinetics. In steady state conditions, the amount of the enzyme-substrate complex (ES) is relatively constant over time. In simple terms, this occurs when the rate of formation of the ES complex equals the rate of conversion of the substrate to product by the enzyme with release of the product. Earlier events Events occurring prior to the conditions of steady state are referred to as pre-steady state. Depending on the enzyme, in as short as a few milliseconds, steady state conditions can be present meaning that if one hopes to study formation of reaction intermediates in pre-steady state, tools for this analysis must work very rapidly. One instrument commonly used for studying pre-steady state kinetics is called a stopped flow instrument. It loads an enzyme solution and a substrate into separate syringes whose output is pointed into a mixing chamber. The solutions are rapidly mixed and measurements of product concentration begin. With a stopped flow instrument, dead times (time between mixing and detection) can be achieved of as small as 0.3 msec. Ribozymes Proteins do not have a monopoly on acting as biological catalysts. Some RNA molecules are also capable of speeding reactions. The most famous of these molecules was discovered by Tom Cech in the early 1980s Studying excision of an intron in Tetrahymena, Cech was puzzled at his inability to find any proteins catalyzing the process. Ultimately, the catalysis was recognized as coming from the intron itself. It was a self-splicing RNA and since then, many other examples of catalytic RNAs have been found. Catalytic RNA molecules are known as ribozymes. Not unusual Ribozymes, however, are not rarities of nature. The protein-making ribosomes of cells are essentially giant ribozymes. The 23S rRNA of the prokaryotic ribosome and the 28S rRNA of the eukaryotic ribosome catalyze the formation of peptide bonds. Ribozymes are also important in our understanding of the evolution of life on Earth. They have been shown to be capable via selection to evolve self-replication. Indeed, ribozymes actually answer a chicken/egg dilemma - which came first, enzymes that do the work of the cell or nucleic acids that carry the information required to produce the enzymes. As both carriers of genetic information and catalysts, ribozymes are likely both the chicken and the egg in the origin of life.
A printable version of this section is here: BiochemFFA_4_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Regulation of enzyme activity Apart from their ability to greatly speed the rates of chemical reactions in cells, enzymes have another property that makes them valuable. This property is that their activity can be regulated, allowing them to be activated and inactivated, as necessary. This is tremendously important in maintaining homeostasis, permitting cells to respond in controlled ways to changes in both internal and external conditions. Inhibition of specific enzymes by drugs can also be medically useful. Understanding the mechanisms that control enzyme activity is, therefore, of considerable importance. Inhibition We will first discuss four types of enzyme inhibition – competitive, non-competitive, uncompetitive, and suicide inhibition. Of these, the first three types are reversible. The last one, suicide inhibition, is not. Competitive inhibition Probably the easiest type of enzyme inhibition to understand is competitive inhibition and it is the one most commonly exploited pharmaceutically. Molecules that are competitive inhibitors of enzymes resemble one of the normal substrates of an enzyme. An example is methotrexate, which resembles the folate substrate of the enzyme dihydrofolate reductase (DHFR). This enzyme normally catalyzes the reduction of folate, an important reaction in the metabolism of nucleotides. Figure 4.33 - Competitive inhibitors resemble the normal substrate and compete for binding at the active site. Image by Aleia Kim Inhibitor binding When the drug methotrexate is present, some of the DHFR enzyme binds to it, instead of to folate, and during the time methotrexate is bound, the enzyme is inactive and unable to bind folate. Thus, the enzyme is inhibited. Notably, the binding site on DHFR for methotrexate is the active site, the same place that folate would normally bind. As a result, methotrexate ‘competes’ with folate for binding to the enzyme. The more methotrexate there is, the more effectively it competes with folate for the enzyme’s active site. Conversely, the more folate there is, the less of an effect methotrexate has on the enzyme because folate outcompetes it. No effect on Vmax How do we study competitive inhibition? It is typically done as follows. First, one performs a set of V0 vs. [S] reactions without inhibitor (20 or so tubes, with buffer and constant amounts of enzyme, varying amounts of substrate, equal reaction times). V0 vs. [S] is plotted (Figure 4.35 red line), as well as 1/V0 vs. 1/[S] (Figure 4.36 green line). Next, a second set of reactions is performed in the same manner as before, except that a fixed amount of the methotrexate inhibitor is added to each tube. At low concentrations of substrate, the methotrexate competes for the enzyme effectively, but at high concentrations of substrate, the inhibitor will have a much reduced effect, since the substrate outcompetes it, due to its higher concentration (remember that the inhibitor is at fixed concentration). Graphically, the results of these inhibitor experiments are shown in Figure 4.35 (blue line) and Figure 4.36 (orange line). Notice that at high substrate concentrations, the competitive inhibitor has essentially no effect, causing the $V_{max}$ for the enzyme to remain unchanged. To reiterate, this is due to the fact that at high substrate concentrations, the inhibitor doesn’t compete well. However, at lower substrate concentrations, it does. Increased $K_m$ In competitively inhibited reactions, the apparent Km of the enzyme for the substrate increases ($-1/K_m$ gets closer to zero - red line in Figure 4.36) when the inhibitor is present compared to when the inhibitor is absent, thus illustrating the better competition of the inhibitor at lower substrate concentrations. It may not be obvious why we call the changed Km the apparent Km of the enzyme. The reason is that the inhibitor doesn’t actually change the enzyme’s affinity for the folate substrate. It only appears to do so. This is because of the way that competitive inhibition works. When the competitive inhibitor binds the enzyme, it is effectively ‘taken out of action.’ Inactive enzymes have NO affinity for substrate and no activity either. We can’t measure Km for an inactive enzyme. The enzyme molecules that are not bound by methotrexate can, in fact, bind folate and are active. Methotrexate has no effect on them and their Km values are unchanged. Why then, does Km appear higher in the presence of a competitive inhibitor? The reason is that the competitive inhibitor is having a greater effect of reducing the amount of active enzyme at lower concentrations of substrate than it does at higher concentrations of substrate. When the amount of enzyme is reduced, one must have more substrate to supply the reduced amount of enzyme sufficiently to get to Vmax/2. It is worth noting that in competitive inhibition, the percentage of inactive enzyme changes drastically over the range of [S] values used. To start, at low [S] values, the greatest percentage of the enzyme is inhibited. At high [S], no significant percentage of enzyme is inhibited. This is not always the case, as we shall see in non-competitive inhibition. Non-competitive inhibition A second type of inhibition employs inhibitors that do not resemble the substrate and bind not to the active site, but rather to a separate site on the enzyme (Figure 4.37). The effect of binding a non-competitive inhibitor is significantly different from binding a competitive inhibitor because there is no competition. In the case of competitive inhibition, the effect of the inhibitor could be reduced and eventually overwhelmed with increasing amounts of substrate. This was because increasing substrate made increasing percentages of the enzyme active. With non-competitive inhibition, increasing the amount of substrate has no effect on the percentage of enzyme that is active. Indeed, in non-competitive inhibition, the percentage of enzyme inhibited remains the same through all ranges of [S].
This means, then, that non-competitive inhibition effectively reduces the amount of enzyme by the same fixed amount in a typical experiment at every substrate concentration used The effect of this inhibition is shown in Figure 4.38 & 4.39. As you can see, $V_{max}$ is reduced in non-competitive inhibition compared to uninhibited reactions. This makes sense if we remember that Vmax is dependent on the amount of enzyme present. Reducing the amount of enzyme present reduces $V_{max}$. In competitive inhibition, this doesn’t occur detectably, because at high substrate concentrations, there is essentially 100% of the enzyme active and the $V_{max}$ appears not to change. Additionally, Km for non-competitively inhibited reactions does not change from that of uninhibited reactions. This is because, as noted previously, one can only measure the $K_m$ of active enzymes and $K_m$ is a constant for a given enzyme. Uncompetitive inhibition A third type of enzymatic inhibition is that of uncompetitive inhibition, which has the odd property of a reduced Vmax as well as a reduced Km. The explanation for these seemingly odd results is rooted in the fact that the uncompetitive inhibitor binds only to the enzyme-substrate (ES) complex (Figure 4.40). The inhibitor-bound complex forms mostly under concentrations of high substrate and the ES-I complex cannot release product while the inhibitor is bound, thus explaining the reduced $V_{max}$. The reduced Km is a bit harder to conceptualize. The reason is that the inhibitor-bound complex effectively reduces the concentration of the ES complex. By Le Chatelier’s Principle, a shift occurs to form additional ES complex, resulting in less free enzyme and more enzyme in the forms ES and ESI (ES with inhibitor). Decreases in free enzyme correspond to an enzyme with greater affinity for its substrate. Thus, paradoxically, uncompetitive inhibition both decreases $V_{max}$ and increases an enzyme’s affinity for its substrate ($K_m$ - Figures 4.41 & 4.42). Suicide inhibition In contrast to the first three types of inhibition, which involve reversible binding of the inhibitor to the enzyme, suicide inhibition is irreversible, because the inhibitor becomes covalently bound to the enzyme during the inhibition. Suicide inhibition rather closely resembles competitive inhibition because the inhibitor generally resembles the substrate and binds to the active site of the enzyme. The primary difference is that the suicide inhibitor is chemically reactive in the active site and makes a bond with it that precludes its removal. Such a mechanism is that employed by penicillin (Figure 4.43), which covalently links to the bacterial enzyme, DD transpeptidase and stops it from functioning. Since the normal function of the enzyme is to make a bond necessary for the peptidoglycan complex of the bacterial cell wall, the cell wall cannot properly form and bacteria cannot reproduce. Control of enzymes It is appropriate to talk at this point about mechanisms cells use to control enzymes. There are four general methods that are employed: 1. allosterism, 2. covalent modification, 3. access to substrate, and 4. control of enzyme synthesis/breakdown. Some enzymes are controlled by more than one of these methods. Allosterism The term allosterism refers to the fact that the activity of certain enzymes can be affected by the binding of small molecules. Molecules causing allosteric effects come in two classifications. Ones that are substrates for the enzymes they affect are called homotropic effectors and those that are not substrates are called heterotropic effectors. The homotropic effectors usually are activators of the enzymes they bind to and the results of their action can be seen in the conversion of the hyperbolic curve typical of a V0 vs. [S] plot for an enzyme (Figure 4.18), being converted to a sigmoidal plot (Figure 4.44). This is due to the conversion of the enzyme from the T-state to the R-state on binding the substrate/homotropic effector. The V0 vs. [S] plot of allosteric enzyme reactions resembles the oxygen binding curve of hemoglobin (see Figure 2.83). Even though hemoglobin is not an enzyme and is thus not catalyzing a reaction, the similarity of the plots is not coincidental. In both cases, the binding of an external molecule is being measured – directly, in the hemoglobin plot, and indirectly by the V0 vs. [S] plot, since substrate binding is a factor in enzyme reaction velocity. Allosteric inhibition Allosterically, regulation of these enzymes works by inducing different physical states (shapes, as it were) that affect their ability to bind to substrate. When an enzyme is inhibited by binding an effector, it is converted to the T-state (T=tight), it has a reduced affinity for substrate and it is through this means that the reaction is slowed. Allosteric activation On the other hand, when an enzyme is activated by effector binding, it converts to the R-state (R=relaxed) and binds substrate much more readily. When no effector is present, the enzyme may be in a mixture of T- and R-states. Feedback inhibition An interesting kind of allosteric control is exhibited by HMG-CoA reductase, which catalyzes an important reaction in the pathway leading to the synthesis of cholesterol. Binding of cholesterol to the enzyme reduces the enzyme’s activity significantly. Cholesterol is not a substrate for the enzyme, so it is therefore a heterotropic effector. Notably, though, cholesterol is the end-product of the pathway that HMG-CoA reductase catalyzes a reaction in. When enzymes are inhibited by an end-product of the pathway in which they participate, they are said to exhibit feedback inhibition. Feedback inhibition always operates by allosterism and further, provides important and efficient control of an entire pathway. By inhibiting an early enzyme in a pathway, the flow of materials (and ATP hydrolysis required for their processing) for the entire pathway is stopped or reduced, assuming there are not alternate supply methods. Pathway control
In the cholesterol biosynthesis pathway, stopping this one enzyme has the effect of shutting off (or at least slowing down) the entire pathway. This is significant because after catalysis by HMG-CoA reductase, there are over 20 further reactions necessary to make cholesterol, many of them requiring ATP energy. Shutting down one reactions stops all of them. Another excellent example of allosteric control and feedback inhibition is the enzyme ATCase, discussed below. ATCase Another interesting example of allosteric control and feedback inhibition is associated with the enzyme Aspartate Transcarbamoylase (ATCase). This enzyme, which catalyzes a step in the synthesis of pyrimidine nucleotides, has 12 subunits. These include six identical catalytic subunits and six identical regulatory subunits. The catalytic subunits bind to substrate and catalyze a reaction. The regulatory subunits bind to either ATP or CTP. If they bind to ATP, the enzyme subunits arrange themselves in the R-state. R-state The R-state of ATCase allows the substrate to have easier access to the six active sites and the reaction occurs more rapidly. For the same amount of substrate, an enzyme in the R-state will have a higher velocity than the same enzyme that is not in the R-state. By contrast, if the enzyme binds to CTP on one of its regulatory subunits, the subunits will arrange in the T-state and in this form, the substrate will not have easy access to the active sites, resulting in a slower velocity for the same concentration of substrate compared to the R-state. ATCase is interesting in that it can also flip into the R-state when one of the substrates (aspartate) binds to an active site within one of the catalytic subunits. Aspartate has the effect of activating the catalytic action of the enzyme by favoring the R-state. Thus, aspartate, which is a substrate of the enzyme is a homotropic effector and ATP and CTP, which are not substrates of the enzyme are heterotropic effectors of ATCase. Allosteric models There are three models commonly used to explain how allosterism regulates multi-subunit enzyme activity. They are known as • the Monod-Wyman-Changeux (MWC) model (also known as the concerted model), • the sequential model (also known as KNF), • and the morpheein model. All models describe a Tense (T) state that is less catalytically active and a Relaxed (R) state that is more catalytically active. The models differ in how the states change. Sequential model In the sequential model, binding of an allosteric effector by one subunit causes it to change from T to R state (or vice versa) and that change makes it easier for adjacent subunits to similarly change state. With this model , there is a cause/effect relationship between binding of an effector by one subunit and change of state by an adjacent subunit. In hemoglobin, for example, binding of one oxygen by one unit of the complex may induce that unit to flip to the R-state and, through interactions with other subunits, cause them to favor adopting the R configuration before they bind to oxygen. In this way, binding of one subunit favors binding of others and cooperativity can be explained by the change in binding affinity as oxygen concentration changes. MWC model The MWC model is less intuitive. In it, the entire complex changes state from T to R (or vice versa) independently of the binding of effectors. Flipping between T-states and R-states is postulated to be in an equilibrium of states in the absence of effector (for example, a 50 to 1 ratio of T/R. This ratio is referred to as L, so L = T/R). Binding of effector by the enzyme complex has the tendency of “locking” the complex in a state. Binding of inhibitors will increase the ratio of T/R whereas binding of activators will increase R and thus decrease the ration of T/R. Morpheein model The morpheein model is similar to the MWC model, but with an added step of dissociation of the subunits. The MWC model proposes that flipping between R and T states occurs by the complex as a whole and occurs on all units simultaneously. The morpheein model instead proposes that the multi-subunit enzyme breaks down to individual units which can then flip in structure and re-form the complex. In the morpheein model, only identically shaped units (all R, for example) can come together in the complex, thus explaining the “all-R-” or “all-T-” state found in the MWC model. A large number of enzymes, including prominent ones like citrate synthase, acetyl-CoA carboxylase, glutamate dehydrogenase, ribonucleotide reductase and lactate dehydrogenase have behavior consistent with the morpheein model. Covalent control of enzymes Some enzymes are synthesized in a completely inactive form and their activation requires covalent bonds in them to be cleaved. Such inactive forms of enzymes are called zymogens. Examples include the proteins involved in blood clotting and proteolytic enzymes of the digestive system, such as trypsin, chymotrypsin, pepsin, and others. Synthesizing some enzymes in an inactive form makes very good sense when an enzyme’s activity might be harmful to the tissue where it is being made. For example, the painful condition known as pancreatitis arises when digestive enzymes made in the pancreas are activated too soon and end up attacking the pancreas. Cascades For both the blood clotting enzymes and the digestive enzymes, the zymogens are activated in a protease cascade. This occurs when activation of one enzyme activates others in a sort of chain reaction. In such a scheme the first enzyme activated proteolytically cleaves the second zymogen, causing it to be activated, which in turn activates a third and this may proceed through several levels of enzymatic action (Figure 4.50). The advantage of cascades is that they allow a large amount of zymogens to become activated fairly quickly, since there is an amplification of the signal at each level of catalysis.
Zymogens are also abundant in blood. Blood clotting involves polymerization of a protein known as fibrin. Since random formation of fibrin is extremely hazardous because it can block the flow of blood, potentially causing heart attack/stroke, the body synthesizes fibrin as a zymogen (fibrinogen) and its activation results from a “cascade” of activations of proteases that arise when a signal is received from a wound. Similarly, the enzyme catalyzing removal of fibrin clots (plasmin) is also synthesized as a zymogen (plasminogen), since random clot removal would also be hazardous (see below also). Phosphorylation/dephosphorylation Another common mechanism for control of enzyme activity by covalent modification is phosphorylation. The phosphorylation of enzymes (on the side chains of serine, threonine or tyrosine residues) is carried out by protein kinases. Enzymes activated by phosphorylation can be regulated by the addition of phosphate groups by kinases or their removal by phosphatases. Thus, this type of covalent modification is readily reversible, in contrast to proteolytic cleavage. Reduction/oxidation An interesting covalent control of enzymes using reduction/oxidation is exhibited in photosynthetic plants. In the light phase of photosynthesis, electrons are excited by light and flow through carriers to NADP+, forming NADPH. Thus, in the light, the NADPH concentration is high. When NADPH concentration is high, the concentration of reduced ferredoxin (a molecule donating electrons to NADP+) is also high. Reduced ferredoxin can transfer electrons to thioredoxin, reducing it. Reduced thioredoxin can, in turn, transfer electrons to proteins to reduce their disulfide bonds. Four enzymes related to the Calvin cycle can receive electrons from thioredoxin and become activated, as a result. These include sedoheptulose 1,7-bisphosphatase, ribulose-5-phosphate kinase, fructose 1,6-bisphosphatase, and glyceraldehyde 3-phosphate dehydrogenase. Thus, in the light, electrons flow, causing NADPH to accumulate and ferredoxin to push electrons in the direction of these enzymes above, activating them and favoring the Calvin cycle. In the dark, the concentration of reduced NADPH, reduced ferredoxin, and reduced thioredoxin fall, resulting in loss of electrons by the Calvin cycle enzymes (oxidations that re-form disulfide bonds) and the Calvin cycle inactivates. Other enzyme control mechanisms Other means of controlling enzymes relate to access to substrate (substrate-level control) and control of enzyme synthesis. Hexokinase is an enzyme that is largely regulated by availability of its substrate, glucose. When glucose concentration is low, the product of the enzyme’s catalysis, glucose-6-phosphate, inhibits the enzyme’s function. Regulation of enzymes by controlling their synthesis is covered later in the book in the discussion relating to control of gene expression.
A printable version of this section is here: BiochemFFA_4_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy The magic of enzymes, as noted, is in their ability to create electronic environments conducive to initiation of a reaction. There are more mechanisms of reaction than we could ever hope to cover in a book like this, and comprehensive discussion of these is not our aim. Instead, we will cite some examples and go into detail on one of them - the mechanism of action of serine proteases. Chymotrypsin We will begin with mechanism of action of one enzyme - chymotrypsin. Found in our digestive system, chymotrypsin’s catalytic activity is cleaving peptide bonds in proteins and it uses the side chain of a serine in its mechanism of catalysis. Many other protein-cutting enzymes employ a very similar mechanism and they are known collectively as serine proteases (Figure 4.52). These enzymes are found in prokaryotic and eukaryotic cells and all use a common set of three amino acids in the active site called a catalytic triad (Figure 4.53). It consists of aspartic acid, histidine, and serine. The serine is activated in the reaction mechanism to form a nucleophile in these enzymes and gives the class their name. With the exception of the recognition that occurs at the substrate binding site, the mechanism shown here for chymotrypsin would be applicable to any of the serine proteases. Specificity As a protease, chymotrypsin acts fairly specifically, cutting not all peptide bonds, but only those that are adjacent to relatively non-polar amino acids in the protein. One of the amino acids it cuts adjacent to is phenylalanine. The enzyme’s action occurs in two phases – a fast phase that occurs first and a slower phase that follows. The enzyme has a substrate binding site that includes a region of the enzyme known as the S1 pocket. Let us step through the mechanism by which chymotrypsin cuts adjacent to phenylalanine. Substrate binding The process starts with the binding of the substrate in the S1 pocket (Figure 4.54). The S1 pocket in chymotrypsin has a hydrophobic hole in which the substrate is bound. Preferred substrates will include amino acid side chains that are bulky and hydrophobic, like phenylalanine. If an ionized side chain, like that of glutamic acid binds in the S1 pocket, it will quickly exit, much like water would avoid an oily interior. Shape change on binding When the proper substrate binds in the S1 pocket, its presence induces an ever so slight change in the shape of the enzyme. This subtle shape change on the binding of the proper substrate starts the steps of the catalysis. Since the catalytic process only starts when the proper substrate binds, this is the reason that the enzyme shows specificity for cutting at specific amino acids in the target protein. Only amino acids with the side chains that interact well with the S1 pocket start the catalytic wheels turning. The slight changes in shape involve changes in the positioning of three amino acids (aspartic acid, histidine, and serine) in the active site known as the catalytic triad. The shift of the negatively charged aspartic acid towards the electron rich histidine ring favors the abstraction of a proton by the histidine from the hydroxyl group on the side chain of serine, resulting in production of a very reactive alkoxide ion in the active site (Figure 4.55). Since the active site at this point also contains the polypeptide chain positioned with the phenylalanine side chain embedded in the S1 pocket, the alkoxide ion performs a nucleophilic attack on the peptide bond on the carboxyl side of phenylalanine sitting in the S1 pocket (Figure 4.56). This reaction breaks the peptide bond (Figure 4.57) and causes two things to happen. First, one end of the original polypeptide is freed and exits the active site (Figure 4.58). The second is that the end containing the phenylalanine is covalently linked to the oxygen of the serine side chain. At this point we have completed the first (fast) phase of the catalysis. Slower second phase The second phase of the catalysis by chymotrypsin is slower. It requires that the covalent bond between phenylalanine and serine’s oxygen be broken so the peptide can be released and the enzyme can return to its original state. The process starts with entry of water into the active site. Water is attacked in a fashion similar to that of the serine side chain in the first phase, creating a reactive hydroxyl group (Figure 4.59) that performs a nucleophilic attack on the phenylalanine-serine bond (Figure 4.60), releasing it and replacing the proton on serine. The second peptide is released in the process and the reaction is complete with the enzyme back in its original state (Figure 4.61). Serine proteases The list of serine proteases is quite long. They are grouped in two broad categories - 1) those that are chymotrypsin-like and 2) those that are subtilisin-like. Though subtilisin-type and chymotrypsin-like enzymes use the same mechanism of action, including the catalytic triad, the enzymes are otherwise not related to each other by sequence and appear to have evolved independently. They are, thus, an example of convergent evolution - a process where evolution of different forms converge on a structure to provide a common function. The serine protease enzymes cut adjacent to specific amino acids and the specificity is determined by the size/shape/charge of amino acid side chain that fits into the enzyme’s S1 binding pocket (Figure 4.62). Examples of serine proteases include trypsin, chymotrypsin, elastase, subtilisin, signal peptidase I, and nucleoporin. Serine proteases participate in many physiological processes, including blood coagulation, digestion, reproduction, and the immune response. Cysteine proteases Cysteine proteases (also known as thiol proteases) catalyze the breakdown of proteins by cleaving peptide bonds using a nucleophilic thiol from a cysteine (Figure 4.63). The cysteine is typically found in a catalytic dyad or triad also involving histidine and (sometimes) aspartic acid (very much like serine proteases). The sulfhydryl group of cysteine proteases is more acidic than the hydroxyl of serine proteases, so the aspartic acid of the triad is not always needed.
The mechanism of action is very similar to that of serine proteases. Binding of proper substrate results in activation of the thiol (removal of the proton by the histidine group). The activated thiol acts as a nucleophile, attacking the peptide bond and causing it break. One peptide is released and the other peptide becomes covalently linked to the sulfur. Hydrolysis by water releases the second peptide and completes the cycle. Examples of cysteine proteases include papain, caspases, hedgehog protein, calpain, and cathepsin K. Caspases Caspases (Cysteine-ASPartic ProteASEs) are a family of cysteine proteases that play important roles in the body. At the cellular level they function in apoptosis and necrosis and in the body, they are involved in inflammation and the immune system. Maturation of lymphocytes is one such role. They are best known, however, for their role in apoptosis, which has given rise to descriptions of them as “executioner” proteins or “suicide proteases” that dismantle cells in programmed cell death. There are 12 known human caspases. The enzymes are synthesized as pro-caspase zymogens with a prodomain and two other subunits. The prodomain contains regions that allow it to interact with other molecules that regulate the enzyme’s activity. The caspases come in two forms. The initiator caspases, when activated, activate the effector caspases. The effector caspases cleave other proteins in the cell. Targets for effector caspase cleavage action include the nuclear lamins (fibrous proteins providing structural integrity to the nucleus), ICAD/DFF45 (an inhibitor of DNAse), PARP (flags areas where DNA repair needed), and PAK2 (apoptotic regulation). The caspase activation cascade can itself be activated by granzyme B (a serine protease secreted by natural killer cells and cytotoxic T-cells), cellular death receptors, and the apoptosome (large protein structure in apoptotic cells stimulated by release of cytochrome C from the mitochondria). Each of these activators is responsible for activating different groups of caspases. Metalloproteases Metalloproteases (Figure 4.64) are enzymes whose catalytic mechanism for breaking peptide bonds involves a metal. Most metalloproteases use zinc as their metal, but a few use cobalt, coordinated to the protein by three amino acid residues with a labile water at the fourth position. A variety of side chains are used - histidine, aspartate, glutamate, arginine, and lysine. The water is the target of action of the metal which, upon binding of the proper substrate, abstracts a proton to create a nucleophilic hydroxyl group that attacks the peptide bond, cleaving it (Figure 4.64). Since the nucleophile here is not attached covalently to the enzyme, neither of the cleaved peptides ends up attached to the enzyme during the catalytic process. Examples of metalloproteases include carboxypeptidases, aminopeptidases, insulinases and thermolysin. Aspartyl proteases As the name suggests, aspartyl proteases use aspartic acid in their catalytic mechanism (Figures 4.63 & 4.65). Like the metalloproteases, aspartyl proteases activate a water to create a nucleophile for catalysis (Figure 4.65). The activated water attacks the peptide bond of the bound substrate and releases the two pieces without the need to release a bound intermediate, since water is not covalently attached to the enzyme. Common aspartyl proteases include pepsin, signal peptidase II, and HIV-1 protease. Threonine proteases Though threonine has an R-group with a hydroxyl like serine, the mechanism of action of this class of proteases differs somewhat from the serine proteases. There are some similarities. First, the threonine’s hydroxyl plays a role in catalysis and that is to act as a nucleophile. The nucleophile is created, however, not by a catalytic triad, but rather as a result of threonine’s own α-amine group abstracting a proton. Because of this, the nucleophilic threonine in a threonine protease must be at the n-terminus of the enzyme. Nucleophilic attack of the peptide bond in the target protease results in breakage of the bond to release one peptide and the other is covalently attached to serine, like the serine proteases. Also, as with the serine proteases, water must come in to release the covalently linked second peptide to conclude the catalytic mechanism. Examples Examples of threonine proteases include the catalytic subunits of the proteasome. Some acyl transferases (such as ornithine acyltransferase) have evolved the same catalytic mechanism by convergent evolution. The latter enzymes use ornithine instead of water to break the enzyme-substrate covalent bond, with the result that the acyl-group becomes attached to ornithine, instead of water. Protease inhibitors Molecules which inhibit the catalytic action of proteases are known as protease inhibitors. These come in a variety of forms and have biological and medicinal uses. Many biological inhibitors are proteins themselves. Protease inhibitors can act in several ways, including as a suicide inhibitor, a transition state inhibitor, a denaturant, and as a chelating agent. Some work only on specific classes of enzymes. For example, most known aspartyl proteases are inhibited by pepstatin. Metalloproteases are sensitive to anything that removes the metal they require for catalysis. Zinc-containing metalloproteases, for example, are very sensitive to EDTA, which chelates the zinc ion. One category of proteinaceous protease inhibitors is known as the serpins. Serpins inhibit serine proteases that act like chymotrypsin. 36 of them are known in humans. Serpins are unusual in acting by binding to a target protease irreversibly and undergoing a conformational change to alter the active site of its target. Other protease inhibitors act as competitive inhibitors that block the active site.
Serpins can be broad in their specificity. Some, for example, can block the activity of cysteine proteases. One of the best known biological serpins is α-1-anti-trypsin (A1AT - Figure 4.66) because of its role in lungs, where it functions to inhibit the elastase protease. Deficiency of A1AT leads to emphysema. This can arise as a result of genetic deficiency or by cigarette smoking. Reactive oxygen species produced by cigarette smoking can oxidize a critical methionine residue (#358 of the processed form) in A1AT, rendering it unable to inhibit elastase. Uninhibited, elastase can attack lung tissue and cause emphysema. Most serpins work extracellularly. In blood, for example, serpins like antithrombin can help to regulate the clotting process. Figure 4.67 - Incidence of α-1-antitrypsin (PiMZ) deficiency in Europe by percent. Wikipedia Anti-viral Agents Protease inhibitors are used as anti-viral agents to prohibit maturation of viral proteins - commonly viral coat proteins. They are part of drug “cocktails” used to inhibit the spread of HIV in the body and are also used to treat other viral infections, including hepatitis C. They have also been investigated for use in treatment of malaria and may have some application in anti-cancer therapies as well.
A printable version of this section is here: BiochemFFA_4_4.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Clotting is a process in which liquid blood is converted into a gelatinous substance that eventually hardens. The aim is to stop the flow of blood from a vessel. The formation of a clot is the result of a series of enzymatic reactions that are triggered upon injury. The process involves: 1. a step of activation (wounding) followed by 2. a cellular response (aggregation of blood platelets) and 3. a molecular response (polymerization of the protein called fibrin to create a meshwork that hardens). Factors released in the cellular response help activate the molecular response. The process is highly conserved across species. Cellular Response Injury to the epithelial lining of a blood vessel begins the process of coagulation almost instantly. The cellular response has an initial action followed by an amplification step. In the cellular response (Figure 4.68), the platelets bind directly to collagen using Ia/IIa collagen-binding surface receptors and glycoprotein VI to form a plug. The signal to the platelets to take this action is exposure of the underlying collagen, something that would not happen in the absence of a wound. Upon injury, platelet integrins get activated and bind tightly to the extracellular matrix to anchor them to the site of the wound. The von Willebrand factor (see below also) assists by forming additional links between the platelets’ glycoprotein Ib/IX/V and the fibrils of the collagen. Amplification In the amplification part of the cellular response, the activated platelets release a large number of factors, including platelet factor 4 (a cytokine stimulating inflammation and moderating action of the heparin anticoagulant) and thromboxane A2, The latter has the effect of increasing the “stickiness” of platelets, favoring their aggregation. In addition, a a Gq-protein linked receptor cascade is activated, resulting in release of calcium from intracellular stores. This will play a role in the molecular response. Molecular response The molecular response results in the creation of a web comprised of polymers of fibrin protein. Like the cellular pathway, the molecular pathway begins with an initiation phase and continues with an amplification phase. Polymerization of fibrin results from convergence of two cascading catalytic pathways. They are the intrinsic pathway (also called the contact activation pathway) and the extrinsic pathway (also referred to as the tissue factor pathway). Of the two pathways, the tissue factor pathway has recently been shown to be the more important. Serine Protease Cascade In both pathways, a series of zymogens of serine proteases are sequentially activated in rapid succession. The advantage of such a cascading system is tremendous amplification of a small signal. At each step of the cascade, activation of a zymogen causes the production of a considerable amount of an active serine protease, which is then able to activate the next zymogen which, in turn, activates an even larger amount of the next zymogen in the system. This results in the ultimate activation of a tremendous amount more fibrin than could be achieved if there were only a single step where an enzyme activated fibrinogen to fibrin. Nomenclature The zymogen factors in the molecular response are generally labeled with Roman numerals. A lowercase, subscripted ‘a’ is used to designate an activated form. The tissue factor pathway functions to create a thrombin burst, a process in which thrombin is activated very quickly. This is the initiation phase. It is fairly straightforward because it has one focus - activation of thrombin. Thrombin, which converts fibrinogen into the fibrin of the clot, is central also to the amplification phase, because it activates some of the factors that activate it, creating an enormous increase in signal and making a lot of thrombin active at once. Initiation phase The initiation phase of the molecular response begins when Factor VII (the letter ‘F’ before the Roman numeral is often used as an abbreviation for ‘factor’) gets activated to FVIIa after damage to the blood vessel (Figure 4.69 & 4.70). This happens as a result of its interaction with Tissue Factor (TF, also called coagulation Factor III) to make a TF-FVIIa complex. The combined efforts of TF-FVIIa, FIXa, and calcium (from the cellular response) inefficiently convert FX to FXa. FXa, FV, and calcium inefficiently convert prothrombin (zymogen) to thrombin (active). A tiny amount of thrombin has been activated at the end of the initiation phase. Figure 4.69 - Intrinsic and extrinsic pathways of blood coagulation. The aim is making a fibrin clot (lower right). Wikipedia Figure 4.70 - Another view of the molecular response of the blood clotting pathway. Wikipedia Amplification phase To make sufficient thrombin to convert enough fibrinogen to fibrin to make a clot, thrombin activates other factors (FV, FXI, FVIII) that help to make more thrombin. This is the amplification phase of the molecular process and is shown in the light blue portion in the upper right part of Figure 4.68. The amplification phase includes factors in both the intrinsic and extrinsic pathways. FVIII is normally bound in a complex with the von Willebrand factor and is inactive until it is released by action of thrombin. Activation of FXI to FXIa helps favor production of more FIXa. FIXa plus FVIIIa stimulate production of a considerable amount of FXa. FVa joins FXa and calcium to make a much larger amount of thrombin. Factors FVa and FVIIIa are critical to the amplification process. FVIIIa stimulates FIXa’s production of FXa by 3-4 orders of magnitude. FVa helps to stimulate FXa’s production of thrombin by about the same magnitude. Thus, thrombin stimulates activation of factors that, in turn, stimulate activation of more thrombin. Transglutaminase In addition to helping to amplify product of itself and conversion of fibrinogen to fibrin, thrombin catalyzes the activation of FXIII to FXIIIa. FXIIIa is a transglutaminase that helps to “harden” the clot (Figure 4.71 & 4.73). It accomplishes this by catalyzing formation of a covalent bond between adjacent glutamine and lysine side chains in the fibrin polymers.
Not all of the factors involved in the clotting process are activated by the pathway, nor are all factors serine proteases. This includes FVIII and FV which are glycoproteins, and FXIII, which is the transglutaminase described above. The blood clotting process must be tightly regulated. Formation of clots in places where no damage has occurred can lead to internal clots (thrombosis) cutting off the flow of blood to critical regions of the body, such as heart or brain. Conversely, lack of clotting can lead to internal bleeding or, in severe cases, death due to unregulated external bleeding. Such is a danger for people suffering from hemophilia. Figure 4.72 - α-thrombin. Wikipedia Diseases of Blood Clotting: Hemophilia Hemophilia is a hereditary genetic disorder affecting the blood clotting process in afflicted individuals. The disease is X-linked and thus occurs much more commonly in males. Deficiency of FVIII leads to Hemophilia A (about 1 in 5000 to 10,000 male births) and deficiency of FIX produces Hemophilia B (about 1 in 20,000 to 35,000 male births). Hemophilia B spread through the royal families of Europe, beginning with Queen Victoria’s son, Leopold. Three of the queen’s grandsons and six of her great-grandsons suffered from the disease. Hemophilia is treated by exogenous provision of missing clotting factors and has improved life expectancy dramatically. In 1960, the life expectancy of a hemophiliac was about 11 years. Today, it is over 60. Diseases of Blood Clotting: von Willebrand’s disease A related disease to hemophilia that is also genetically linked is von Willebrand’s Disease. The von Willebrand factor plays a role in both the cellular and the molecular responses in blood clotting. First, the factor is a large multimeric glycoprotein present in blood plasma and also is produced in the endothelium lining blood vessels. The von Willebrand factor helps to anchor platelets near the site of the wound in the cellular response. It binds to several things. First, it binds to platelets’ Ib glycoprotein. Second, it binds to heparin and helps moderate its action. Third, it binds to collagen and fourth, the factor binds to FVIII in the molecular response, playing a protective role for it. In the absence of the von Willebrand factor, FVIII is destroyed. Fifth, the von Willebrand factor binds to integrin of platelets, helping them to adhere together and form a plug. Defects of the von Willebrand factor lead to various various bleeding disorders. Blood “thinners” The clotting of blood is essential for surviving wounds that cause blood loss. However, some people have conditions that predispose them to the formation of clots that can lead to stroke, heart attack, or other problems, like pulmonary embolism. For these people, anti-clotting agents (commonly called blood thinners) are used to reduce the likelihood of undesired clotting. The first, and more common of these is aspirin. Aspirin is an inhibitor of the production of prostaglandins. Prostaglandins are molecules with 20 carbons derived from arachidonic acid that have numerous physiological effects. Metabolically, the prostaglandins are precursors of a class of molecules called the thromboxanes. Thromboxanes play roles in helping platelets to stick together in the cellular response to clotting. By inhibiting the production of prostaglandins, aspirin reduces the production of thromboxanes and reduces platelet stickiness and the likelihood of clotting. Vitamin K action Another approach to preventing blood clotting is one that interferes with an important molecular action of Vitamin K. A pro-clotting factor found in the blood, vitamin K is necessary for an important modification to prothrombin and other blood clotting proteins. Vitamin K serves as an enzyme cofactor that helps to catalyze addition of an extra carboxyl group onto the side chain of glutamic acid residues of several clotting enzymes (see HERE). This modification gives them the ability to bind to calcium (Figure 4.77), which is important for activating the serine protease cascade. During the reaction that adds carboxyl groups to glutamate, the reduced form of vitamin K becomes oxidized. In order for vitamin K to stimulate additional carboxylation reactions to occur, the oxidized form of vitamin K must be reduced by the enzyme vitamin K epoxide reductase. Figure 4.77 - γ-carboxylglutamic acid (left) has a calcium binding Site. Unmodified glutamic acid (right) does not. Warfarin blocks reduction The compound known as warfarin (brand name = coumadin - Figure 4.78) interferes with the action of vitamin K epoxide reductase and thus, blocks recycling of vitamin K. As a consequence, fewer prothrombins (and other blood clotting proteins) get carboxylated, and less clotting occurs. Vitamin K-mediated carboxylation of glutamate occurs on the γ carbon of the amino acid’s side chain, for 16 different proteins, 7 of which are involved in blood clotting, including prothrombin. When the carboxyl group is added as described, the side chain is able to efficiently bind to calcium ions. In the absence of the carboxyl group, the side chain will not bind to calcium. Calcium released near the site of the wound in the cellular response to clotting helps to stimulate activation of proteins in the serine protease cascade of the molecular response. Vitamin K comes in several forms. It is best described chemically as a group of 2-methyl-1,4-naphthoquinone derivatives. There are five different forms recognized as vitamin Ks (K1, K2, K3, K4, and K5). Of these, vitamins K1 and K2 come from natural sources and the others are synthetic. Vitamin K2, which is made from vitamin K1 by gut microorganisms, has several forms, with differing lengths of of isoprenoid side-chains. The various forms are commonly named as MK-X, where X is a number, and MK stands for menaquinone, which is the name given to this form of vitamin K. Figure 4.79 shows a common form known as MK-4 (menatetrenone). Figure 4.79 - MK-4 (menatetrenone) Hemorrhaging danger
It is very critical that the proper amount of warfarin be given to patients. Too much can result in hemorrhaging. Patients must have their clotting times checked regularly to ensure that they are taking the right dose of anti-coagulant medication. Diet and the metabolism of Vitamin K in the body can affect the amount of warfarin needed. Vitamin K is synthesized in plants and plays a role in photosynthesis. It can be found in the highest quantities in vegetables that are green and leafy. Patients whose diet is high in these vegetables may require a different dose than those who rarely eat greens. Dietary vitamin K is also, as mentioned earlier, metabolized by bacteria in the large intestine, where they convert vitamin K1 into vitamin K2. Plasmin Clots, once made in the body, do not remain there forever. Instead, a tightly regulated enzyme known as plasmin is activated, when appropriate, to break down the fibrin-entangled clot. Like many of the enzymes in the blood clotting cascade, plasmin is a serine protease. It is capable of cleaving a wide range of proteins. They include polymerized fibrin clots, fibronectin, thrombospondin, laminin, and the von Willebrand factor. Plasmin plays a role in activating collagenases and in the process of ovulation by weakening the wall of the Graafian follicle in the ovary. Plasmin is made in the liver as the zymogen known as plasminogen. Several different enzymes can activate it. Tissue plasminogen activator (tPA), using fibrin as a co-factor, is one. Others include urokinase plasminogen activator (using urokinase plasminogen activator receptor as a co-factor), kallikrein (plasma serine protease with many forms and blood functions), and FXIa and FXIIa from the clotting cascade. Plasmin inhibition Plasmin’s activity can also be inhibited. Plasminogen activator inhibitor, for example, can inactivate tPA and urokinase. After plasmin has been activated, it can also be inhibited by α2-antiplasmin and α2-macroglobulin (Figure 4.80). Thrombin also plays a role in plasmin’s inactivation, stimulating activity of thrombin activatable fibrinolysis inhibitor. Angiostatin is a sub-domain of plasmin produced by auto-proteolytic cleavage. It blocks the growth of new blood vessels and is being investigated for its anti-cancer properties. Figure 4.80 - Regulation of fibrin breakdown. Activators in blue. Inhibitors in red. Wikipedia Fibronectin Fibronectin is a large (440 kDa) glycoprotein found in the extracellular matrix that binds to integral cellular proteins called integrins and to extracellular proteins, including collagen, fibrin, and heparan sulfate. It comes in two forms. The soluble form is found in blood plasma and is made by the liver. It is found in high concentration in the blood stream (300 µg/ml). The insoluble form is found abundantly in the extracellular matrix. The protein is assembled in the extracellular matrix and plays roles in cellular growth, adhesion, migration, and differentiation. It is very important in wound healing. Figure 4.82 - Fibronectin 1. Wikipedia Assists in blood clot formation Fibronectin from the blood plasma is localized to the site of the wound, assisting in formation of the blood clot to stop bleeding. In the initial stages of wound healing, plasma fibronectin interacts with fibrin in clot formation. It also protects tissue surrounding the wound. Later in the repair process, remodeling of the damaged area begins with the action of fibroblasts and endothelial cells at the wound site. Their task is to degrade proteins of the blood clot matrix, replacing them with a new matrix like the undamaged, surrounding tissue. Fibroblasts act on the temporary fibronectin-fibrin matrix, remodeling it to replace the plasma fibronectin with cellular fibronectin. This may cause the phenomenon of wound contraction, one of the steps in wound healing. Secretion of cellular fibronectin by fibroblasts is followed by fibronectin assembly and integration with the extracellular matrix. Embryogenesis Fibronectin is essential for embryogenesis. Deleting the gene in mice causes lethality before birth. This is likely due to its role in migration and guiding the attachment of cells as the embryo develops. Fibronectin also has a role in the mouth. It is found in saliva and is thought to inhibit colonization of the mouth by pathogenic bacteria. Platelet activating factor Platelet Activating Factor (PAF) is a compound (Figure 4.83) produced primarily in cells involved in host defense. These include platelets, macrophages, neutrophils, and monocytes, among others. It is produced in greater quantities in inflammatory cells upon proper stimulation. The compound acts like a hormone and mediates platelet aggregation/degranulation, inflammation, and anaphylaxis. It can transmit signals between cells to trigger and amplify inflammatory and clotting cascades. When unregulated, signaling by PAF can cause severe inflammation resulting in sepsis and injury. Inflammation in allergic reactions arises partly as a result of PAF and is an important factor in bronchoconstriction in asthma. In fact, at a concentration of only 10 picomolar, PAF can cause asthmatic inflammation of the airways that is life threatening. Figure 4.83 - Platelet Activating Factor. Wikipedia I’m feeling so sad ‘Cuz I cut . . . . myself bad Now I’m all worried ‘bout . . . . consequences It’s starting to bleed There’s some clo . . . . sure I need So the body kicks . . . . in its defenses It’s happened all so many times before The blood flows out and then it shuts the door Thank goodness my blood is clotting Enmeshing the fibrin chains Thank goodness my blood is clotting The zymogens Are activating and all is well So I’ll stop bleeding again The vitamin K’s Help to . . . . bind to cee-ays Adding C-O-. . . . O-H to amend things Um-m-um-um-um-um It hardens and stays When a glu. . . . taminase Creates co. . . . valent bonds . . . . for cementing In just a moment, things are good to go The clot’s in place and it has stopped the flow But what about clot dissolving? Untangling fibrin chains? This calls for some problem solving There is a way Just activate up some t-PA Get plasmin active in veins Oh, oh, oh. And thanks to the dis-enclotting’ As part of repairin’ veins It’s part of my body’s plotting The wound is gone I’m back where I started and Nothing’s wrong My blood flow is normal again. Thank Goodness My Blood is Clotting
• 5.1: Basics of Energy Living organisms are made up of cells, and cells contain a horde of biochemical components. Living cells, though, are not random collections of these molecules. They are extraordinarily organized or "ordered". By contrast, in the nonliving world, there is a universal tendency to increasing disorder. Maintaining and creating order in cells takes the input of energy. Without energy, life is not possible. • 5.3: Energy - Photophosphorylation The third type of phosphorylation to make ATP is found only in cells that carry out photosynthesis. This process is similar to oxidative phosphorylation in several ways. A primary difference is the ultimate source of the energy for ATP synthesis. In oxidative phosphorylation, the energy comes from electrons produced by oxidation of biological molecules. In photosynthesis, the energy comes from the light of the sun. • 5.2: Electron Transport and Oxidative Phosphorylation In eukaryotic cells, the vast majority of ATP synthesis occurs in the mitochondria in a process called oxidative phosphorylation. Even plants, which generate ATP by photophosphorylation in chloroplasts, contain mitochondria for the synthesis of ATP through oxidative phosphorylation. 05: Energy Source: BiochemFFA_5_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Living organisms are made up of cells, and cells contain a horde of biochemical components. Living cells, though, are not random collections of these molecules. They are extraordinarily organized or "ordered". By contrast, in the nonliving world, there is a universal tendency to increasing disorder. Maintaining and creating order in cells takes the input of energy. Without energy, life is not possible. Oxidative Energy The primary mechanism used by non-photosynthetic organisms to obtain energy is oxidation and carbon is the most commonly oxidized energy source. The energy released during the oxidative steps is “captured” in ATP and can be used later for energy coupling. The more reduced a carbon atom is, the more energy can be realized from its oxidation. Fatty acids are highly reduced, whereas carbohydrates are moderately so. Complete oxidation of both leads to carbon dioxide, which has the lowest energy state. Conversely, the more oxidized a carbon atom is, the more energy it takes to reduce it. In the series shown in Figure $1$, the most reduced form of carbon is on the left. The energy of oxidation of each form is shown above it. The reduction states of fatty acids and carbohydrates can be seen by their formulas. • Palmitic acid: $\ce{C16H34O2}$ • Glucose: $\ce{C6H12O6}$ Palmitic acid only contains two oxygens per sixteen carbons, whereas glucose has six oxygen atoms per six carbons. Consequently, when palmitic acid is fully oxidized, it generates more ATP per carbon (128/16) than glucose (38/6). It is because of this that we use fat (contains fatty acids) as our primary energy storage material. Figure $2$: Photosynthesis: The primary source of biological energy. Image by Aleia Kim Oxidation vs. Reduction in Metabolism Biochemical processes that break things down from larger to smaller are called catabolic processes. Catabolic processes are often oxidative in nature and energy releasing. Some, but not all, of that energy is captured as ATP. If not all of the energy is captured as ATP, what happens to the rest of it? The answer is simple. It is released as heat and it is for this reason we get hot when we exercise. By contrast, synthesizing large molecules from smaller ones (for example, making proteins from amino acids) is referred to as anabolism. Anabolic processes are often reductive in nature (Figures 5.3 & 5.4) and require energy input. By themselves, they would not occur, as they are reversing oxidation and decreasing entropy (making many small things into a larger one). To overcome this energy barrier, cells must expend energy. For example, if one wishes to reduce $\ce{CO2}$ to carbohydrate, energy must be used to do so. Plants do this during the dark reactions of photosynthesis (Figure $3$). The energy source for the reduction is ultimately the sun. The electrons for the reduction come from water, and the $\ce{CO2}$ is removed from the atmosphere and gets incorporated into a sugar. Energy Coupling The synthesis of the many molecules needed by cells needs the input of energy to occur. Cells overcome this energy obstacle by using ATP to “drive” the reaction (Figure $6$). The energy needed to drive reactions is harvested in very controlled conditions in enzymes. This involves a process called ‘coupling’. Coupled reactions rely on linking an energetically favorable reaction (i.e., one with a negative ∆G°’) with the reaction requiring an energy input, which has a positive ∆G°’. As long as the overall ∆G°’ of the two reactions together is negative, the reaction can proceed. Hydrolysis of ATP is a very energetically favorable reaction that is commonly linked to many energy requiring reactions in cells. Without the hydrolysis of ATP (or GTP, in some cases), the reaction would not be feasible. Entropy and energy Most students who have had some chemistry know about the Second Law of Thermodynamics with respect to increasing disorder of a system. Cells are very organized or ordered structures, leading some to mistakenly conclude that life somehow violates the second law. In fact, that notion is incorrect. The second law doesn’t say that entropy always increases, just that, left alone, it tends to do so, in an isolated system. Cells are not isolated systems, though, in that they obtain energy, either from the sun, if they are autotrophic, or food, if they are heterotrophic. To counter the universal tendency towards disorder on a local scale requires energy. As an example, take a fresh deck of cards which is neatly aligned with Ace-King-Queen . . . . 4,3,2 for each suit. Throw the deck into the air, letting the cards scatter. When you pick them up, they will be more disordered than when they started. However, if you spend a few minutes (and expend a bit of energy), you can reorganize the same deck back to its previous, organized state. If entropy always increased everywhere, you could not do this. However, with the input of energy, you overcame the disorder. This illustrates an important concept: the cost of fighting disorder is energy. Biological energy
There are, of course, other reasons that organisms need energy. Muscular contraction, synthesis of molecules, neurotransmission, signaling, thermoregulation, and subcellular movements are examples. Where does this energy come from? The currencies of energy are generally high-energy phosphate-containing molecules. ATP is the best known and most abundant, but GTP is also an important energy source (energy source for protein synthesis). CTP is involved in synthesis of glycerophospholipids and UTP is used for synthesis of glycogen and other sugar compounds. In each of these cases, the energy is in the form of potential chemical energy stored in the multi-phosphate bonds. Hydrolyzing those bonds releases the energy in them. Of the triphosphates, ATP is the primary energy source, acting to facilitate the synthesis of the others by action of the enzyme NDPK. ATP is made by three distinct types of phosphorylation – oxidative phosphorylation (in mitochondria), photophosphorylation (in chloroplasts of plants), and substrate level phosphorylation (in enzymatically catalyzed reactions). Gibbs free energy in Biology ATP is generally considered the “storage battery” of cells (See also ‘Molecular Battery Backups for Muscles HERE). In order to understand how energy is captured, we must first understand Gibbs free energy and in doing so, we begin to see the role of energy in determining the directions chemical reactions take. Gibbs free energy may be thought of as the energy available to do work in a thermodynamic system at constant temperature and pressure. Mathematically, the Gibbs free energy is given as: $G = H – TS$ where $H$ is the enthalpy, $T$ is the temperature in Kelvin, and $S$ is the entropy. At standard temperature and pressure, every system seeks to achieve a minimum of free energy. Thus, increasing entropy, $S$, will reduce Gibbs free energy. Similarly, if excess heat is available (reducing the enthalpy, $H$), the free energy can also be reduced. Cells must work within the laws of thermodynamics, as noted, so all of their biochemical reactions, too, are ruled by these laws. Now we shall consider energy in the cell. The change in Gibbs free energy ($∆G$) for a reaction is crucial, for it, and it alone, determines whether or not a reaction goes forward. $∆G = ∆H – T ∆S.$ There are three cases • ∆G < 0: the reaction proceeds as written • ∆G = 0: the reaction is at equilibrium • ∆G > 0: the reaction runs in reverse For a reaction $\ce{aA <=> bB}$ (where ‘a’ and ‘b’ are integers and A and B are molecules) at pH 7, ∆G can be determined by the following equation, $∆G = ∆G°’ + RT \ln(\frac{[B]^b}{[A]^a})$ For multiple substrate reactions, such as $\ce{aA + cC <=> bB + dD}$ $∆G = ∆G°’ + RT \ln(\frac{[B]^b [D]^d}{[A]^a[C]^c})$ The ∆G°’ term is called the change in Standard Gibbs Free energy, which is the change in energy that occurs when all of the products and reactants are at standard conditions and the pH is 7.0. It is a constant for a given reaction. In simple terms, we can collect all of the terms of the numerator together and call them {Products} and all of the terms of the denominator together and call them {Reactants}, $∆G = ∆G°’ + RT \ln(\frac{\rm{\{Products\}}}{\rm{\{Reactants\}}})$ For most biological systems, the temperature, T, is a constant for a given reaction. Since ∆G°’ is also a constant for a given reaction, the ∆G is changed almost exclusively as the ratio of {Products}/{Reactants} changes. Importance of ∆G°’ If one starts out at standard conditions, where everything except protons is at 1M, the RTln({Products}/{Reactants}) term is zero, so the ∆G°’ term equals the ∆G, and the ∆G°’ determines the direction the reaction will take (only under those conditions). This is why people say that a negative ∆G°’ indicates an energetically favorable reaction, whereas a positive ∆G°’ corresponds to an unfavorable one. Increasing the ratio of {Products}/{Reactants} causes the value of the natural log (ln) term to become more positive (less negative), thus making the value of ∆G more positive. Conversely, as the ratio of {Products}/{Reactants} decreases, the value of the natural log term becomes less positive (more negative), thus making the value of ∆G more negative. System response to stress Intuitively, this makes sense and is consistent with Le Chatelier’s Principle – a system responds to stress by acting to alleviate the stress. If we examine the ∆G for a reaction in a closed system, we see that it will always move to a value of zero (equilibrium), no matter whether it starts with a positive or negative value. Another type of free energy available to cells is that generated by electrical potential. For example, mitochondria and chloroplasts partly use Coulombic energy (based on charge) from a proton gradient across their membranes to provide the necessary energy for the synthesis of ATP. Similar energies drive the transmission of nerve signals (sodium and potassium gradients) and the movement of some molecules in secondary active transport processes across membranes (e.g., H+ differential driving the movement of lactose). From the Gibbs free energy change equation, $∆G = ∆H – T∆S$ it should be noted that an increase in entropy will help contribute to a decrease in ∆G. This happens, for example when a large molecule is being broken into smaller pieces or when the rearrangement of a molecule increases the disorder of molecules around it. The latter situation arises in the hydrophobic effect, which helps drive the folding of proteins. Chemical and electrical potential It is said that absence makes the heart grow fonder. We won’t tackle that philosophical issue here, but we will say that separation provides potential energy that cells can and do harvest. The lipid bilayer of cell and (in eukaryotic cells) organelle membranes provide the necessary barrier for separation. Impermeable to most ions and polar compounds, biological membranes are essential for processes that generate cellular energy. Consider Figure 5.8. A lipid bilayer separates two solutions with different concentrations of a solute. There is a greater concentration of negative ions in the bottom and a greater concentration of positive ions on the top.
Whenever there is a difference in concentration of molecules across a membrane, there is said to be a concentration gradient across it. A difference in concentration of ions across a membrane also creates a charge (or electrical) gradient. Because there is a difference in both the chemical concentration of the ions and in the charge on the two sides of the membrane, this is described as an electrochemical gradient (Figures 5.8 -5.10). Potential energy Such gradients function like batteries and contain potential energy. When the potential energy is harvested by cells, they can create ATP, transmit nerve signals, pump molecules across membranes, and more. It is important, therefore, to understand how to calculate the potential energy of electrochemical gradients. First, we consider chemical (solute) gradients. In Figure 5.9, two concentrations of glucose are separated by a lipid bilayer. Let’s assume C2 be the concentration of glucose inside the cell (bottom) and C1 be the glucose concentration outside (top). The Gibbs free energy associated with moving glucose in the direction of C2 (into the cell) is given by ∆G = RTln[C2/C1] To move it in the direction of C1 (to the outside of the cell) the expression would be $∆G = RT\ln[C_1/C_2]$ Since C2 is smaller than C1 (i.e., there are fewer glucose molecules inside the cell) then the ∆G is negative and diffusion would be favored into the cell, if the glucose could traverse the bilayer. Conversely, if C2 was greater than C1 (more glucose was in the cell than outside) the ∆G would be positive, so movement in the direction of C2 would not be favored and instead the glucose would tend to move towards C1 , that is, out of the cell. If C2 = C1, with the same concentration of glucose inside and outside, then the ∆G would be zero and there would be no net movement, as the system would be at equilibrium. In the example above, we considered glucose, which is an uncharged molecule. When ions are involved, their charges must also be taken into consideration. Figure $1$0 depicts a similar situation across a lipid bilayer. In this case, a difference of concentration and charge exists. There are more positive charges inside the cell than outside. Using C2 to indicate the concentration of materials inside the cell and C1 for the concentration outside the cell (as before), then the free energy for movement of an ion from top to bottom is given by the following equation $∆G = RT\ln[C_2/C_1] + ZF∆ψ$ Note here that this equation must take into consideration both the concentration differences and the charge differences. Z refers to the charge of the transported species, F is the Faraday constant (96,485 Coulombs/mol), and ∆ψ is the electrical potential difference (voltage difference) across the membrane. If we were to calculate the ∆G for movement of the potassium ion from top to bottom, it would be positive, since [C2/C1] is greater than 1 (making for a positive ln term), and the ZF∆ψ is positive because positively charged ions (Z) are moving against a positive charge gradient given by ∆ψ (greater concentration at the target (bottom) than the starting point (top)). If we were to calculate the concentration of ions moving from bottom to top, then the ln term would be negative (C2<C1) and the ZF∆ψ would be negative as well (Z=positive, but ∆ψ negative). Reduction Potential In discussing chemical potential, we must also consider reduction potential. Reduction potential measures the tendency of a chemical to be reduced by electrons. It is also designated by several other names/variables. These include redox potential, oxidation/reduction potential, ORP, pE, ε, E, and Eh. Reduction potential is measured in volts, or millivolts. A substance with a higher reduction potential will have a greater tendency to accept electrons and be reduced. If two substances are mixed in an aqueous solution, the one with the greater (more positive) reduction potential will tend to take electrons away, thus being reduced, from the one with the lower reduction potential, which becomes oxidized. Relative measures Absolute reduction potentials are difficult to measure, so reduction potentials are typically defined relative to a reference electrode. In aqueous solutions, reduction potentials are measured as the potential difference between an inert sensing electrode (typically platinum) in contact with the test solution and a stable reference electrode (measured as a Standard Hydrogen Electrode: SHE) as shown in Figure $1$1. The standard of reference for measurement is the half-reaction H+ + e→ ½ H2 The electrode where this reaction occurs (referred to as a half-cell) is given the value of E° (Standard Reduction Potential) of 0.00 volts. The hydrogen electrode is connected via an external circuit to another half cell containing a mixture of the reduced and oxidized species of another molecule (for example, Fe++ and Fe+++) at 1M each and standard conditions of temperature (25°C) and pressure (1 atmosphere). Direction and voltage measured The direction and magnitude of electron movement is then measured. If the test mixture takes electrons from the hydrogen electrode, the sign of the voltage is positive and if the direction is reversed, the voltage is negative. Thus, compounds which have greater affinity for electrons than hydrogen will register a positive voltage and negative voltages correspond to compounds with lesser affinity for electrons than hydrogen. Movement of electrons Under standard conditions, electrons will move from compounds generating lower voltages to ones generating higher (more positive) voltages. Just as the standard Gibbs free energy change is the Gibbs free energy change under standard conditions, so, too, is the standard reduction potential E° the reduction potential E under standard conditions. The actual reduction potential of a half cell will vary with the concentration of each chemical species in the cell. The relationship between the reduction potential E and the standard reduction potential E° is given by the following equation (also called the Nernst equation) where F is the Faraday constant (96,480 J/(Voltsmoles), R is the gas constant (8.315 J/(molesK), n is the number of moles of electrons being transferred, and T is the absolute temperature in Kelvin. At 25°C, this equation becomes
As for Gibbs free energy, it is useful to measure values at conditions found in cells. This means doing measurements at pH = 7, which differs from having all species at 1M. Adjustment Because of this adjustment, a slightly different standard reduction potential is defined and we designate it by E°’, just as we defined a special standard Gibbs free energy change at pH 7 as ΔG°’. There is a relationship between the change in Gibbs free energy ΔG and the change in reduction potential (ΔE). It is $ΔG = -nFΔE$ Similarly, the relation between the change in standard Gibbs free energy and the change in standard reduction potential is \]ΔG°’ = -nFΔE°’\] Energy Storage in Triphosphates Movie 5.1: ATP: The fuel of the cell Formation of triphosphates, like ATP, is essential to meeting the cell’s energy needs for synthesis, motion, and signaling. In a given day, an average human body makes and breaks down more than its weight in triphosphates. This is especially remarkable considering that there is only about 250 g of the molecule present in the body at any given time. Energy in ATP is released by hydrolysis of a phosphate from the molecule. The three phosphates, starting with the one closest to the sugar are referred to as α, β, and γ (Figure $1$2). It is the γ phosphate that is cleaved in hydrolysis and the product is ADP. In a few reactions, the bond between the α and β is cleaved. When this happens, a pyrophosphate (β linked to γ) is released and AMP is produced. This latter reaction to produce AMP releases more energy (ΔG°’ = -45.6 kJ/mol) than the first reaction which produces ADP (ΔG°’ = -30.5 kJ/mol). Since triphosphates are the “currency” that meet immediate needs of the cell, it is important to understand how triphosphates are made. There are three phosphorylation mechanisms – 1) substrate level; 2) oxidative; and 3) photophosphorylation. We consider them here individually. Substrate level phosphorylation The easiest type of phosphorylation to understand is that which occurs at the substrate level. This type of phosphorylation involves the direct synthesis of ATP from ADP and a high energy intermediate, typically a phosphate-containing molecule. Substrate level phosphorylation is a relatively minor contributor to the total synthesis of triphosphates by cells. An example substrate phosphorylation comes from glycolysis. Phosphoenolpyruvate (PEP) + ADP ⇌ Pyruvate + ATP This reaction has a very negative ∆G°’ (-31.4 kJ/mol), indicating that the PEP contains more energy than ATP, thus tending to energetically favor ATP’s synthesis. Other triphosphates can be made by substrate level phosphorylation, as well. For example, GTP can be synthesized by the following citric acid cycle reaction. Succinyl-CoA + GDP + Pi ⇌ Succinate + GTP + CoA-SH Triphosphates can be interchanged readily in substrate level phosphorylations catalyzed by the enzyme Nucleoside Diphosphate Kinase (NDPK). A generalized form of the reactions catalyzed by this enzyme is as follows: XTP + YDP ⇌ XDP + YTP where X = adenosine, cytidine, uridine, thymidine, or guanosine and Y can be any of these as well. Further, XTP and YDP can be any of the deoxynucleotides as well. Last, an unusual way of synthesizing ATP by substrate level phosphorylation is via the reaction catalyzed by adenylate kinase 2 ADP ⇌ ATP + AMP ATP source This reaction is an important means of generating ATP when the cell doesn’t have other sources of energy. Accumulation of AMP resulting from this reaction activates enzymes, such as phosphofructokinase, of glycolysis, which will catalyze reactions to give the cell additional, needed energy. It is important to note that enzymes cannot make reactions happen that are energetically unfavorable. Enzymes speed reactions, but do not change their direction. Cells are thus bound by the rules of Gibbs free energy. So, how do energetically unfavorable reactions happen in a cell? Reaction coupling Reactions that are energetically unfavorable, can be made favorable by coupling them with the hydrolysis of ATP, a very energetically favorable reaction. There are numerous parallels in the “real world.” Movement of automobiles is energetically unfavorable, but coupling movement of the automobile to oxidation of gasoline makes an unfavorable process favorable. Another approach to making an unfavorable reaction favorable is to manipulate the concentration of reactants and products. Consider the reaction below, which occurs in pyrimidine nucleotide metabolism: orotate + PRPP ⇌ OMP + PPi The ΔG°’ for this reaction is -0.8 kJ/mol, meaning that if one starts with equal concentrations of reactants and products, at equilibrium, there will be a small excess of products. In the cell, however, this reaction moves strongly to the right (ΔG = very negative). Given that the ΔG°’ is very close to zero, a very negative ΔG can only occur if the concentrations of reactants and products are altered, since $ΔG = ΔG°’ + RT \ln(\frac{[\rm{OMP}][\rm{PP_i}]}{[\rm{Orotate}][\rm{PRPP}]})$ Manipulation is exactly what happens here. The key item whose concentration is adjusted in this reaction is the pyrophosphate (PPi). This is possible because cells contain an enzyme called pyrophosphorylase that catalyzes the following reaction PPi + H2O ⇌ 2 Pi Hydrolysis of pyrophosphate is very energetically favored, causing the PPi produced in the reaction to be quickly hydrolyzed. As a result, the concentration of PPi in the cell is kept very low. A low concentration of a product (PPi) causes the natural log (ln) term of the orotate equation to become more negative, driving the ΔG term for the overall reaction to become much more negative. Pushing and pulling Reactions that yield pyrophosphate as a product are produced in the synthesis of DNA and RNA, as well as many other molecules. As shown in the previous example, this pyrophosphate is rapidly hydrolyzed, causing the overall reaction to move in the direction of pyrophosphate production. When reactants are removed/reduced in a metabolic reaction to decrease the concentration of a product, we say that the reaction is “pulled”, to represent the increase in the forward reaction as a result of product depletion.
Source: BiochemFFA_5_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Photophosphorylation The third type of phosphorylation to make ATP is found only in cells that carry out photosynthesis. This process is similar to oxidative phosphorylation in several ways. A primary difference is the ultimate source of the energy for ATP synthesis. In oxidative phosphorylation, the energy comes from electrons produced by oxidation of biological molecules. In photosynthesis, the energy comes from the light of the sun. Photons from the sun interact with chlorophyll molecules in reaction centers in the chloroplasts (Figures $1$ and $2$) of plants or membranes of photosynthetic bacteria. The similarities of photophosphorylation to oxidative phosphorylation include: • a membrane associated electron transport chain • creation of a proton gradient • harvesting energy of the proton gradient by making ATP with the help of an ATP synthase. Some of the differences include : • the source of the electrons – H2O for photosynthesis versus NADH/FADH2 for oxidative phosphorylation • direction of proton pumping – into the thylakoid space of the chloroplasts versus outside the matrix of the mitochondrion • movement of protons during ATP synthesis – out of the thylakoid space in photosynthesis versus into the mitochondrial matrix in oxidative phosphorylation • nature of the terminal electron acceptor – NADP+ in photosynthesis versus O2 in oxidative phosphorylation. Electron transport: chloroplasts vs mitochondria In some ways, the movement of electrons in chloroplasts during photosynthesis is opposite that of electron transport in mitochondria. In photosynthesis, water is the source of electrons and their final destination is NADP+ to make NADPH. In mitochondria, NADH/FADH2 are electron sources and H2O is their final destination. How do biological systems get electrons to go both ways? It would seem to be the equivalent of going to and from a particular place while always going downhill, since electrons will move according to potential. Solar power The answer is the captured energy of the photons from the sun (Figure 5.59), which elevates electrons to an energy where they move “downhill” to their NADPH destination in a Z-shaped scheme. The movement of electrons through this scheme in plants requires energy from photons in two places to “lift” the energy of the electrons sufficiently. Last, it should be noted that photosynthesis actually has two phases, referred to as the light cycle (described above) and the dark cycle, which is a set of chemical reactions that captures CO2 from the atmosphere and “fixes” it, ultimately into glucose. The dark cycle is also referred to as the Calvin Cycle and is discussed HERE. Photosynthesis Photosynthesis is an energy capture process found in plants and other organisms to harvest light energy and convert it into chemical energy. This photochemical energy is stored ultimately in carbohydrates which are made using ATP (from the energy harvesting), carbon dioxide and water. In most cases, a byproduct of the process is oxygen, which is released from water in the capture process. Photosynthesis is responsible for most of the oxygen in the atmosphere and it supplies the organic materials and most of the energy used by life on Earth. Steps The steps in the photosynthesis process varies slightly between organisms. In a broad overview, it always starts with energy capture from light by protein complexes, containing chlorophyll pigments, called reaction centers. Plants sequester these proteins in chloroplasts, but bacteria, which don’t have organelles, embed them in their plasma membranes. Energy from the light is used to strip electrons away from electron donors (usually water) and leave a byproduct (oxygen, if water was used). Electrons are donated to a carrier and ultimately are accepted by NADP+, to become NADPH. As electrons travel towards NADP+, they generate a proton gradient across the thylakoid membrane, which is used to drive synthesis of ATP. Thus NADPH, ATP, and oxygen are the products of the first phase of photosynthesis called the light reactions. Energy from ATP and electrons from NADPH are used to reduce CO2 and build sugars, which are the ultimate energy storage directly arising from photosynthesis. Chloroplasts Chloroplasts are found in almost all aboveground plant cells, but are primarily concentrated in leaves. The interior of a leaf, below the epidermis is made up of photosynthesis tissue called mesophyll, which can contain up to 800,000 chloroplasts per square millimeter. The chloroplast’s membrane has a phospholipid inner membrane, a phospholipid outer membrane, and a region between them called the intermembrane space (Figure 5.61). Within the inner chloroplast membrane is the stroma, in which the chloroplast DNA and the enzymes of the Calvin cycle are located. Also within the stroma are stacked, flattened disks known as thylakoids which are defined by their thylakoid membranes. The space within the thylakoid membranes are termed the thylakoid spaces or thylakoid lumen. The protein complexes containing the light-absorbing pigments, known as photosystems, are located on the thylakoid membrane. Besides chlorophylls, carotenes and xanthophylls are also present, allowing for absorption of light energy over a wider range. The same pigments are used by green algae and land plants. Brown algae and diatoms add fucoxanthin (a xanthophyll) and red algae add phycoerythrin to the mix. In plants and algae, the pigments are held in a very organized fashion complexes called antenna proteins that help funnel energy, through resonance energy transfer, to the reaction center chlorophylls. A system so organized is called a light harvesting complex. The electron transport complexes of photosynthesis are also located on the thylakoid membranes. Figure $6$: Complexes in the thylakoid membrane. Image by Aleia Kim Light reactions of photosynthesis In chloroplasts, the light reactions of photosynthesis involving electron transfer occur in the thylakoid membranes (Figure $6$). Separate biochemical reactions involving the assimilation of carbon dioxide to make glucose are referred to as the Calvin cycle, also sometimes referred to as the “dark reactions”. This will be discussed elsewhere in the section on metabolism (HERE).
The chloroplasts are where the energy of light is captured, electrons are stripped from water, oxygen is liberated, electron transport occurs, NADPH is formed, and ATP is generated. The thylakoid membrane corresponds to the inner membrane of the mitochondrion for transport of electrons and proton pumping (Figure $4$). The thylakoid membrane does its magic using four major protein complexes. These include Photosystem II (PS II), Cytochrome b6f complex (Cb6f), Photosystem I (PS I), and ATP synthase. The roles of these complexes, respectively, are to capture light energy, create a proton gradient from electron movement, capture light energy (again), and use proton gradient energy from the overall process to synthesize ATP. Light harvesting Harvesting the energy of light begins in PS II with the absorption of a photon of light at a reaction center. PS II performs this duty best with light at a wavelength of 680 nm and it readily loses an electron to excitation when this occurs, leaving PS II with a positive charge. This electron must be replaced. The ultimate replacement source of electrons is water, but water must lose four electrons and PS II can only accept one at a time. Manganese centers An intermediate Oxygen Evolving Complex (OEC) contains four manganese centers that provide the immediate replacement electron that PSII requires. After four electrons have been donated by the OEC to PS II, the OEC extracts four electrons from two water molecules, liberating oxygen and dumping four protons into the thylakoid space, thus contributing to the proton gradient. The excited electron from PS II must be passed to another carrier very quickly, lest it decay back to its original state. It does this, giving its electron within picoseconds to pheophytin (Figure $8$). Pheophytin passes the electron on to protein-bound plastoquinones . The first is known as PQA. PQA hands the electron off to a second plastoquinone (PQB), which waits for a second electron and collects two protons to become PQH2, also known as plastoquinol (Figure $9$). PQH2 passes these to the Cytochrome b6f complex (Cb6f) which uses passage of electrons through it to pump protons into the thylakoid space. ATP synthase makes ATP from the proton gradient created in this way. Cb6f drops the electron off at plastocyanin, which holds it until the next excitation process begins with absorption of another photon of light at 700 nm by PS I. Absorption of light at PS I With absorption of a photon of light by PS I, a process begins, that is similar to the process in PS II. PS I gains a positive charge as a result of the loss of an excited electron and pulls the electron in plastocyanin away from it. Meanwhile, the excited electron from PS I passes through an iron-sulfur protein, which gives the electron to ferredoxin (another iron sulfur protein). Ferredoxin then passes the electron off to the last protein in the system known as Ferredoxin:NADP+ oxidoreductase, which gives the electron and a proton to NADP+, creating NADPH. Note that reduction of NADP+ to NADPH requires two electrons and one proton, so the four electrons and two protons from oxidation of water will result in production of two molecules of NADPH. At this point, the light cycle is complete - water has been oxidized, ATP has been created, and NADPH has been made. The electrons have made their way from water to NADPH via carriers in the thylakoid membrane and their movement has released sufficient energy to make ATP. Energy for the entire process came from four photons of light. The two photosystems performing all of this magic are protein complexes that are similar in structure and means of operation. They absorb photons with high efficiency so that whenever a pigment in the photosynthetic reaction center absorbs a photon, an electron from the pigment is excited and transferred to another molecule almost instantaneously. This reaction is called photo-induced charge separation and it is a unique means of transforming light energy into chemical forms. Cyclic photophosphorylation Besides the path described above for movement of electrons through PS I, plants have an alternative route that electrons can take. Instead of electrons going through ferredoxin to form NADPH, they instead take a backwards path through the the proton-pumping b6f complex. This system, called cyclic photophosphorylation (Figure $8$) which generates more ATP and no NADPH, is similar to a system found in green sulfur bacteria. The ability of plants to switch between non-cyclic and cyclic photosystems allows them to make the proper ratio of ATP and NADPH they need for assimilation of carbon in the dark phase of photosynthesis. This ratio turns out to be 3 ATPs to 2 NADPHs. Figure $9$ - Photosystem II of cyanobacteria. Wikipedia Photosynthetic energy The output of the photophosphorylation part of photosynthesis (O2, NADPH, and ATP), of course, is not the end of the process of photosynthesis. For the growing plant, the NADPH and ATP are used to capture carbon dioxide from the atmosphere and convert it (ultimately) into glucose and other important carbon compounds. This, as noted previously, occurs in the Calvin Cycle (see HERE) in what is called the dark phase of the process. The oxygen liberated in the process is a necessary for respiration of all aerobic life forms on Earth. Indeed, it is believed that essentially all of the oxygen in the atmosphere today is the result the splitting of water in photosynthesis over the many eons that the process has existed.
Source: BiochemFFA_5_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy In eukaryotic cells, the vast majority of ATP synthesis occurs in the mitochondria in a process called oxidative phosphorylation. Even plants, which generate ATP by photophosphorylation in chloroplasts, contain mitochondria for the synthesis of ATP through oxidative phosphorylation. Oxidative phosphorylation is linked to a process known as electron transport (Figure 5.14). The electron transport system, located in the inner mitochondrial membrane, transfers electrons donated by the reduced electron carriers NADH and FADH2 (obtained from glycolysis, the citric acid cycle or fatty acid oxidation) through a series of electrons acceptors, to oxygen. As we shall see, movement of electrons through complexes of the electron transport system essentially “charges” a battery that is used to make ATP in oxidative phosphorylation. In this way, the oxidation of sugars and fatty acids is coupled to the synthesis of ATP, effectively extracting energy from food. Chemiosmotic model Dr. Peter Mitchell introduced a radical proposal in 1961 to explain the mechanism by which mitochondria make ATP. It is known as the chemiosmotic hypothesis and has been shown over the years to be correct. Mitchell proposed that synthesis of ATP in mitochondria depends on an electrochemical gradient, across the mitochondrial inner membrane, that arises ultimately from the energy of reduced electron carriers, NADH and FADH2. Electron transport Further, the proposal states that the gradient is created when NADH and FADH2 transfer their electrons to an electron transport system (ETS) located in the inner mitochondrial membrane. Movement of electrons through a series of of electron carriers is coupled to the pumping of protons out of the mitochondrial matrix across the inner mitochondrial membrane into the space between the inner and outer membranes. The result is creation of a gradient of protons whose potential energy can be used to make ATP. Electrons combine with oxygen and protons at the end of the ETS to make water. ATP synthase In oxidative phosphorylation, ATP synthesis is accomplished as a result of protons re-entering the mitochondrial matrix via the transmembrane ATP synthase complex, which combines ADP with inorganic phosphate to make ATP. Central to the proper functioning of mitochondria through this process is the presence of an intact mitochondrial inner membrane impermeable to protons. Tight coupling When this is the case, tight coupling is said to exist between electron transport and the synthesis of ATP (called oxidative phosphorylation). Chemicals which permeabilize the inner mitochondrial membrane to protons cause uncoupling, that is, they allow the protons to leak back into the mitochondrial matrix, rather than through the ATP synthase, so that the movement of electrons through the ETS is no longer linked to the synthesis of ATP. Power plants Mitochondria are called the power plants of the cell because most of a cell’s ATP is produced there in the process of oxidative phosphorylation. The mechanism by which ATP is made in oxidative phosphorylation is one of the most interesting in all of biology. Considerations The process has three primary considerations. The first is electrical – electrons from reduced electron carriers, such as NADH and FADH2, enter the electron transport system via Complex I and II, respectively. As seen in Figure 5.16 and Figure 5.17, electrons move from one complex to the next, not unlike the way they move through an electrical circuit. Such movement occurs a a result of a set of reduction-oxidation (redox) reactions with electrons moving from a more negative reduction potential to a more positive one. One can think of this occurring as a process where carriers “take” electrons away from complexes with lower reduction potential, much the way a bully takes lunch money from a smaller child. In this scheme, the biggest “bully” is oxygen in Complex IV. Electrons gained by a carrier cause it to be reduced, whereas the carrier giving up the electrons is oxidized. Entry of electrons to system Movement of electrons through the chain begins either by 1) transfer from NADH to Complex I (Figure 5.16) or 2) movement of electrons through a covalently bound FADH2 (Figure 5.17) in the membrane-bound succinate dehydrogenase (Complex II). (An alternate entry point for electrons from FADH2 is the Electron Transferring Flavoprotein via the electron-transferring-flavoprotein dehydrogenase, not shown). Traffic cop Both Complex I and II pass electrons to the inner membrane’s coenzyme Q (CoQ - Figures 5.18 & 5.19). In each case, coenzyme Q accepts electrons in pairs and passes them off to Complex III (CoQH2-cytochrome c reductase) singly. Coenzyme Q thus acts as a traffic cop, regulating the flow of electrons through the ETS. Docking station Complex III is a docking station or interchange for the incoming electron carrier (coenzyme Q) and the outgoing carrier (cytochrome c). Movement of electrons from Coenzyme Q to Complex III and then to cytochrome C occurs as a result of what is referred to as the Q-cycle (see below). Complex III acts to ferry electrons from CoQ to cytochrome c. Cytochrome c takes one electron from Complex III and passes it to Complex IV (cytochrome oxidase). Complex IV is the final protein recipient of the electrons. It passes them to molecular oxygen (O2) to make two molecules of water. Making two water molecules requires four electrons, so Complex IV must accept, handle, and pass to molecular oxygen four separate electrons, causing the oxidation state of oxygen to be sequentially changed with addition of each electron. Proton pumping As electrons pass through complexes I, III, and IV, there is a release of a small amount of energy at each step, which is used to pump protons from the mitochondrial matrix (inside of mitochondrion) and deposit them in the intermembrane space (between the inner and outer membranes of the mitochondrion). The effect of this redistribution is to increase the electrical and chemical potential across the membrane. Potential energy
As discussed earlier, electrochemical gradients have potential energy. Students may think of the process as “charging the battery.” Just like a charged battery, the potential arising from the proton differential across the membrane can be used to do things. In the mitochondrion, what the proton gradient does is facilitate the production of ATP from ADP and Pi. This process is known as oxidative phosphorylation, because the phosphorylation of ADP to ATP is dependent on the oxidative reactions occurring in the mitochondria. Having understood the overall picture of the synthesis of ATP linked to the movement of electrons through the ETS, we will take a closer look at the individual components of the ETS. Complex I Complex I (also called NADH:ubiquinone oxidoreductase or NADH dehydrogenase (ubiquinone)) is the electron acceptor from NADH in the electron transport chain and the largest complex found in it. Complex I contains 44 individual polypeptide chains, numerous iron-sulfur centers, a molecule of flavin mononucleotide (FMN) and has an L shape with about 60 transmembrane domains. In the process of electron transport through it, four protons are pumped across the inner membrane into the intermembrane space and electrons move from NADH to coenzyme Q, converting it from ubiquinone (no electrons) to ubiquinol (gain of two electrons). An intermediate form, ubisemiquinone (gain of one electron), is found in the Q-cycle. Electrons travel through the complex via seven primary iron sulfur centers. The best known inhibitor of the complex, rotenone, works by binding to the CoQ binding site. Other inhibitors include ADP-ribose (binds to the NADH site) and piericidin A (rotenone analog). The process of electron transfer through complex I is reversible and when this occurs, superoxide (a reactive oxygen species) may be readily generated. Complex II Complex II (also called succinate dehydrogenase or succinate-coenzyme Q reductase ) is a membrane bound enzyme of the citric acid cycle that plays a role in the electron transport process, transferring electrons from its covalently bound FADH2 to coenzyme Q. The process occurs, as shown in Figure 5.20 and Figure 5.21, with transfer of electrons from succinate to FAD to form FADH2 and fumarate. FADH2, in turn, donates electrons to a relay system of iron-sulfur groups and they ultimately reduce ubiquinone (CoQ) along with two protons from the matrix to ubiquinol. The role of the heme group in the process is not clear. Inhibitors of the process include carboxin, malonate, malate, and oxaloacetate. The role of citric acid cycle intermediates as inhibitors is thought to be due to inhibition of the reversal of the transfer process which can produce superoxide. Coenzyme Q Coenzyme Q (Figure 5.23) is a 1,4 benzoquinone whose name is often given as Coenzyme Q10, CoQ, or Q10. The 10 in the name refers to the number of isoprenyl units it contains that anchor it to the mitochondrial inner membrane. CoQ is a vitamin-like lipid substance found in most eukaryotic cells as a component of the electron transport system. The requirement for CoQ increases with increasing energy needs of cells, so the highest concentrations of CoQ in the body are found in tissues that are the most metabolically active - heart, liver, and kidney. Three forms CoQ is useful because of its ability to carry and donate electrons and particularly because it can exist in forms with two extra electrons (fully reduced - ubiquinol), one extra electron (semi-reduced - ubisemiquinone), or no extra electrons (fully oxidized - ubiquinone). This ability allows CoQ to provide transition between the first part of the electron transport system that moves electrons in pairs and the last part of the system that moves electrons one at a time. Complex III Complex III (also known as coenzyme Q : cytochrome c — oxidoreductase or the cytochrome bc1 complex - Figure 5.24) is the third electron accepting complex of the electron transport system. It is a transmembrane protein with multiple subunits present in the mitochondria of all aerobic eukaryotic organisms and and the cell membrane of almost all bacteria. The complex contains 11 subunits, a 2-iron ferredoxin, cytochromes b and c1 and belongs to the family of oxidoreductase enzymes. It accepts electrons from coenzyme Q in electron transport and passes them off to cytochrome c. In this cycle, known as the Q cycle, electrons arrive from CoQ in pairs, but get passed to cytochrome c individually. In the overall process, two protons are consumed from the matrix and four protons are pumped into the intermembrane space. Movement of electrons through the complex can be inhibited by antimycin A, myxothiazol, and stigmatellin. Complex III is also implicated in creation of superoxide (a reactive oxygen species) when electrons from it leak out of the chain of transfer. The phenomenon is more pronounced when antimycin A is present. Q-cycle In the Q-cycle, electrons are passed from ubiquinol (QH2) to cytochrome c using Complex III as an intermediary docking station for the transfer. Two pair of electrons enter from QH2 and one pair is returned to another CoQ to re-make QH2. The other pair is donated singly to two different cytochrome c molecules. Step one The Q-cycle happens in a two step process. First, a ubiquinol (CoQH2) and a ubiquinone (CoQ) dock at Complex III. Ubiquinol transfers two electrons to Complex III. One electron goes to a docked cytochrome c, reducing it and it exits (replaced by an oxidized cytochrome c). The other goes to the docked uniquinone to create the semi-reduced semiubiquinone (CoQ.-) and leaving behind a ubiquinone, which exits. This is the end of step 1. Step two The gap left behind by the ubiquinone (Q) that departed is replaced by another ubiquinol (QH2). It too donates two electrons to Complex III, which splits them. One goes to the newly docked oxidized cytochrome c, which is reduced and exits. The other goes to the ubisemiquinone. Two protons from the matrix combine with it to make another ubiquinol. It and the ubiquinone created by the electron donation exit Complex III and the process starts again. In the overall process, two protons are consumed from the matrix and four protons are pumped into the intermembrane space. Cytochrome c
Cytochrome c (Figure 5.26) is a small (12,000 Daltons), highly conserved protein, from unicellular species to animals, that is loosely associated with the inner mitochondrial membrane where it functions in electron transport. It contains a heme group which is used to carry a single electron from Complex III to Complex IV. Cytochrome c also plays an important role in apoptosis in higher organisms. Damage to the mitochondrion that results in release of cytochrome c can stimulate assembly of the apoptosome and activation of the caspase cascade that leads to programmed cell death. Complex IV Complex IV, also known as cytochrome c oxidase is a 14 subunit integral membrane protein at the end of the electron transport chain (Figure 5.27). It is responsible for accepting one electron each from four cytochrome c proteins and adding them to molecular oxygen (O2) along with four protons from the mitochondrial matrix to make two molecules of water. Four protons from the matrix are also pumped into the intermembrane space in the process. The complex has two molecules of heme, two cytochromes (a and a3), and two copper centers (called CuA ad CuB). Cytochrome c docks near the CuA and donates an electron to it. The reduced CuA passes the electron to cytochrome a, which turns it over to the a3-CuB center where the oxygen is reduced. The four electrons are thought to pass through the complex rapidly resulting in complete reduction of the oxygen-oxygen molecule without formation of a peroxide intermediate or superoxide, in contrast to previous predictions. Respirasome There has been speculation for many years that a supercomplex of electron carriers in the inner membrane of the mitochondrion may exist in cells with individual carriers making physical contact with each other. This would make for more efficient transfer reactions, minimize the production of reactive oxygen species and be similar to metabolons of metabolic pathway enzymes, for which there is some evidence. Now, evidence appears to be accumulating that complexes I, III, and IV form a supercomplex, which has been dubbed the respirasome1. Oxidative phosphorylation The process of oxidative phosphorylation uses the energy of the proton gradient established by the electron transport system as a means of phosphorylating ADP to make ATP. The establishment of the proton gradient is dependent upon electron transport. If electron transport stops or if the inner mitochondrial membrane’s impermeability to protons is compromised, oxidative phosphorylation will not occur because without the proton gradient to drive the ATP synthase, there will be no synthesis of ATP. ATP synthase The protein complex harvesting energy from the proton gradient and using it to make ATP from ADP is an enzyme that has several names - Complex V, PTAS (Proton Translocating ATP Synthase), and ATP synthase (Figure 5.29). Central to its function is the movement of protons through it (from the intermembrane space back into the matrix). Protons will only provide energy to make ATP if their concentration is greater in the intermembrane space than in the matrix and if ADP is available. It is possible, in some cases, for the concentration of protons to be greater inside the matrix than outside of it. When this happens, the ATP synthase can run backwards, with protons moving from inside to out, accompanied by conversion of ATP to ADP + Pi. This is usually not a desirable circumstance and there are some controls to reduce its occurrence. Normally, ATP concentration will be higher inside of the mitochondrion and ADP concentration be higher outside the mitochondrion. However, when the rate of ATP synthesis exceeds the rate of ATP usage, then ATP concentrations rise outside the mitochondrion and ADP concentrations fall everywhere. This may happen, for example, during periods of rest. It has the overall effect of reducing transport and thus lowering the concentration of ADP inside the matrix. Reducing ADP concentration in the matrix reduces oxidative phosphorylation and has effects on respiratory control (see HERE). Another important consideration is that when ATP is made in oxidative phosphorylation, it is released into the mitochondrial matrix, but must be transported into the cytosol to meet the energy needs of the rest of the cell. This is accomplished by action of the adenine nucleotide translocase, an antiport that moves ATP out of the matrix in exchange for ADP moving into the matrix. This transport system is driven by the concentrations of ADP and ATP and ensures that levels of ADP are maintained within the mitochondrion, permitting continued ATP synthesis. One last requirement for synthesis of ATP from ADP is that phosphate must also be imported into the matrix. This is accomplished by action of the phosphate translocase, which is a symport that moves phosphate into the mitochondrial matrix along with a proton. There is evidence that the two translocases and ATP synthase may exist in a complex, which has been dubbed the ATP synthasome. In summary, the electron transport system charges the battery for oxidative phosphorylation by pumping protons out of the mitochondrion. The intact inner membrane of the mitochondrion keeps the protons out, except for those that re-enter through ATP Synthase. The ATP Synthase allows protons to re-enter the mitochondrial matrix and harvests their energy to make ATP. ATP synthase mechanism In ATP Synthase, the spinning components, or rotor, are the membrane portion (c ring) of the F0 base and the γ-ε stalk, which is connected to it. The γ-ε stalk projects into the F1 head of the mushroom structure. The F1 head contains the catalytic ability to make ATP. The F1 head is hexameric in structure with paired α and β proteins arranged in a trimer of dimers. ATP synthesis occurs within the β subunits. Rotation of γ unit Turning of the γ shaft (caused by proton flow) inside the α-β trimer of the F1 head causes each set of β proteins to change structure slightly into three different forms called Loose, Tight, and Open (L,T,O - Figure 5.31). Each of these forms has a function. The Loose form binds ADP + Pi. The Tight form “squeezes” them together to form the ATP. The Open form releases the ATP into the mitochondrial matrix. Thus, as a result of the proton flow through the ATP synthase, from the intermembrane space into the matrix, ATP is made from ADP and Pi. Respiratory control
When a mitochondrion has an intact inner membrane and protons can only return to the matrix by passing through the ATP synthase, the processes of electron transport and oxidative phosphorylation are said to be tightly coupled. Interdependence In simple terms, tight coupling means that the processes of electron transport and oxidative phosphorylation are interdependent. Without electron transport going on in the cell, oxidative phosphorylation will soon stop. The reverse is also true, because if oxidative phosphorylation stops, the proton gradient will not be dissipated as it is being built by the electron transport system and will grow larger and larger. The greater the gradient, the greater the energy needed to pump protons out of the mitochondrion. Eventually, if nothing relieves the gradient, it becomes too large and the energy of electron transport is insufficient to perform the pumping. When pumping stops, so too does electron transport. ADP dependence Another relevant point is that ATP synthase is totally dependent upon a supply of ADP. In the absence of ADP, the ATP synthase stops functioning and when it stops, so too does movement of protons back into the mitochondrion. With this information, it is possible to understand the link between energy usage and metabolism. The root of this, as noted, is respiratory control. At rest To illustrate these links, let us first consider a person, initially at rest, who then suddenly jumps up and runs away. At first, the person’s ATP levels are high and ADP levels are low (no exercise to burn ATP), so little oxidative phosphorylation is occurring and thus the proton gradient is high. Electron transport is moving slowly, if at all, so it is not using oxygen and the person’s breathing is slow, as a result. Exercise When running starts, muscular contraction, which uses energy, causes ATP to be converted to ADP. Increasing ADP in muscle cells favors oxidative phosphorylation to attempt to make up for the ATP being burned. ATP synthase begins working and protons begin to come back into the mitochondrial matrix. The proton gradient decreases, so electron transport re-starts. Electron transport needs an electron acceptor, so oxygen use increases and when oxygen use increases, the person starts breathing more heavily to supply it. When the person stops running, ATP concentrations get rebuilt by ATP synthase. Eventually, when ATP levels are completely restored, ADP levels fall and ATP synthase stops or slows considerably. With little or no proton movement, electron transport stops because the proton gradient is too large. When electron transport stops, oxygen use decreases and the rate of breathing slows down. Electron transport critical The really interesting links to metabolism occur relative to whether or not electron transport is occurring. From the examples, we can see that electron transport will be relatively slowed when not exercising and more rapid when exercise (or other ATP usage) is occurring. Remember that electron transport is the way in which reduced electron carriers, NADH and FADH2, donate their electrons to the ETS , becoming oxidized to NAD+ and FAD, respectively. Oxidized carriers, such as NAD+ and FAD are needed by catabolic pathways, like glycolysis, the citric acid cycle, and fatty acid oxidation. Anabolic pathways, such as fatty acid/fat synthesis and gluconeogenesis rely on reduced electron carriers, such as FADH2, NADH, and the related carrier, NADPH. Links to exercise High levels of NADH and FADH2 prevent catabolic pathways from operating, since NAD+ and FAD levels will be low and these are needed to accept the electrons released during catabolism by the oxidative processes. Thanks to respiratory control, when one is exercising, NAD+ and FAD levels increase (because electron transport is running), so catabolic pathways that need NAD+ and FAD can function. The electrons lost in the oxidation reactions of catabolism are captured by NAD+ and FAD to yield NADH and FADH2, which then supply electrons to the electron transport system and oxidative phosphorylation to make more needed ATP. Thus, during exercise, cells move to a mode of quickly cycling between reduced electron carriers (NADH/FADH2) and oxidized electron carriers (NAD+/FAD). This allows rapidly metabolizing tissues to transfer electrons to NAD+/FAD and it allows the reduced electron carriers to rapidly become oxidized, allowing the cell to produce ATP. Rest When exercise stops, NADH and FADH2 levels rise (because electron transport is slowing) causing catabolic pathways to slow/stop. If one does not have the proper amount of exercise, reduced carriers remain high in concentration for long periods of time. This means we have an excess of energy and then anabolic pathways, particularly fatty acid synthesis, are favored, so we get fatter. Altering respiratory control One might suspect that altering respiratory control could have some very dire consequences and that would be correct. Alterations can take the form of either inhibiting electron transport/oxidative phosphorylation or uncoupling the two . These alterations can be achieved using compounds with specific effects on particular components of the system. All of the chemicals described here are laboratory tools and should never be used by people. The first group for discussion are the inhibitors. In tightly coupled mitochondria, inhibiting either electron transport or oxidative phosphorylation has the effect of inhibiting the other one as well. Electron transport inhibitors Common inhibitors of electron transport include rotenone and amytal, which stop movement of electrons past Complex I, malonate, malate, and oxaloacetate, which inhibit movement of electrons through Complex II, antimycin A which stops movement of electrons past Complex III, and cyanide, carbon monoxide, azide, and hydrogen sulfide, which inhibit electron movement through Complex IV (Figure 5.33). All of these compounds can stop electron transport directly (no movement of electrons) and oxidative phosphorylation indirectly (proton gradient will dissipate). While some of these compounds are not commonly known, almost everyone is aware of the hazards of carbon monoxide and cyanide, both of which can be lethal. ATP synthase inhibitor
It is also possible to use an inhibitor of ATP synthase to stop oxidative phosphorylation directly (no ATP production) and electron transport indirectly (proton gradient not relieved so it becomes increasingly difficult to pump protons out of matrix). Oligomycin A (Figure 5.34) is an inhibitor of ATP synthase. Rotenone Rotenone, which is a plant product, is used as a natural insecticide that is permitted for organic farming. When mitochondria are treated with this, electron transport will stop at Complex I and so, too, will the pumping of protons out of the matrix. When this occurs, the proton gradient rapidly dissipates, stopping oxidative phosphorylation as a consequence. There are other entry points for electrons than Complex I, so this type of inhibition is not as serious as using inhibitors of Complex IV, since no alternative route for electrons is available. It is for this reason that cyanide, for example, is so poisonous. 2,4-DNP Imagine a dam holding back water with a turbine generating electricity through which water must flow. When all water flows through the turbine, the maximum amount of electricity can be generated. If one pokes a hole in the dam, though, water will flow through the hole and less electricity will be created. The generation of electricity will thus be uncoupled from the flow of water. If the hole is big enough, the water will all drain out through the hole and no electricity will be made. Bypassing ATP synthase Imagine, now, that the proton gradient is the equivalent of the water, the inner membrane is the equivalent of the dam and the ATP synthase is the turbine. When protons have an alternate route, little or no ATP will be made because protons will pass through the membrane’s holes instead of spinning the turbine of ATP synthase. It is important to recognize, though, that uncoupling by 2,4 DNP works differently from the electron transport inhibitors or the ATP synthase inhibitor. In those situations, stopping oxidative phosphorylation resulted in indirectly stopping electron transport, since the two processes were coupled and the inhibitors did not uncouple them. Similarly, stopping electron transport indirectly stopped oxidative phosphorylation for the same reason. Such is not the case with 2,4 DNP. Stopping oxidative phosphorylation by destroying the proton gradient allows electron transport to continue unabated (it actually stimulates it), since the proton gradient cannot build no matter how much electron transport runs. Consequently, electron transport runs like crazy but oxidative phosphorylation stops. When that happens, NAD+ and FAD levels rise, and catabolic pathways run unabated with abundant supplies of these electron acceptors. The reason such a scenario is dangerous is because the body is using all of its nutrient resources, but no ATP is being made. Lack of ATP leads to cellular (and organismal) death. In addition, the large amounts of heat generated can raise the temperature of the body to unsafe levels. Thermogenin One of the byproducts of uncoupling electron transport is the production of heat. The faster metabolic pathways run, the more heat is generated as a byproduct. Since 2,4 DNP causes metabolism to speed up, a considerable amount of heat can be produced. Controlled uncoupling is actually used by the body in special tissues called brown fat. In this case, brown fat cells use the heat created to help thermoregulate the temperature of newborn children. Permeabilization of the inner membrane is accomplished in brown fat by the synthesis of a protein called thermogenin (also known as uncoupling protein). Thermogenin binds to the inner membrane and allows protons to pass through it, thus bypassing the ATP synthase. As noted for 2,4 DNP, this results in activation of catabolic pathways and the more catabolism occurs, the more heat is generated. Dangerous drug In uncoupling, whether through the action of an endogenous uncoupling protein or DNP, the energy that would have normally been captured in ATP is lost as heat. In the case of uncoupling by thermogenin, this serves the important purpose of keeping newborn infants warm. But in adults, uncoupling merely wastes the energy that would have been harvested as ATP. In other words, it mimics starvation, even though there is plenty of food, because the energy is dissipated as heat. This fact, and the associated increase in metabolic rate, led to DNP being used as a weight loss drug in the 1930s. Touted as an effortless way to lose weight without having to eat less or exercise more, it was hailed as a magic weight loss pill. It quickly became apparent, however, that this was very dangerous. Many people died from using this drug before laws were passed to ban the use of DNP as a weight loss aid. Alternative oxidase Another approach to generating heat that doesn’t involve breaking respiratory control is taken by some fungi, plants, and protozoa. They use an alternative electron transport. In these organisms, there is an enzyme called alternative oxidase (Figure 5.36). Alternative oxidase is able to accept electrons from CoQ and pass them directly to oxygen. The process occurs in coupled mitochondria. Its mechanism of action is to reduce the yield of ATP, since fewer protons are being pumped per reduced electron carrier. Thus NAD+ concentrations increase, oxygen consumption increases, and the efficiency of ATP production decreases. Organisms using this method must activate catabolic pathways by the increase in NAD+ concentration. This, in turn produces quantities of NADH and FADH2 necessary to make sufficient amounts of ATP. The byproduct of this increased catabolism is more heat. Not surprisingly, the alternative oxidase pathway can be activated by cold temperatures. Energy efficiency
Cells are not 100% efficient in energy use. Nothing we know is. Consequently, cells do not get as much energy out of catabolic processes as they put into anabolic processes. A good example is the synthesis and breakdown of glucose, something liver cells are frequently doing. The complete conversion of glucose to pyruvate in glycolysis (catabolism) yields two pyruvates plus 2 NADH plus 2 ATPs. Conversely, the complete conversion of two pyruvates into glucose by gluconeogenesis (anabolism) requires 4 ATPs, 2 NADH, and 2 GTPs. Since the energy of GTP is essentially equal to that of ATP, gluconeogenesis requires a net of 4 ATPs more than glycolysis yields. This difference must be made up in order for the organism to meet its energy needs. It is for this reason that we eat. In addition, the inefficiency of our capture of energy in reactions results in the production of heat and helps to keep us warm, as noted. You can read more about glycolysis (HERE) and gluconeogenesis (HERE). Metabolic controls of energy It is also noteworthy that cells do not usually have both catabolic and anabolic processes for the same molecules occurring simultaneously inside of them (for example, breakdown of glucose and synthesis of glucose) because the cell would see no net production of anything but heat and a loss of ATPs with each turn of the cycle. Such cycles are called futile cycles and cells have controls in place to limit the extent to which they occur. Since futile cycles can, in fact, yield heat, they are used as sources of heat in some types of tissue. Brown adipose tissue of mammals uses this strategy, as described earlier. See also HERE for more on heat generation with a futile cycle. Reactive oxygen species Endogenous production of ROS is directed towards intracellular signaling (H2O2 and nitric oxide, for example) and defense. Many cells, for example, have NADPH oxidase (Figure 5.38) embedded in the exterior portion of the plasma membranes, in peroxisomes, and endoplasmic reticulum. It produces superoxides in the reaction below to kill bacteria . In the immune system, cells called phagocytes engulf foreign cells and then use ROS to kill them. ROS can serve as signals for action. In zebrafish, damaged tissues have increased levels of H2O2 and this is thought to be a signal for white blood cells to converge on the site. In fish lacking the genes to produce hydrogen peroxide, white blood cells do not converge at the damage site. Sources of hydrogen peroxide include peroxisomes, which generate it as a byproduct of oxidation of long chain fatty acids. Aging Reactive oxygen species are at the heart of the free radical theory of aging, which states that organisms age due to the accumulation of damage from free radicals in their cells. In yeast and Drosophila, there is evidence that reducing oxidative damage can increase lifespan. In mice, increasing oxidative damage decreases life span, though in Caenorhabditis, blocking production of superoxide dismutase actually increases lifespan, so the role of ROS in aging is not completely clear. It is clear, though, that accumulation of mitochondrial damage is problematic for individual cells. Bcl-2 proteins on the surface of mitochondria monitor damage and if they detect it, will activate proteins called Bax to stimulate the release of cytochrome c from the mitochondrial membrane, stimulating apoptosis (programmed cell death). Eventually the dead cell will be phagocytosed. A common endogenous source of superoxide is the electron transport chain. Superoxide can be produced when movement of electrons into and out of the chain don’t match well. Under these circumstances, semi-reduced CoQ can donate an electron to O2 to form superoxide (O2-). Superoxide can react with many molecules, including DNA where it can cause damage leading to mutation. If it reacts with the aconitase enzyme, ferrous iron (Fe++) can be released which, in turn, can react in the Fenton reaction to produce another reactive oxygen species, the hydroxyl radical (Figure 5.39) . Countering the effects of ROS are enzymes, such as catalase, superoxide dismutase, and anti-oxidants, such as glutathione and vitamins C and E. Glutathione protects against oxidative damage by being a substrate for the enzyme glutathione peroxidase. Glutathione peroxidase catalyzes the conversion of hydrogen peroxide to water (next page). Catalase 2 H2O2 <=> 2 H2O + O2 The enzyme, which employs four heme groups in its catalysis, works extremely rapidly, converting up to 40,000,000 molecules of hydrogen peroxide to water and oxygen per enzyme per second. It is abundantly found in peroxisomes. In addition to catalase’s ability to break down hydrogen peroxide, the enzyme can also use hydrogen peroxide to oxidize a wide variety organic compounds, including phenols, formic acid, formaldehyde, acetaldehyde, and alcohols, but with much lower efficiency. Health The importance of catalase for health is uncertain. Mice deficient in the enzyme appear healthy and humans with low levels of the enzyme display few problems. On the other hand, mice engineered to produce higher levels of catalase, in at least one study, lived longer. The ability of organisms to live with lower levels or no catalase may arise from another group of enzymes, the peroxiredoxins, which also act on hydrogen peroxide and may make up for lower quantities of catalase. Last, there is evidence that reduced levels of catalase with aging may be responsible for the graying of hair. Higher levels of H2O2 with reduced catalase results in a bleaching of hair follicles. Superoxide dismutase Another important enzyme for protection against reactive oxygen species is superoxide dismutase (SOD), which is found, like catalase, in virtually all organisms living in an oxygen environment. Superoxide dismutase, also like catalase, has a very high Kcat value and, in fact, has the highest Kcat/Km known for any known enzyme. It catalyzes the reactions at the top of the next column (superoxides shown in red): The enzyme thus works by a ping-pong (double displacement) mechanism (see HERE), being converted between two different forms. The hydrogen peroxide produced in the second reaction is easily handled by catalase and is also less harmful than superoxide, which can react with nitric oxide (NO) to form very toxic peroxynitrite ions (Figure 5.43). Peroxynitrite has negative effects on cells, as shown in Figure 5.45.
In addition to copper, an ion of Zn++ is also bound by the enzyme and likely plays a role in the catalysis. Forms of superoxide dismutase that use manganese, nickel, or iron are also known and are mostly found in prokaryotes and protists, though a manganese SOD is found in most mitochondria. Copper/zinc enzymes are common in eukaryotes. Three forms of superoxide dismutase are found in humans and localized to the cytoplasm (SOD1 - Figure 5.45), mitochondria (SOD2 - Figure 5.46), and extracellular areas (SOD3 - Figure 5.47). Mice lacking any of the three forms of the enzyme are more sensitive to drugs, such as paraquat. Deficiency of SOD1 in mice leads to hepatocellular carcinoma and early loss of muscle tissue related to aging. Drosophila lacking SOD2 die before birth and those lacking SOD1 prematurely age. In humans, superoxide dismutase mutations are associated with the genetically-linked form of Amyotrophic Lateral Sclerosis (ALS) and over-expression of the gene is linked to neural disorders associated with Down syndrome. Mixed function oxidases Other enzymes catalyzing reactions involving oxygen include the mixed function oxidases. These enzymes use molecular oxygen for two different purposes in one reaction. The mixed function part of the name is used to indicate reactions in which two different substrates are being oxidized simultaneously. Monooxygenases are examples of mixed function oxidases. An example of a mixed function oxidase reaction is shown below. AH + BH2 + O2 <=> AOH + B + H2O In this case, the oxygen molecule has one atom serve as an electron acceptor and the other atom is added to the AH, creating an alcohol. Cytochrome P450 enzymes Cytochrome P450 enzymes (called CYPs) are family of heme-containing mixed function oxidase enzymes found in all domains of life. Over 21,000 CYP enzymes are known. The most characteristic reaction catalyzed by these enzymes follows Monooxygenase reactions such as this are relatively rare in the cell due to their use of molecular oxygen. CYPs require an electron donor for reactions like the one shown here and frequently require a protein to assist in transferring electrons to reduce the heme iron. There are six different classes of P450 enzymes based on how they get electrons 1. Bacterial P450 - electrons from ferredoxin reductase and ferredoxin 2. Mitochondrial P450 - electrons from adrenodoxin reductase and adrenodoxin 3. CYB5R/cyb5 - electrons come from cytochrome b5 4. FMN/Fd - use a fused FMN reductase 5. Microsomal P450 - NADPH electrons come via cytochrome P450 reductase or from cytochrome b5 and cytochrome b5 reductase 6. P450 only systems - do not require external reducing power The CYP genes are abundant in humans and catalyze thousand of reactions on both cellular and extracellular chemicals. There are 57 human genes categorized into 18 different families of enzymes. Some CYPs are specific for one or a few substrates, but others can act on many different substrates. CYP enzymes are found in most body tissues and perform important functions in synthesis of steroids (cholesterol, estrogen, testoterone, Vitamin D, e.g.), breakdown of endogenous compounds (bilirubin), and in detoxification of toxic compounds including drugs. Because they act on many drugs, changes in CYP activity can produce unexpected results and cause problems with drug interactions. Bioactive compounds, for example, in grapefruit juice, can inhibit CYP3A4 activity, leading to increased circulating concentrations of drugs that would normally have been acted upon by CYP3A4. This is the reason that patients prescribed drugs that are known to be CYP3A4 substrates are advised to avoid drinking grapefruit juice while under treatment. St. Johns Wort, an herbal treatment, on the other hand, induces CYP3A4 activity, but inhibits CYP1A1, CYP1B1, and CYP2D6. Tobacco smoke induces CYP1A2 and watercress inhibits CYP2E1. Cytochromes Cytochromes are heme-containing proteins that play major roles in the process of electron transport in the mitochondrion and in photosynthesis in the chloroplast. They exist either as monomers (cytochrome c) or as subunits within large redox complexes (Complex III and Complex IV of electron transport. An atom of iron at the center of the heme group plays a central role in the process, flipping between the ferrous (Fe++) and ferric (Fe+++) states as a result of the movement of electrons through it. There are several different cytochromes. Cytochrome c (Figure 5.47) is a soluble protein loosely associated with the mitochondrion. Cytochromes a and a3 are found in Complex IV. Complex III has cytochromes b and c1 and the plastoquinol-plastocyanin reductase of the chloroplast contains cytochromes b6 and f. Another important class of enzymes containing cytochromes is the cytochrome P450 oxidase group (see above). They get their name from the fact that they absorb light at 450 nm when their heme iron is reduced. Iron-Sulfur Proteins Iron-sulfur proteins contain iron-sulfur clusters in a variety of formats, including sulfide-linked di-, tri-, and tetrairon centers existing in different oxidation states (Figures 5.48 & 5.49). The clusters play a variety of roles, but the best known ones are in electron transport where they function in the redox reactions involved in the movement of electrons. Complexes I and Complex II contain multiple Fe-S centers. Iron-sulfur proteins, though, have many other roles in cells. Aconitase uses an iron-sulfur center in its catalytic action and the ability of the enzyme to bind iron allows it to function as a barometer of iron concentration in cells. Iron-sulfur centers help to generate radicals in enzymes using S-Adenosyl Methionine (SAM) and can serve as a source of sulfur in the synthesis of biotin and lipoic acid. Some iron-sulfur proteins even help to regulate gene expression. Ferredoxin
Ferredoxins are iron-sulfur containing proteins performing electron transfer in a wide variety of biological systems and processes. They include roles in photosynthesis in chloroplasts. Ferredoxins are classified structurally by the iron-sulfur clustered centers they contain. Fe2S2 clusters (Figure 5.50) are found in chloroplast membranes and can donate electrons to glutamate synthase, nitrate reductase, and sulfite reductase and serve as electron carriers between reductase flavoproteins and bacterial dioxygenase systems. Adrenodoxin is a soluble human Fe2S2 ferredoxin (also called ferredoxin 1) serving as an electron carrier (to cytochrome P450) in mitochondrial monooxygenase systems. Fe4S4 ferredoxins are subdivided as low and high potential ferredoxins, with the latter ones functioning in anaerobic electron transport chains. Ferritin Ferritin is an intracellular iron-storage protein found in almost all living organisms, from bacteria to higher plants and animals. It is a globular protein complex with 24 subunits and is the primary intracellular iron-storage protein in eukaryotes and prokaryotes. Ferritin functions to keep iron in a soluble and non-toxic form. Its ability to safely store iron and release it in a controlled fashion allow it to act like the prime iron buffer and solubilizer in cells - keeping the concentration of free iron from going to high or falling too low. Ferritin is located in the cytoplasm in most tissues, but it is also found in the serum acting as an iron carrier. Ferritin that doesn’t contain any iron is known as apoferritin. Monoamine oxidases Monoamine oxidases are enzymes that catalyze the oxidative deamination of monoamines, such as serotonin, epinephrine, and dopamine. Removal of the amine with oxygen results in the production of an aldehyde and ammonia. The enzymes are found inside and outside of the central nervous system. There are two types of monoamine oxidase enzymes - MAO-A and MAO-B. MAO-A is particularly important for oxidizing monoamines consumed in the diet. Both MAO-A and MAO-B play important roles in inactivating monoaminergic neurotransmitters. Both enzymes act on dopamine, tyramine (Figure 5.50), and tryptamine. MAO-A is the primary enzyme for metabolizing melatonin, serotonin, norepinephrine, and epinephrine, while MAO-B is the primary enzyme for phenethylamine (Figure 5.51) and benzylamine. MAO-B levels have been reported to be considerably reduced with tobacco usage. Actions of monoamine oxidases thus affects levels of neurotransmitters and consequently are thought to play roles in neurological and/or psychiatric disorders. Aberrant levels of MAOs have been linked to numerous psychological problems, including depression, attention deficit disorder (ADD), migraines, schizophrenia, and substance abuse. Medications targeting MAOs are sometimes used to treat depression as a last resort - due to potential side effects. Excess levels of catecholamines, such as epinephrine, norepinephrine, and dopamine, can result in dangerous hypertension events. DNA damage theory of aging The DNA Damage Theory of Aging is based on the observation that, over time, cells are subject to extensive oxidative events. As already noted, these afford opportunities for the formation of ROS that can damage cellular molecules, and it follows that accumulation of such damage, especially to the DNA would be deleterious to the cell. The build-up of DNA damage could, thus, be responsible for the changes in gene expression that we associate with aging. Numerous damage events The amount of DNA damage that can occur is considerable. In mice, for example, it is estimated that each cell experiences 40,000 to 150,000 damage events per day. The damage, which happens to nuclear as well as to mitochondrial DNA, can result in apoptosis and/or cellular senescence. DNA repair systems, of course, protect against damage to DNA, but over time, unrepairable damage may accumulate. Oxidative damage DNA damage can occur in several ways. Oxidation can damage nucleotides and alter their base-pairing tendencies. Oxidation of guanine by reactive oxygen species, for example, can produce 8-oxo-guanine (Figures 5.52 and 5.53). This oxidized nucleobase commonly produced lesion in DNA arising from action of reactive oxygen species like superoxides. 8-oxoguanine is capable of forming a stable base pairing interaction within a DNA duplex with adenine, potentially giving rise to a mutation when DNA replication proceeds. 8-oxoguanine can be repaired if recognized in time by a DNA glycosylase, which acts to clip out the damaged base and it can then be replaced by the proper one. Polycyclic aromatic hydrocarbons from cigarette smoke, diesel exhaust, or overcooked meat can covalently bind to DNA and, if unrepaired, lead to mutation. Chemical damage to DNA can result in broken or cross-linked DNAs. Diseases of DNA repair The importance of DNA repair in the aging process is made clear by diseases affecting DNA repair that lead to premature aging. These include Werner syndrome, for whom the life expectancy is 47 years. It arises as a result of loss of two enzymes in base excision repair. People suffering from Cockayne syndrome have a life expectancy of 13 years due to mutations that alter transcription-coupled nucleotide excision repair, which is an important system for fixing oxidative damage. Further, the life expectancies of 13 species of mammalian organisms correlates with the level of expression of the PARP DNA repair-inducing protein. Interestingly, people who lived past the age of 100 had a higher level of PARP than younger people in the population. Antioxidants There is a growing interest in the subject of antioxidants because of health concerns raised by our knowledge of problems created as a result of spontaneous oxidation of biomolecules by Reactive Oxygen Species (ROS), such as superoxide. Antioxidants have the chemical property of protecting against oxidative damage by being readily oxidized themselves, preferentially to other biomolecules. Biologically, cells have several lines of antioxidant defense. They include molecules, such as vitamins C, A, and E, glutathione, and enzymes that destroy ROS such as superoxide dismutase, catalase, and peroxidases. Health effects
Oxidation by ROS is mutagenic and has been linked to atherosclerosis. Nonetheless, randomized studies of oral supplementation of various vitamin combinations have shown no protective effect against cancer and supplementation of Vitamin E and selenium has revealed no decrease in the risk of cardiovascular disease. Further, no reduction in mortality rates as a result of supplementation with these materials has been found, so the protective effects, if any, of antioxidants on ROS in human health remain poorly understood. Glutathione The thiol group of cysteine is a reducing agent that reduces disulfide bonds to sulfhydryls in cytoplasmic proteins. This, in turn, is the bridge when two glutathiones get oxidized and form a disulfide bond with each other (Figure 5.56). Glutathione’s two oxidative states are abbreviated as follows: GSH (reduced) and GSSG (oxidized). Disulfide-joined glutathiones can be separated by reduction of their bonds with glutathione reductase, using electrons from NADPH for the reduction. Non-ribosomal synthesis Glutathione is not made by ribosomes. Rather, two enzymes catalyze its synthesis. The enzyme γ-glutamylcysteine synthetase catalyzes the joining of the glutamate to the cysteine and then glutathione synthetase catalyzes the peptide bond formation between the cysteine and the glycine. Each step requires energy from ATP. Essential for life Glutathione is important for life. Mice lacking the first enzyme involved in its synthesis in the liver die in the first month after birth. In healthy cells, 90% of glutathione is in the GSH state. Higher levels of GSSG correspond to cells that are oxidatively stressed. Besides reducing disulfide bonds in cells, glutathione is also important for the following: • Neutralization of free radicals and reactive oxygen species. • Maintenance of exogenous antioxidants such as vitamins C and E in their reduced forms. Regulation of the nitric oxide cycle Resveratrol Some data indicates resveratrol may improve the functioning of mitochondria. It also acts as an antioxidant and causes concentration of another anti-oxidant, glutathione, to increase. The compound appears to induce expression of manganese superoxide dismutase (protects against reactive oxygen species) and inhibits several phosphodiesterases. This causes an increase in cAMP which results in increases in oxidation of fatty acids, mitochondria formation, gluconeogenesis, and glycogen breakdown. It has been claimed to be the cause of the French Paradox in which drinking of red wine is supposed to give protection for the cardiovascular system. Research data is lacking in support of the claim, however. Resveratrol is known to activate Sirtuin proteins, which play roles in gene inactivation. Summary In summary, energy is needed for cells to perform the functions that they must carry out in order to stay alive. At its most basic level, this means fighting a continual battle with entropy, but it is not the only need for energy that cells have. References 1. Winge, D.R., Mol Cell Biol. 2012 Jul; 32(14): 2647–2652. doi: 10.1128/MCB.00573-12 Energy: Electron Transport & Oxidative Phosphorylation 429 YouTube Lectures by Kevin HERE & HERE YouTube Lectures by Kevin HERE & HERE 430 Figure 5.14 - Overview of electron transport (bottom left and top right) and oxidative phosphorylation (top left - yellow box) in the mitochondrion 431 Figure 5.15 - Loss of electrons by NADH to form NAD+. Relevant reactions occur in the top ring of the molecule. 432 Figure 5.16 - Flow of electrons from NADH into the electron transport system. Entry is through complex I Image by Aleia Kim Figure 5.17 - Flow of electrons from FADH2 into the electron transport chain. Entry is through complex II. Image by Aleia Kim Interactive Learning Module HERE 433 Figure 5.18 - Complex I embedded in the inner mitochondrial membrane. The mitochondrial matrix at at the top Wikipedia 434 Figure 5.19 - Complex II embedded in inner mitochondrial membrane. Matrix is up. Wikipedia YouTube Lectures by Kevin HERE & HERE 435 Figure 5.20 - Movement of electrons through complex I from NADH to coenzyme Q. The mitochondrial matrix is at the bottom Image by Aleia Kim Figure 5.21 - Movement of electrons from succinate through complex II (A->B->C->D->Q). Mitochondrial matrix on bottom. Image by Aleia Kim 436 Figure 5.22 - Complex II in inner mitochondrial membrane showing electron flow. Matrix is up. Wikipedia Figure 5.23 - Coenzyme Q 437 Movie 5.2 - The Q-cycle Wikipedia Figure 5.24 - The Q-Cycle Image by Aleia Kim Figure 5.24 - Complex III Wikipedia 438 YouTube Lectures by Kevin HERE & HERE Figure 5.25 - The Q-cycle. Matrix is down. Image by Aleia Kim 439 Figure 5.26 - Movement of electrons and protons through complex IV. Matrix is down Image by Aleia Kim Figure 5.25 - Cytochrome c with bound heme Group Wikipedia 440 Figure 5.27 - Mitochondrial anatomy. Electron transport complexes and ATP synthase are embedded in the inner mitochondrial membrane Image by Aleia Kim 441 Figure 5.28 - ATP synthase. Protons pass from intermembrane space (top) through the complex and exit in the matrix (bottom). Image by Aleia Kim Interactive Learning Module HERE 442 Movie 5.3 - ATP Synthase - ADP + Pi (pink) and ATP (red). The view is end-on from the cytoplasmic side viewing the β subunits Movie 5.3 - ATP Synthase - ADP + Pi (pink) and ATP (red). The view is end-on from the cytoplasmic side viewing the β subunits 443 Figure 5.29 - Important structural features of the ATP synthase Image by Aleia Kim 444 Figure 5.30 - Loose (L), Tight (T), and Open (O) structures of the F1 head of ATP synthase. Change of structure occurs by rotation of γ-protein (purple) in center as a result of proton movement. Individual α and β units do not rotate Image by Aleia Kim 445 Figure 5.31 - Respiration overview in eukaryotic cells Wikipedia YouTube Lectures by Kevin HERE & HERE 446 Rest ATP High / ADP Low Oxidative Phosphorylation Low Electron Transport Low Oxygen Use Low NADH High / NAD+ Low Citric Acid Cycle Slow Exercise ATP Low / ADP High Oxidative Phosphorylation High Electron Transport High Oxygen Use High NADH Low / NAD+ High Citric Acid Cycle Fast Interactive Learning Module HERE 447 Figure 5.32 - Three inhibitors of electron transport Image by Aleia Kim 448 Figure 5.33 - Oligomycin A - An inhibitor of ATP synthase
Figure 5.34 - 2,4 DNP - an uncoupler of respiratory control 449 In Cells With Tight Coupling O2 use depends on metabolism NAD+ levels vary with exercise Proton gradient high with no exercise Catabolism depends on energy needs ETS runs when OxPhos runs and vice versa In Cells That Are Uncoupled O2 use high NAD+ Levels high Little or no proton gradient Catabolism high OxPhos does not run, but ETS runs rapidly YouTube Lectures by Kevin HERE & HERE 450 451 Figure 5.35 - Alternative oxidase (AOX) of fungi, plants, and protozoa bypasses part of electron transport by taking electrons from CoQ and passing them to oxygen. 452 Figure 5.36 - Structure of an oxygen free radical Wikipedia NADPH + 2O2 NADP+ + 2O2− + H+ Figure 5.37 - Three sources of reactive oxygen species (ROS) in cells Wikipedia 453 454 YouTube Lectures by Kevin HERE & HERE Figure 5.38 A hydroxyl radical Wikipedia 455 Reduced Glutathione (GSH) + H2O2 Oxidized Glutathione (GSSG) + H2O Figure 5.40 - Detoxifying reactive oxygen species Figure 5.39 - Catalase 456 1. O2- + Enzyme-Cu++ O2 + Enzyme-Cu+ 2. O2- + Enzyme-Cu+ + 2H+ H2O2 + Enzyme-Cu++ Figure 5.41 - SOD2 of humans Figure 5.42 3 - Peroxynitrite Ion Figure 5.44 - SOD1 of humans Wikipedia Figure 5.45 - SOD3 of humans 457 Figure 5.43 - Peroxynitrite’s effects on cells lead to necrosis or apoptosis Wikipedia 458 RH + O2 + NADPH + H+ ROH + H2O + NADP+ 459 Figure 5.46 - Cytochrome c with its heme group 460 YouTube Lectures by Kevin HERE & HERE Figure 5.47 - Fe2S2 Cluster Figure 5.48 - Redox reactions for Fe4S4 clusters 461 Figure 5.49 - Tyramine Figure 5.50 - Phenethylamine 462 Figure 5.51 - Guanine and 8-oxo-guanine Figure 5.52 - Adenine-8-oxo-guanine base pair. dR = deoxyribose 463 Figure 5.53 - Good antioxidant sources 464 Figure 5.55 - Oxidized glutathiones (GSSG) joined by a disulfide bond Wikipedia Figure 5.54 - Structure of reduced glutathione (GSH) 465 Figure 5.56 - Resveratrol YouTube Lectures by Kevin HERE & HERE 466 Graphic images in this book were products of the work of several talented students. Links to their Web pages are below Click HERE for Martha Baker’s Web Page Click HERE for Pehr Jacobson’s Web Page Click HERE for Aleia Kim’s Web Page Click HERE for Penelope Irving’s Web Page Problem set related to this section HERE Point by Point summary of this section HERE To get a certificate for mastering this section of the book, click HERE Kevin Ahern’s free iTunes U Courses - Basic / Med School / Advanced Biochemistry Free & Easy (our other book) HERE / Facebook Page Kevin and Indira’s Guide to Getting into Medical School - iTunes U Course / Book To see Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 To register for Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 Biochemistry Free For All Facebook Page (please like us) Kevin Ahern’s Web Page / Facebook Page / Taralyn Tan’s Web Page Kevin Ahern’s free downloads HERE OSU’s Biochemistry/Biophysics program HERE OSU’s College of Science HERE Oregon State University HERE Email Kevin Ahern / Indira Rajagopal / Taralyn Tan I'm a little mitochondrion Who gives you energy I use my proton gradient To make the ATPs He's a little mitochondrion Who gives us energy He uses proton gradients To make some ATPs Electrons flow through Complex II To traffic cop Co-Q Whenever they arrive there in An FADH-two Electrons flow through Complex II To traffic cop Co-Q Whenever they arrive there in An FADH-two Tightly coupled is my state Unless I get a hole Created in my membrane by Some di-ni-tro-phe-nol Yes tightly coupled is his state Unless he gets a hole Created in his membrane by Some di-ni-tro-phenol Both rotenone and cyanide Stop my electron flow And halt the calculation of My "P" to "O" ratio Recording by Tim Karplus Lyrics by Kevin Ahern Recording by Tim Karplus Lyrics by Kevin Ahern I’m a Little Mitochondrion To the tune of “I’m a Lumberjack” Metabolic Melodies Website HERE In the catabolic pathways that our cells employ Oxidations help create the ATP While they lower Gibbs free energy Thanks to enthalpy If a substrate is converted from an alcohol To an aldehyde or ketone it is clear Those electrons do not disappear They just rearrange – very strange N-A-D is in my ears and in my eyes Help-ing mol-e-cules get oxidized Making N-A-D-H then And the latter is a problem anaerobically ‘Cuz accumulations of it muscles hate They respond by using pyruvate To produce lactate Catalyzing is essential for the cells to live So the enzymes grab their substrates eagerly If they bind with high affinity Low Km you see, just as me N-A-D is in my ears and in my eyes Help-ing mol-e-cules get oxidized Making N-A-D-H then N-A-D To the tune of “Penny Lane” Metabolic Melodies Website HERE Recorded by Tim Karplus Lyrics by Kevin Ahern Recorded by Tim Karplus Lyrics by Kevin Ahern When oxygen’s electrons all are in the balanced state There’s twelve of them for oh-two. The molecule is great But problems sometimes happen on the route to complex IV Making reactive species that the cell cannot ignore Oh superoxide dismutase is super catalytic Keeping cells from getting very peroxynitritic Faster than a radical, its actions are terrific Superoxide dismutase is super catalytic Enzyme, enzyme deep inside Blocking all the bad oxides The enzyme’s main advantage is it doesn’t have to wait By binding superoxide in a near-transition state It turns it to an oxygen in mechanism one Producing “h two oh two” when the cycle is all done Oh superoxide dismutase you’re faster than all them You’ve got the highest ratio of kcat over KM This means that superoxide cannot cause too much mayhem Superoxide dismutase is faster than all them Superoxide dismutase Stopping superoxide’s ways The enzyme’s like a ping-pong ball that mechanistic-ly Bounces between two copper states, plus one and two you see So S-O-D behaves just like an anti-oxidant Giving as much protection as a cell could ever want Oh superoxide dismutase, the cell’s in love with you Because you let electron transport do what it must do Without accumulation of a radical oh two Superoxide dismutase - that’s why a cell loves you Superoxide Dismutase To the tune of “Supercalifragilistiexpialidocious” Metabolic Melodies Website HERE Lyrics by Kevin Ahern No Recording Yet For This Song
• 6.1: Metabolism - Sugars • 6.2: Citric Acid Cycle & Related Pathways The primary catabolic pathway in the body is the citric acid cycle because it is here that oxidation to carbon dioxide occurs for breakdown products of the cell’s major building blocks - sugars, fatty acids, and amino acids. The pathway is cyclic and thus, does not really have a starting or ending point. All of the reactions occur in mitochondria, though one enzyme is embedded in the organelle’s inner membrane. Cells may use a subset of the reactions of the cycle to produce a desired molecule. • 6.3: Fats and Fatty Acids There is a tremendous amount of interest in the metabolism of fat and fatty acids. Fat is the most important energy storage form of animals, storing considerably more energy per carbon than carbohydrates, but its insolubility in water requires the body to package it specially for transport. Surprisingly, fat/fatty acid metabolism is not nearly as tightly regulated as that of carbohydrates. Neither are the metabolic pathways of breakdown and synthesis particularly complicated, either. • 6.4: Other Lipids Sugars are the building blocks of carbohydrates, amino acids are the building blocks of proteins and nucleotides are the building blocks of the nucleic acids - DNA and RNA. Another crucial building block is acetyl-CoA, which is used to build many lipid substances, including fatty acids, cholesterol, fat soluble vitamins, steroid hormones, prostaglandins, endocannabinoids, and the bile acids. Indeed, acetyl-CoA goes into more different classes of molecule than any other building block. • 6.5: Amino Acids and the Urea Cycle In contrast to some of the metabolic pathways described to this point, amino acid metabolism is not a single pathway. The 20 amino acids have some parts of their metabolism that overlap with each other, but others are very different from the rest. In discussing amino acid metabolism, we will group metabolic pathways according to common metabolic features they possess (where possible). • 6.6: Nucleotides Nucleotides are most often thought of as the building blocks of the nucleic acids, DNA and RNA. While this, is, of course, a vital function, nucleotides also play other important roles in cells. Ribonucleoside triphosphates like ATP, CTP, GTP and UTP are necessary, not just for the synthesis of RNA, but as part of activated intermediates like UDP-glucose in biosynthetic pathways. ATP is also the universal “energy currency” of cells. Thumbnail: Metabolic Metro Map. Image used with permission (CC BY-SA 4.0; Chakazul). 06: Metabolism Glycolysis Carbohydrates, whether synthesized by photosynthetic organisms, stored in cells as glycogen, or ingested by heterotrophs, must be broken down to obtain energy for the cell’s activities as well as to synthesize other molecules required by the cell. Starch and glycogen, polymers of glucose, are the main energy storage forms of carbohydrates in plants and animals, respectively. To use these sources of energy, cells must first break down the polymers to yield glucose. The glucose is then taken up by cells through transporters in cell membranes. The metabolism of glucose, as well as other six carbon sugars (hexoses) begins with the catabolic pathway called glycolysis. In this pathway, sugars are oxidized and broken down into pyruvate molecules. The corresponding anabolic pathway by which glucose is synthesized is termed gluconeogenesis. Neither glycolysis nor gluconeogenesis is a major oxidative/reductive process, with one step in each one involving loss/gain of electrons, but the product of glycolysis, pyruvate, can be completely oxidized to carbon dioxide (Figure 6.2). Indeed, without production of pyruvate from glucose in glycolysis, a major energy source for the cell would not be available. Glucose is the most abundant hexose in nature and is traditionally used to illustrate the reactions of glycolysis, but fructose (in the form of fructose-6- phosphate) is also readily metabolized, while galactose can easily be converted into glucose for catabolism in the pathway as well. The end metabolic products of glycolysis are two molecules of ATP, two molecules of NADH and two molecules of pyruvate (Figure 6.3), which, in turn, can be oxidized further in the citric acid cycle. Entry points for glycolysis Glucose and fructose are the sugar ‘funnels’ serving as entry points to the glycolytic pathway. Other sugars must be converted to either of these forms to be metabolized in glycolysis. Some pathways, including the Calvin Figure 6.2 - Metabolic fates of glucose Image by Aleia Kim Your cells may have a mounting crisis Should they not go through glyco-lye-sis No glucose energy releases Until it’s fractured into pieces Figure 6.3 - Glycolysis and its Regulators Image by Ben Carson Cycle and the Pentose Phosphate Pathway (PPP) contain intermediates in common with glycolysis, so in that sense, almost any cellular sugar can be metabolized here. Other pathways Intermediates of glycolysis and gluconeogenesis that are common to other pathways include glucose-6-phosphate (PPP, glycogen metabolism), Fructose-6-phosphate (Calvin Cycle, PPP), Glyceraldehyde-3- phosphate (Calvin Cycle, PPP), dihydroxyacetone phosphate (PPP, glycerol metabolism, Calvin Cycle), 3- phosphoglycerate (Calvin Cycle, PPP), phosphoenolpyruvate (C4 plant metabolism, Calvin Cycle), and pyruvate (fermentation, acetyl-CoA genesis, amino acid metabolism). It is worth noting that glycerol from the breakdown of fat can readily be metabolized to dihydroxyacetone phosphate (DHAP) and thus enter the glycolysis pathway. It is the only part of a fat that is used in these pathways. Reaction 1 Glucose gets a phosphate from ATP to make glucose-6-phosphate (G6P) in a reaction catalyzed by the enzyme hexokinase, a transferase enzyme. Glucose + ATP ⇄ G6P + ADP + H+ Hexokinase is one of three regulated enzymes in glycolysis and is inhibited by one of the products of its action - G6P. Hexokinase has flexibility in its substrate binding and is able to phosphorylate a variety of hexoses, including fructose, mannose, and galactose. Why phosphorylate glucose?
Phosphorylation of glucose serves two important purposes. First, the addition of a phosphate group to glucose effectively traps it in the cell, as G6P cannot diffuse across the lipid bilayer. Second, the reaction decreases the concentration of free glucose, favoring additional import of the molecule. G6P is a substrate for the pentose phosphate pathway and can also be converted to glucose-1-phosphate (G1P) for use in glycogen synthesis and galactose metabolism (Figure 6.5). It is worth noting that the liver has an enzyme like hexokinase called glucokinase, which Figure 6.4 - Reaction #1 - Phosphorylation of glucose - catalyzed by hexokinase has a much higher Km (lower affinity) for glucose. This is important, because the liver is a site of glucose synthesis (gluconeogenesis) where cellular concentrations of glucose can be relatively high. With a lower affinity glucose phosphorylating enzyme, glucose is not converted to G6P unless glucose concentrations get high, so the liver is able to release the glucose it makes into the bloodstream for the rest of the body to use. Reaction 2 Next, G6P is converted to fructose-6-phosphate (F6P), in a reaction catalyzed by the enzyme \[\ce{G6P ⇄ F6P}\] The reaction has a low ΔG°’ , so it is readily favorable in either direction with Figure 6.6 - Mechanism of conversion of G6P to F6P in reaction #2 Figure 6.5 - The centrality of glucose-6-phosphate in metabolism Image by Aleia Kim only slight changes in concentration of reactants. Reaction 3 \[ce{F6P + ATP ⇄ F1,6BP + ADP + H+}\] The second input of energy occurs when F6P gets another phosphate from ATP in a reaction catalyzed by the enzyme phosphofructokinase-1 (PFK-1 - another transferase) to make fructose-1,6- bisphosphate (F1,6BP). PFK-1 is a very important enzyme regulating glycolysis, with several allosteric activators and inhibitors (see HERE). Like the hexokinase reaction the energy from ATP is needed to make the reaction energetically favorable. PFK-1 is the most important regulatory enzyme in the pathway and this reaction is the ratelimiting step. It is also one of three essentially irreversible reactions in glycolysis. A variant enzyme found in plants and some bacterial uses pyrophosphate rather than ATP as the energy source and due to the lower energy input from hydrolysis of the pyrophosphate, that reaction is reversible. Reaction 4 \[\ce{F1,6BP ⇄ D-GLYAL3P + DHAP}\] With the glycolysis pump thus primed, the pathway proceeds to split the F1,6BP into two 3-carbon intermediates. This reaction catalyzed by the lyase known as aldolase is energetically a “hump” to overcome in the glycolysis direction (∆G°’ = +24 kJ/mol Figure 6.7 - Reaction #3 - Conversion of F6P to F1,6BP by PFK Wikipedia Figure 6.8 - Reaction #4 - Breakdown of F1,6BP into GLYAL3P (left) and DHAP (right) by aldolase °K) so to get over the energy hump, cells must increase the concentration the reactant (F1,6BP) and decrease the concentration of the products, which are D-glyceraldehyde- 3-phosphate (D-GLYAL3P) and dihydroxyacetone phosphate (DHAP). A novel scheme facilitates decreasing concentration of the products (see below). Aldolases cut the ketose ring by two different mechanisms and these enzymes are grouped as Class I (in animals and plants) and Class II (in fungi and bacteria). Reaction 5 \[\ce{DHAP ⇄ D-GLYAL3P}\] In the next step, DHAP is converted to DGLYAL3P in a reaction catalyzed by the enzyme triosephosphate isomerase. At this point, the six carbon glucose molecule has been broken down to two units of three carbons each - D-GLYAL3P. From this point forward each reaction of glycolysis contains two of each molecule. Reaction #5 is fairly readily reversible in cells. The enzyme is of note because it is one example of a “perfect enzyme.” Enzymes in this category have very high ratios of Kcat/Km that approach a theoretical maximum limited only by the diffusion of substrate into the active site of the enzyme. The apparent reason for the enzyme evolving in this way is that the mechanism of the reaction produces an unstable, toxic intermediate (Figure 6.9). With the reaction proceeding as rapidly as it does, there is less chance of the intermediate escaping and causing damage in the cell. Reaction 6 \[\ce{D-GLYAL3P + NAD+ + Pi D-1,3BPG + NADH + H+}\] Figure 6.9 - Reaction #5 - Triose phosphate isomerase with unstable, toxic intermediate (methyl glyoxal) Image by Ben Carson In this reaction, D-GLYAL3P is oxidized in the only oxidation step of glycolysis catalyzed by the enzyme glyceraldehyde-3- phosphate dehydrogenase, an oxidoreductase. The aldehyde in this reaction is oxidized, then linked to a phosphate to make an ester - D-1,3-bisphospho-glycerate (D- 1,3BPG). Electrons from the oxidation are donated to NAD+, creating NADH. NAD+ is a critical constituent in this reaction and is the reason that cells need a fermentation option at the end of the pathway (see below). Note here that ATP energy was not required to put the phosphate onto the oxidized D-GLYAL3P. The reason for this is because the energy provided by the oxidation reaction is sufficient for adding the phosphate. Reaction 7 \[\ce{D-1,3BPG + ADP ⇄ 3PG + ATP}\] The two phosphates in the tiny 1,3BPG molecule repel each other and give the molecule high potential energy. This energy is utilized by the enzyme phosphoglycerate kinase (another transferase) to phosphorylate ADP and make ATP, as well as the product, 3-phosphoglycerate (3-PG). This is an example of a substrate-level phosphorylation. Such mechanisms for making ATP require an intermediate with a high enough energy to phosphorylate ADP to make ATP. Figure 6.10 - Reaction #6 - Oxidation of GLYAL3P, catalyzed by glyceraldehyde-3-phosphate dehydrogenase Figure 6.11 - Reaction #7 - Substrate-level Phosphorylation by 1,3-BPG Though there are a few substrate level phosphorylations in cells (including another one at the end of glycolysis), the vast major of ATP is made by oxidative phosphorylation in the mitochondria (in animals). In addition to oxidative phosphorylation, plants also make ATP by photophosphorylation in their chloroplasts. Since there are two 1,3 BPGs produced for every glucose, the two ATPs produced in this reaction replenish the two ATPs used to start the cycle and the net ATP count at this point of the pathway is zero. Reaction 8 \[\ce{3-PG ⇄ 2-PG }\]
Conversion of the 3-PG intermediate to 2-PG (2- phosphoglycerate) occurs by an important mechanism. An intermediate in this readily reversible reaction (catalyzed by phosphoglycerate mutase - a mutase enzyme) is 2,3-BPG. This intermediate, which is stable, is released with low frequency by the enzyme instead of being con- Figure 6.13 - Two routes to formation of 2,3-BPG Figure 6.14 - 2,3- Bisphosphoglycerate (2,3-BPG) Figure 6.12 - Reaction #8 - Conversion of 3-PG to 2-PG verted to 2-PG. 2,3BPG is important because it binds to hemoglobin and stimulates release of oxygen. The molecule can also be made from 1,3-BPG as a product of a reaction catalyzed by bisphophglycerate mutase (Figure 6.13). Cells which are metabolizing glucose rapidly release more 2,3-BPG and, as a result, get more oxygen, supporting their needs. Notably, cells which are metabolizing rapidly are using oxygen more rapidly and are more likely to be deficient in it. Reaction 9 \[\ce{2-PG ⇄ PEP + H2O}\] 2-PG is converted by enolase (a lyase) to phosphoenolpyruvate (PEP) by removal of water, creating a very high energy intermediate. The reaction is readily reversible, but with PEP, the cell has one of its highest energy molecules and that is important for the next reaction. Reaction 10 \[\ce{PEP + ADP + H+ ⇄ PYR + ATP}\] Conversion of PEP to pyruvate by pyruvate kinase is the second substrate level phosphorylation of glycolysis, creating ATP. This reaction is what some refer to as the “Big Bang” of glycolysis because there is almost enough energy in PEP to stimulate production of a second ATP (ΔG°’ = 31.6 kJ/ mol), but it is not used. Consequently, this energy is lost as heat. If you wonder why you get hot when you exercise, the heat produced in the breakdown of glucose is a prime contributor and the pyruvate kinase reaction is a major source. Figure 6.16 - Reaction #10 - The big bang - PEP phosphorylates ADP with a lot of energy to spare Wikipedia Figure 6.15 - Reaction #9 - Enolase-catalyzed removal of water Wikipedia Pyruvate kinase is the third and last enzyme of glycolysis that is regulated (see below). The primary reason this is the case is to be able to prevent this reaction from occurring when cells are making PEP while going through gluconeogenesis (see more HERE). Catabolism of other sugars Though glycolysis is a pathway focused on the metabolism of glucose and fructose, the fact that other sugars can be readily metabolized into glucose means that glycolysis can be used for extracting energy from them as well. Galactose is a good example. It is commonly produced in the produced in the body as a result of hydrolysis of lactose, catalyzed by the enzyme known as lactase (Figure 6.17). Deficiency of lactase is the cause of lactose intolerance. Galactose begins preparation for entry into glycolysis by being converted to galactose-1- phosphate (catalyzed by galactokinase - Figure 6.18). Galactose-1-phosphate swaps with glucose-1-phosphate from UDP-glucose to make UDP-galactose (Figure 6.19). An epimerase converts UDPgalactose back to UDP-glucose and the cycle is complete. Each turn of the cycle thus takes in one galactose-1-phosphate and releases one glucose-1-phosphate. Deficiency of galactose conversion enzymes results in accumulation of galactose (from breakdown of lactose). Excess galactose is converted to galactitol, a sugar alcohol. Galactitol in the human eye lens causes it to absorb water and this may be a factor in formation of cataracts.Figure 6.17 - Breakdown of lactose to glucose and galactose by lactase Image by Pehr Jacobson Figure 6.18 - Galactokinase Reaction Image by Penelope Irving Free fructose can also enter glycolysis by two mechanisms. First, it can be phosphorylated to fructose-6-phosphate by hexokinase. A more interesting alternate entry point is that shown in Figure 6.20. Phosphorylation of fructose by fructokinase produces fructose-1-phosphate and cleavage of that by fructose-1- phosphate aldolase yields DHAP and glyceraldehyde. Phosphorylation of glyceraldehyde by triose kinase yields GLYAL3P. This alternative entry means for fructose may have important implications because DHAP and GLYAL3P are introduced into the glycolysis pathway while bypassing PFK-1 regulation. Some have proposed this may be important when considering metabolism of high fructose corn syrup, since it forces production of pyruvate, a precursor of acetyl-CoA, which is itself a precursor of fatty acids when ATP levels are high. Mannose metabolism Mannose can also be metabolized in glycolysis. In this case, it enters via fructose by the following two-step process - 1) phosphoryla- Figure 6.19 - Conversion of galactose-1-phosphate into glucose-6-phosphate Image by Aleia Kim tion by hexokinase to make mannose-6- phosphate followed by its conversion to fructose-6-phosphate, catalyzed by phosphomannoisomerase (Figure 6.21). Glycerol metabolism Glycerol is an important molecule for the synthesis of fats, glycerophospholipids, and other membrane lipids. Most commonly it is made into glycerol-3- phosphate (Figure 6.22) and the glycolysis/gluconeogenesis pathways are important both for producing the compound and for metabolizing it. The relevant intermediate in these pathways both for producing and for using glycerol-3-phosphate is DHAP. The enzyme glycerol-3-phosphate dehydrogenase reversibly converts glycerol-3- phosphate into DHAP (Figure 6.22). This reaction, which is an oxidation, transfers electrons to NAD+ to produce NADH. In the reverse reaction, production of glycerol-3- phosphate from DHAP, of course, requires electrons from NADH for the reduction. Both glycolysis and gluconeogenesis are sources DHAP, meaning when the cell needs glycerol- 3-phosphate that it can use sugars (glucose, fructose, mannose, or galactose) as sources in glycolysis. For gluconeogenesis, sources include pyruvate, alanine and Figure 6.20 - Entry of fructose into glycolysis, bypassing PFK-1 Image by Penelope Irving Figure 6.21 - Entry of other sugars into glycolysis Image by Penelope Irving lactate (both can easily be made into pyruvate), oxaloacetate, aspartic acid (which can be made into oxaloacetate by transamination), and others. All of the intermediates of the citric acid cycle (and glyoxylate cycle) can be converted ultimately to oxaloacetate, which is a gluconeogenesis intermediate, as well.
It is worth noting that animals are unable to use fatty acids as materials for gluconeogenesis in net amounts, but they can, in fact, use glycerol in both glycolysis and gluconeogenesis. It is the only part of the fat molecule that can be so used. Pyruvate metabolism As noted, pyruvate produced in glycolysis can be oxidized to acetyl-CoA, which is itself oxidized in the citric acid cycle to carbon dioxide. That is not the only metabolic fate of pyruvate, though (Figure 6.23). Pyruvate is a “starting” point for gluconeogenesis, being converted to oxaloacetate in the mitochondrion in the first step. Pyruvate in animals can also be reduced to lactate by adding electrons from NADH (Figure 6.24). This reaction produces NAD+ and is critical for generating the latter molecule to keep the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis (reaction #6) going under conditions when there is no oxygen. This is because oxygen is necessary for the electron transport system (ETS) to operate and it performs the important function of converting NADH back to NAD+. When the ETS is running, NADH donates electrons to Complex I and is oxidized to NAD+ in the process, generating the intermediate needed for oxidizing GLYAL-3P. In the absence of oxygen, however, NADH cannot be converted to Figure 6.22 - Reactions in glycerol metabolism Image by Penelope Irving NAD+ by the ETS, so an alternative means of making NAD+ is necessary for keeping glycolysis running under low oxygen conditions (fermentation). Bacteria and yeast generate NAD+ under oxygen deprived conditions by doing fermentation in a different way (Figure 6.25). They use NADH-requiring reactions that regenerate NAD+ while producing ethanol from pyruvate instead of making lactate. Thus, fermentation of pyruvate is essential to keep glycolysis operating when oxygen is limiting. It is also for these reasons that brewing of beer (using yeast) involves depletion of oxygen and muscles low in oxygen produce lactic acid (animals). Pyruvate is a precursor of alanine which can be easily synthesized by transfer of a nitrogen from an amine donor, such as glutamic acid. Pyruvate can also be converted into oxaloacetate by carboxylation in the process of gluconeogenesis (see below). The enzymes involved in pyruvate metabolism include pyruvate dehydrogenase (makes acetyl-CoA), lactate dehydrogenase (makes lactate), transaminases (make alanine), pyruvate carboxylase (makes ox- Figure 6.23 - Pyruvate metabolism. When oxygen is absent, pyruvate is converted to lactate (animals) or ethanol (bacteria and yeast). When oxygen is present, pyruvate is converted to acetyl-CoA. Not shown - Pyruvate transamination to alanine or carboxylation to form oxaloacetate. aloacetate), and pyruvate decarboxylase (a part of pyruvate dehydrogenase that makes acetaldehyde in bacteria and yeast). Catalytic action and regulation of the pyruvate dehydrogenase complex is discussed in the section on the citric acid cycle (HERE). Gluconeogenesis The anabolic counterpart to glycolysis is gluconeogenesis (Figure 6.26), which occurs mostly in the cells of the liver and kidney and virtually no other cells in the body. In seven of the eleven reactions of gluconeogenesis (starting from pyruvate), the same enzymes are used as in glycolysis, but the reaction directions are reversed. Notably, the ∆G values of these reactions in the cell are typically near zero, meaning their direction can be readily controlled by changing substrate and product concentrations by small amounts. The three regulated enzymes of glycolysis all catalyze reactions whose cellular ∆G values are not close to zero, making manipulation of reaction direction for their reac- Figure 6.24 - Formation of lactate in animal fermentation produces NAD+ for G3PDH Image by Ben Carson Figure 6.25 - Formation of ethanol in microbial fermentation produces NAD+ for G3PDH Image by Ben Carson tions non-trivial. Consequently, cells employ “work-around” reactions catalyzed by four different enzymes to favor gluconeogenesis, when appropriate. Bypassing pyruvate kinase Two of the enzymes (pyruvate carboxylase and PEP carboxykinase - PEPCK) catalyze reactions that bypass pyruvate kinase. F1,6BPase bypasses PFK-1 and G6Pase bypasses hexokinase. Notably, pyruvate carboxylase and G6Pase are found in the mitochondria and endoplasmic reticulum, respectively, whereas the other two are found in the cytoplasm along with all of the enzymes of glycolysis. Biotin An important coenzyme used by pyruvate carboxylase is biotin (Figure 6.27). Biotin is commonly used by carboxylases to carry CO2 to incorporate into the substrate. Also known as vitamin H, biotin is a water soluble B vitamin (B7) needed for many metabolic processes, including fatty acid synthesis, gluconeogenesis, and amino acid metabolism. Deficiency of the vitamin is rare, since it is readily produced by gut Gluconeogenesis and glycolysis. Only the enzymes differing in gluconeogenesis are shown Image by Aleia Kim teria. There are many claims of advantages of taking biotin supplements, but there is no strong indication of benefits in most cases. Deficiencies are associated with inborn genetic errors, alcoholism, burn patients, and people who have had a gastrectomy. Some pregnant and lactating women may have reduced levels due to increased biotin catabolism. Reciprocal regulation All of the enzymes of glycolysis and nine of the eleven enzymes of gluconeogenesis are all in the cytoplasm, necessitating a coordinated means of controlling them. Cells generally need to minimize the extent to which paired anabolic and catabolic pathways are occurring simultaneously, lest they produce a futile cycle, resulting in wasted energy with no tangible product except heat. The mechanisms of controlling these pathways have opposite effects on catabolic and anabolic processes. This method of control is called reciprocal regulation (see above). Reciprocal regulation is a coordinated means of simultaneously controlling metabolic pathways that do opposite things. In reciprocal regulation, a single molecule (allosteric regulation) or a single covalent modification (phosphorylation/dephosphorylation, Allosteric Regulation of Glycolysis & Gluconeogenesis Reciprocal Regulation AMP - Activates PFK-1, Inhibits F1,6BPase F2,6BP - Activates PFK-1, Inhibits F1,6BPase Citrate - Activates PFK-1, Inhibits F1,6BPase Glycolysis Only ATP - Inhibits PFK-1 and Pyruvate Kinase
Alanine - Inhibits Pyruvate Kinase Gluconeogenesis Only ADP - Inhibits Pyruvate Carboxylase and PEPCK Acetyl-CoA - Activates Pyruvate Carboxylase Figure 6.27 - Biotin carrying carbon dioxide (red) Wikipedia for example) has opposite effects on the different pathways. Reciprocal allosteric effects For example, in glycolysis, the enzyme known as phosphofructokinase (PFK-1) is allosterically activated by AMP and a molecule known as F2,6BP (Figure 6.28). The corresponding enzyme from gluconeogenesis catalyzing a reversal of the glycolysis reaction is known as F1,6BPase. F1,6BPase is inhibited by both AMP and F2,6BP. Reciprocal covalent effects In glycogen metabolism, the enzymes phosphorylase kinase and glycogen phosphorylase catalyze reactions important for the breakdown of glycogen. The enzyme glycogen synthase catalyzes the synthesis of glyco- Directional velocity Inverts with reciprocity If glycolysis is flowing Glucose synthesis awaits But when the latter is a-going Sugar breakdown then abates Figure 6.28 - Regulation of glycolysis (orange path) and gluconeogenesis (black path) Image by Aleia Kim gen. Each of these enzymes is, at least partly, regulated by attachment and removal of phosphate. Phosphorylation of phosphorylase kinase and glycogen phosphorylase has the effect of making them more active, whereas phosphorylation of glycogen synthase makes it less active. Conversely, dephosphorylation has the reverse effects on these enzymes - phosphorylase kinase and glycogen phosphorylase become less active and glycogen synthase becomes more active. Simple and efficient The advantage of reciprocal regulation schemes is that they are very efficient. It doesn’t take separate molecules or separate treatments to control two pathways simultaneously. Further, its simplicity ensures that when one pathway is turned on, the other is turned off. This is especially important with catabolic/ anabolic regulation, because having both pathways going on simultaneously in a cell is not very productive, leading only to production of heat in a futile cycle. A simple futile cycle is shown on Figure 6.29. If unregulated, the cyclic pathway in the figure (shown in black) will make ATP in creating pyruvate from PEP and will use ATP to make oxaloacetate from pyruvate. It will also use GTP to make PEP from oxaloacetate. Thus, each turn of the cycle will make one ATP, use one ATP and use one GTP for a net loss of energy. The process will start with pyruvate and end with pyruvate, so there is no net production of molecules. (see HERE for one physiological use of a futile cycle). Specific gluconeogenesis controls Besides reciprocal regulation, other mechanisms help control gluconeogenesis. First, PEPCK is controlled largely at the level of synthesis. Overexpression of PEPCK (stimulated by glucagon, glucocorticoid hormones, and cAMP and inhibited by insulin) produces symptoms of diabetes. Pyruvate carboxylase is sequestered in the mitochondrion (one means of regulation) Figure 6.29 - A simple futile cycle - follow the black lines Image by Aleia Kim Interactive Learning Module HERE and is sensitive to acetyl-CoA, which is an allosteric activator. Acetyl-CoA concentrations increase as the citric acid cycle activity decreases. Glucose-6- phosphatase is present in low concentrations in many tissues, but is found most abundantly and importantly in the major gluconeogenic organs – the liver and kidney cortex. Specific glycolysis controls Control of glycolysis and gluconeogenesis is unusual for metabolic pathways, in that regulation occurs at multiple points. For glycolysis, this involves three enzymes: 1. Hexokinase (Glucose ⇄ G6P) 2. Phosphofructokinase-1 (F6P ⇄ F1,6BP) 3. Pyruvate kinase (PEP ⇄ Pyruvate). Regulation of hexokinase is the simplest of these. The enzyme is unusual in being inhibited by its product, glucose-6-phosphate. This ensures when glycolysis is slowing down hexokinase is also slowing down to reduce feeding the pathway. Pyruvate kinase It might also seem odd that pyruvate kinase, the last enzyme in the pathway, is regulated (Figure 6.30), but the reason is simple. Pyruvate kinase catalyzes the most energetically rich reaction of glycolysis. The reaction is favored so strongly in the forward direction that cells must do a ‘two-step’ around it in the reverse direction when making glucose in the gluconeogenesis pathway. In other words, it takes two enzymes, two reactions, and two triphosphates (ATP and GTP) to go from one pyruvate back to one PEP in gluconeogenesis. When cells are needing to make glu- igure 6.30 - Regulation of pyruvate kinase For cells a glucose cycling’s cost Is energy in reams Four ATPs each time is lost From breaking/making schemes So use for metabolic heat To make it warm inside your feet Else it’s of no utility To practice such futility cose, they can’t be sidetracked by having the PEP they have made in gluconeogenesis be converted directly back to pyruvate by pyruvate kinase. Consequently, pyruvate kinase must be inhibited during gluconeogenesis or a futile cycle will occur and no glucose will be made. Another interesting control mechanism called feedforward activation involves pyruvate kinase. Pyruvate kinase is activated allosterically by the glycolysis intermediate, F1,6BP. This molecule is a product of the PFK-1 reaction and a substrate for the aldolase reaction. Reactions pulled As noted above, the aldolase reaction is energetically unfavorable (high positive ∆G°’), thus allowing F1,6BP to accumulate. When this happens, some of the excess F1,6BP binds to pyruvate kinase, which activates and jump- Figure 6.31 - Regulation of Synthesis and Breakdown of F2,6BP Image by Penelope Irving starts the conversion of PEP to pyruvate. The resulting drop in PEP levels has the effect of “pulling” on the reactions preceding pyruvate kinase. As a consequence, the concentrations of GLYAL3P and DHAP fall, helping to pull the aldolase reaction forward. PFK-1 regulation PFK-1 has a complex regulation scheme. First, it is reciprocally regulated (relative to F1,6BPase) by three molecules. F2,6BP activates PFK-1 and inhibits F1,6BPase. PFK-1 is also allosterically activated by AMP, whereas F1,6BPase is inhibited. On the other hand, citrate inhibits PFK-1, but activates F1,6BPase.
PFK-1 is also inhibited by ATP and is exquisitely sensitive to proton concentration, easily losing activity when the pH drops only slightly. PFK- 1’s inhibition by ATP is noteworthy and odd at first glance because ATP is also a substrate whose increasing concentration should favor the reaction instead of inhibit it. The root of this conundrum is that PFK-1 has two ATP binding sites - one at an allosteric site that binds ATP relatively inefficiently and one that the active site that binds ATP with high affinity. Thus, only when ATP concentration is high is binding at the allosteric site favored and only then can ATP turn off the enzyme. F2,6BP regulation Regulation of PFK-1 by F2,6BP is simple at the PFK-1 level, but more complicated at the level of synthesis of F2,6BP. Despite having a name sounding like a glycolysis/ gluconeogenesis intermediate (F1,6BP), F2,6BP is not an intermediate in either pathway. Instead, it is made from fructose-6-phosphate and ATP by the enzyme known as phosphofructokinase-2 (PFK- 2 - Figure 6.31). Cori cycle With respect to energy, the liver and muscles act complementarily. The liver is the major or- Figure 6.32 - The Cori cycle Image by Aleia Kim gan in the body for the synthesis of glucose. Muscles are major users of glucose to make ATP. Actively exercising muscles use oxygen faster than the blood can deliver it. As a consequence, the muscles go anaerobic and produce lactate. This lactate is of no use to muscle cells, so they dump it into the blood. Lactate travels in the blood to the liver, which takes it up and reoxidizes it back to pyruvate, catalyzed by the enzyme lactate dehydrogenase (Figure 6.32). Pyruvate in the liver is then converted to glucose by gluconeogenesis. The glucose thus made by the liver is dumped into the bloodstream where it is taken up by muscles and used for energy, completing the important intercellular pathway known as the Cori cycle. Glucose alanine cycle The glucose alanine cycle (also known as the Cahill Cycle), has been described as the amine equivalent of the Cori cycle (Figure 6.33). The Cori cycle, of course, exports lac- Figure 6.33 - Overlap between the Cori cycle and the glucose alanine cycle tate from muscles (when oxygen is limiting) to the liver via the bloodstream. The liver, in turn, converts lactate to glucose, which it ships back to the muscles via the bloodstream. The Cori Cycle is an essential source of glucose energy for muscles during periods of exercise when oxygen is used faster than it can be delivered. In the glucose-alanine cycle, cells are generating toxic amines and must export them. This is accomplished by transaminating pyruvate (the product of glycolysis) to produce the amino acid alanine. The glucose-alanine process requires the enzyme alanine aminotransferase, which is found in muscles, liver, and intestines. Alanine is exported in the process to the blood and picked up by the liver, which deaminates it to release the amine for synthesis of urea and excretion. The pyruvate left over after the transamination is a substrate for gluconeogenesis. Glucose produced in the liver is then exported to the blood for use by cells, thus completing the cycle. Polysaccharide metabolism Sugars are metabolized rapidly in the body and that is one of the primary reasons they are used. Managing levels of glucose in the body is very important - too much leads to complications related to diabetes and too little gives rise to hypoglycemia (low blood sugar). Sugars in the body are maintained by three processes - 1) diet; 2) synthesis (gluconeogenesis); and 3) storage. The storage forms of sugars are, of course, the polysaccharides and their metabolism is our next topic of discussion. Amylose and amylopectin The energy needs of a plant are much less dynamic than those of animals. Muscular contraction, nervous systems, and information processing in the brain require large amounts of quick energy. Because of this, the polysaccharides stored in plants are somewhat less complicated than those of animals. Plants store glucose for energy in the form of amylose (Figure 6.34 and see HERE) and amylopectin and for structural integrity in the form of cellulose (see HERE). These structures differ in that cellulose contains glucose units solely joined by β-1,4 bonds, whereas amylose has only α-1,4 bonds and amylopectin has α-1,4 and α-1,6 bonds. Figure 6.34 Amylose, a polymer of glucose in plants Glycogen Animals store glucose primarily in liver and muscle in the form of a compound related to amylopectin known as glycogen. The structural differences between glycogen and amylopectin are solely due to the frequency of the α-1,6 branches of glucoses. In glycogen they occur about every 10 residues instead of every 30-50, as in amylopectin (Figure 6.35). Glycogen provides an additional source of glucose besides that produced via gluconeogenesis. Because glycogen contains so many glucoses, it acts like a battery backup for the body, providing a quick source of glucose when needed and providing a place to store excess glucose when glucose concentrations in the blood rise. The branching of glycogen is an important feature of the molecule metabolically as well. Since glycogen is broken down from the "ends" of the molecule, more branches translate to more ends, and more glucose that can be released at once. Just as in gluconeogenesis, the cell has a separate mechanism for glycogen synthesis that is distinct from glycogen breakdown. As noted previously, this allows the cell to separately control the reactions, avoiding futile cycles, and enabling a process to occur efficiently (synthesis of glycogen) that would not occur if Figure 6.35 - Glycogen Structure - α-1,4 links with α-1,6 branches every 7-10 residues it were simply the reversal of glycogen breakdown. Glycogen breakdown Breakdown of glycogen involves 1) release of glucose-1-phosphate (G1P), 2) rearranging the remaining glycogen (as necessary) to permit continued breakdown, and 3) conversion of G1P to G6P for further metabolism. G6P can be 1) used in glycolysis, 2) converted to glucose by gluconeogenesis, or 3) oxidized in the pentose phosphate pathway.
Glycogen phosphorylase (sometimes simply called phosphorylase) catalyzes breakdown of glycogen into glucose-1- Phosphate (G1P - Figure 6.36). The reaction that produces G1P from glycogen is a phosphorolysis, not a hydrolysis reaction. The distinction is that hydrolysis reactions use water to cleave bigger molecules into smaller ones, but phosphorolysis reactions use phosphate instead for the same purpose. Note that the phosphate is just that - it does NOT come from ATP. Since ATP is not used to put phosphate on G1P, the reaction saves the cell energy. Glycogen debranching enzyme Glycogen phosphorylase will only act on nonreducing ends of a glycogen chain that are at least 5 glucoses away from a branch point. A second enzyme, Glycogen Debranching Enzyme (GDE) (also called debranching enzyme), is therefore needed to convert α (1-6) branches to α (1-4) branches. GDE acts on glycogen branches that have reached their limit of phosphorylysis with glycogen phosphorylase. Figure 6.36 - Breaking of α-1,4 bonds of glycogen by glycogen phosphorylase Image by Aleia Kim Interactive Learning Module HERE GDE acts to transfer a trisaccharide from an α-1,6 branch onto an adjacent α-1,4 branch, leaving a single glucose at the 1,6 branch. Note that the enzyme also catalyzes the hydrolysis of the remaining glucose at the 1,6 branch point (Figure 6.37). Thus, the breakdown products from glycogen are G1P and glucose (mostly G1P). Glucose can, of course, be converted to Glucose-6-Phosphate (G6P) as the first step in glycolysis by either hexokinase or glucokinase. G1P can be converted to G6P by action of an enzyme called phosphoglucomutase. This reaction is readily reversible, allowing G6P and G1P to be interconverted as the concentration of one or the other increases. This is important, because phosphoglucomutase is needed to form G1P for glycogen synthesis. Regulation of glycogen metabolism Regulation of glycogen metabolism is complex, occurring both allosterically and via hormone-receptor controlled events that result in protein phosphorylation or dephosphorylation. In order to avoid a futile cycle of glycogen synthesis and breakdown simultaneously, cells have evolved an elaborate set of controls that ensure only one pathway is primarily active at a time. Regulation of glycogen metabolism is managed by the enzymes glycogen phosphorylase and glycogen synthase. Glycogen phosphorylase is regulated by both allosteric factors (ATP, G6P, AMP, and glucose) and by covalent modification (phosphorylation / dephosphorylation). Its regulation is consistent with the energy needs of the cell. High energy molecules (ATP, G6P, glucose) al- Figure 6.37 - Catalytic activity of debranching enzyme losterically inhibit glycogen phosphorylase, while the low energy molecule AMP allosterically activates it. GPa/GPb allosteric regulation Glycogen phosphorylase exists in two different covalent forms – one form with phosphate (called GPa here) and one form lacking phosphate (GPb here). GPb is converted to GPa by phosphorylation by an enzyme known as phosphorylase kinase. GPa and GPb can each exist in an 'R' state and a 'T' state (Figure 6.38). For both GPa and GPb, the R state is the more active form of the enzyme. GPa's negative allosteric effector (glucose) is usually not abundant in cells, so GPa does not flip into the T state often. There is no positive allosteric effector of GPa. When glucose is absent, GPa automatically flips into the R (more active) state (Figure 6.39). It is for this reason that people tend to think of GPa as being the more active covalent form of the enzyme. GPb can convert from the GPb T state to the GPb R state by binding AMP. Unless a cell is low in energy, AMP concentration is low. Thus GPb is not converted Figure 6.38 - Glycogen phosphorylase regulation - covalent (horizontal) and allosteric (vertical) Image by Aleia Kim to the R state very often. This is why people think of the GPb form as less active than GPa. On the other hand, ATP and/or G6P are usually present at high enough concentration in cells that GPb is readily flipped into the T state (Figure 6.40). GPa/GPb covalent regulation The relative amounts of GPa and GPb largely govern the overall process of glycogen breakdown, since GPa tends to be active more often than GPb. It is i Phosphorylase kinase itself has two covalent forms – phosphorylated (active) and dephosphorylated (inactive). It is phosphorylated by the enzyme Protein Kinase A (PKA - ). Another way to activate the enzyme is allosterically with calcium (Figure 6.41). Phosphory- Figure 6.39 - Allosteric regulation of GPa Image by Aleia Kim Figure 6.40 - Allosteric regulation of GPb Image by Aleia Kim lase kinase is dephosphorylated by phosphoprotein phosphatase, the same enzyme that removes phosphate from GPa. PKA and cAMPcAMP PKA is activated by cAMP, which is, in turn, produced by adenylate cyclase after activation by a G-protein (See HERE for overview). G-proteins are activated ultimately by binding of ligands to specific membrane receptors called 7-TM receptors, also known as Gprotein coupled receptors. These are discussed in greater detail HERE. Common ligands for 7-TM receptors include epinephrine (binds β- adrenergic receptor) and glucagon (binds glucagon receptor). Epinephrine exerts its greatest effects on muscle and glucagon works preferentially on the liver. Thus, epinephrine and glucagon can activate glycogen breakdown by stimulating synthesis of cAMP followed by the cascade of events described above. Turning off glycogen breakdown Turning off signals is as important, if not more so, than turning them on. Glycogen is a precious resource. If its breakdown is not controlled, a lot of energy used in its synthesis is wasted. The steps in the glycogen breakdown regulatory pathway can be reversed at every level. First, the ligand (epinephrine or glucagon) can leave the receptor, turning off the stimulus. Second, the G-proteins have an inherent GTPase activity. GTP, of course, is what activates Gproteins, so a GTPase activity converts the GTP it is carrying to GDP and the G-protein becomes inactive. Thus, G-proteins turn off Figure 6.41 - Activation of phosphorylase kinase Image by Aleia Kim their own activity. Interfering with their ability to convert GTP to GDP can have dire consequences, including cancer in some cases.
Third, cells have phosphodiesterase enzymes (inhibited by caffeine) for breaking down cAMP. cAMP is needed to activate PKA, so breaking it down stops PKA from activating phosphorylase kinase. Fourth, the enzyme known as phosphoprotein phosphatase (also called PP1) plays a major role. It can remove phosphates from phosphorylase kinase (inactivating it) and form GPa, converting it to the less likely to be active GPb. Regulation of phosphoprotein phosphatase activity occurs at several levels. Two of these are shown in Figures 6.42 & 6.43. In Figure 6.42, phosphoprotein phosphatase is shown being inactivated by phosphorylation of an inhibitor (called PI-1 - see below). This happens as a result of cascading actions from binding of epinephrine (or glucagon) to a cell’s β-adrenergic receptor. Reversal of these actions occurs when insulin binds to the cell’s insulin receptor, resulting in activation of phosphoprotein phosphatase. PI-1 The inhibitor PI-1 can block activity of phosphpoprotein phosphatase only if it (PI-1) is phosphorylated. When PI-1 gets dephosphorylated, it no longer functions as an inhibitor, so phosphoprotein phosphatase be- Figure 6.42 - Inactivation of phosphoprotein phosphatase by protein kinase A via phosphorylation of PI-1 (Inhibitor) and the GM binding protein Image by Pehr Jacobson Interactive Learning Module HERE comes active. Now, here is the clincher - PI-1 gets phosphorylated by PKA (thus, when epinephrine or glucagon binds to a cell) and gets dephosphorylated when insulin binds to a cell. Another regulatory mechanism Another way to regulate phosphoprotein phosphatase in the liver involves GPa directly (Figure 6.43). In liver cells, phosphoprotein phosphatase is bound to a protein called GL. GL can also bind to GPa. As shown in the figure, if the three proteins are complexed together (top of figure), then PP1 (phosphoprotein phosphatase) is inactive. When glucose is present (such as when the liver has made too much glucose), then the free glucose binds to the GPa and causes GPa to be released from the GL. This has the effect of activating phosphoprotein phosphatase, which begins dephosphorylating enzymes. As shown in the figure, two such enzymes are GPa (making GPb) and glycogen synthase b, making glycogen synthase a. These dephosphorylations have opposite effects on the two enzymes, making GPb, which is less active and glycogen synthase a, which is much more active. Glycogen synthesis The anabolic pathway opposing glycogen breakdown is that of glycogen synthesis. Just Figure 6.43 - Regulation of phosphoprotein phosphatase (PP-1) activity by GPa Image by Penelope Irving as cells reciprocally regulate glycolysis and gluconeogenesis to prevent a futile cycle between these pathways, so too do cells use reciprocal schemes to regulate glycogen breakdown and synthesis. Synthesis of glycogen starts with G1P, which is converted to an 'activated' intermediate, UDPglucose. This activated intermediate is what 'adds' the glucose to the growing glycogen chain in a reaction catalyzed by the enzyme known as glycogen synthase (Figure 6.44). Once the glucose is added to glycogen, the glycogen molecule may need to have branches inserted in it by the enzyme known as branching enzyme (Figure 6.45). Steps Let us first consider the steps in glycogen synthesis. 1) Glycogen synthesis from glucose involves phosphorylation to form G6P, and isomerization to form G1P (using phospho- igure 6.45 - Branch formation in glycogen by branching enzyme Image by Penelope Irving Figure 6.44 - Catalytic activity of glycogen synthase Image by Penelope Irving glucomutase, common to glycogen breakdown). G1P is reacted with UTP to form UDP-glucose in a reaction catalyzed by UDP-glucose pyrophosphorylase. Glycogen synthase catalyzes synthesis of glycogen by joining carbon #1 of the UDP-derived glucose onto the carbon #4 of the non-reducing end of a glycogen chain, to form the familiar α(1,4) glycogen links. Another product of the reaction is UDP. “Primer” requirements It is also worth noting, in passing, that glycogen synthase will only add glucose units from UDP-Glucose onto a preexisting glycogen chain that has at least four glucose residues. Linkage of the first few glucose units to form the minimal "primer" needed for glycogen synthase recognition is catalyzed by a protein called glycogenin, which attaches to the first glucose and catalyzes linkage of the first eight glucoses by α(1,4) bonds. 3) The characteristic α(1,6) branches of glycogen are the products of the enzyme known as branching enzyme. Branching enzyme breaks α(1,4) chains and carries the broken chain to the carbon #6 and forms an α(1,6) linkage (Figure 6.45). Regulation of glycogen synthesis The regulation of glycogen biosynthesis is reciprocal to that of glycogen breakdown. It also has a cascading covalent modification system similar to the glycogen breakdown system described above. In fact, part of the system is identical to glycogen breakdown. Epinephrine or glucagon signaling stimulates adenylate cyclase to make cAMP, which activates PKA. Figure 6.46 - Reciprocal regulation by the phosphorylation cascade - glycogen breakdown activated / glycogen synthesis inhibited Image by Penelope Irving Effect of phosphorylation In glycogen synthesis, protein kinase A phosphorylates the active form of glycogen synthase (GSa), and converts it into the usually inactive b form (called GSb). Note the conventions for glycogen synthase and glycogen phosphorylase. For both enzymes, the more active forms are called the 'a' forms (GPa and GSa) and the less active forms are called the 'b' forms (GPb and GSb). The major difference, however, is that GPa has a phosphate, but GSa does not and GPb has no phosphate, but GSb does. Thus phosphorylation and dephosphorylation have opposite effects on the enzymes of glycogen metabolism (Figure 6.46). This is the hallmark of reciprocal regulation. It is of note that the less active glycogen synthase form, GSb, can be activated by G6P. Recall that G6P had the exactly opposite effect on GPb. Glycogen synthase, glycogen phosphorylase (and phosphorylase kinase) can all be dephosphorylated by the same enzyme - phosphoprotein phosphatase - and it is activated when insulin binds to its receptor in the cell membrane. Big picture
In the big picture, binding of epinephrine or glucagon to appropriate cell receptors stimulates a phosphorylation cascade which simultaneously activates breakdown of glycogen by glycogen phosphorylase and inhibits synthesis of glycogen by glycogen synthase. Epinephrine, is also known as adrenalin, and the properties that adrenalin gives arise from a large temporary increase of blood glucose, which powers muscles. On the other hand, insulin stimulates dephosphorylation by activating phosphoprotein phosphatase. Dephosphorylation reduces action of glycogen phosphorylase (less glycogen breakdown) and activates glycogen synthase (starts glycogen synthesis). Our bodies make glycogen when blood glucose levels rise. Since high blood glucose levels are harmful, insulin stimulates cells to take up glucose. In the liver and in muscle cells, the uptaken glucose is made into glycogen. Figure 6.47 - Cotton - the purest natural form of cellulose Wikipedia Interactive Learning Module HERE Cellulose synthesis Cellulose is synthesized as a result of catalysis by cellulose synthase. Like glycogen synthesis it requires an activated intermediate to add glucose residues and there are two possible ones - GDP-glucose and UDPglucose, depending on which cellulose synthase is involved. In plants, cellulose provides support to cell walls. The reaction catalyzed is shown next where Cellulosen = a polymer of [(1→4)-β-Dglucosyl] n units long. The GDP-glucose reaction is the same except with substitution of GDP-glucose for UDP-Figure 6.48 - The Pentose Phosphate Pathway - Enzymes - 1 = G6P dehydrogenase / 2 = 6-Phosphogluconolactonase / 3 = 6-PG dehydrogenase / 4a = Ribose 5- phosphate isomerase / 4b = Ribulose 5-phosphate 3-epimerase / 5,7 = Transketolase / 6 = Transaldolase UDP-glucose + Cellulosen UDP + Cellulosen+1 glucose. UDP-glucose for the reaction is obtained by catalysis of sucrose synthase. The enzyme is named for the reverse reaction. Pentose phosphate pathway The pentose phosphate pathway (PPP - also called the hexose monophosphate shunt) is an oxidative pathway involving sugars that is sometimes described as a parallel pathway to glycolysis. It is, in fact, a pathway with multiple inputs and outputs (Figure 6.48). PPP is also a major source of NADPH for biosynthetic reactions and can provide ribose-5-phosphate for nucleotide synthesis. Though when drawn out, the pathway’s “starting point” is often shown as glucose-6-phosphate (G6P), in fact there are multiple entry points including other glycolysis intermediates, such as fructose-6-phosphate (F6P) and glyceraldehyde-3-phosphate (GLYAL-3-P), as well as less common sugar compounds with 4,5, and 7 carbons. The multiple entry points and multiple outputs gives the cell tremendous flexibility to meet its needs by allowing it to use a variety of materials to make any of these products. Oxidation #1 Beginning with G6P, PPP proceeds through its oxidative phase as follows: The enzyme catalyzing the reaction is G6P dehydrogenase. It is the rate limiting step of the pathway and the enzyme is inhibited both by NADPH and acetyl-CoA. NADPH is important for anabolic pathways, such as fatty acid synthesis and also for maintaining glutathione in a reduced state. The latter is important in protection against damage from reactive oxygen species. Deficiency of the G6P dehydrogenase enzyme is not rare, leading to acute hemolytic anemia, due to reduced NADPH concentration, and a reduced ability of the cell to disarm reactive oxygen species with glutathione. Reduced activity of the enzyme appears to have a protective effect against malarial infection, likely due to the increased fragility of the red blood cell membrane, which is then unable to sustain an infection by the parasite. Hydrolysis Reaction #2 is a hydrolysis and it is catalyzed by Hydrolysis Reaction #2 is a hydrolysis and it is catalyzed by 6-phosphogluconolactonase. The reac- Sucrose + UDP UDP-glucose + Fructose G6P + NADP+ 6-Phosphoglucono-δ-lactone + NADPH tion converts the circular 6-phosphoglucono- δ-lactone into the linear 6- phosphogluconate (6-PG) in preparation for oxidation in the next step. Decarboxylation Reaction #3 is the only decarboxylation in the PPP and the last oxidative step. It is catalyzed by 6-phosphogluconate dehydrogenase. Mutations disabling the protein made from this gene negatively impact red blood cells. At this point, the oxidative phase of PPP is complete and the remaining reactions involve molecular rearrangements. Ru5P has two possible fates and these are each described below. Isomerization Reaction 4a: The enzyme catalyzing this reversible reaction is Ru5P isomerase (top of next column). It is important because this is the way cells make R-5-P for nucleotide synthesis. The R-5-P can also be used in other PPP reactions shown elsewhere. Epimerization Reaction 4b (catalyzed by Ru-5-P epimerase) is another source of a pentose sugars and provides an important substrate for subsequent reactions. Transketolase reactions The other reactions don’t really have an order to them and whether they occur or not depends on cellular needs. The first enzyme, transketolase, is flexible in terms of its substrate/product combinations and is used not only in PPP, but also in the Calvin cycle of plants. It catalyzes the next two reactions In the first reaction (above), two phosphorylated sugars of 5 carbons each are converted into one phosphorylated sugar of 3 carbons and one of 7 carbons. In the second (next page), a five carbon sugar phosphate and aRu-5-P Xylulose-5-phosphate (Xu-5-P) Xu-5-P + R-5-P GLYAL-3-P + Sedoheptulose-7-phosphate (S-7-P) 6-PG + NADP+ Ribulose-5-phosphate (Ru-5-P) + NADPH + CO2 6-Phosphoglucono-δ-lactone + H2O 6-phosphogluconate (6-PG) + H+ Ru-5-P Ribose-5-phosphate (R-5-P) four carbon sugar phosphate are rearranged into sugar phosphates with 3 and 6 carbons. Glycolysis intermediates In the reversible reactions of the pentose phosphate pathway, one can see how glycolysis intermediates can easily be rearranged and made into other sugars. Thus, GLYAL-3-P and F6P can be readily made into Ribose-5- phosphate for nucleotide synthesis. Involvement of F6P in the pathway permits cells to continue making nucleotides (by making R-5-P) or tryptophan (by making E- 4-P) even if the oxidative reactions of PPP are inhibited. The last reaction is catalyzed by the enzyme known as transaldolase. TPP co-factor
Transketolase uses thiamine pyrophosphate (TPP) to catalyze reactions. TPP’s thiaFigure 6.49 - Intermediates of the pentose phosphate pathway Xu-5-P + Erythrose-4-phosphate (E-4-P) GLYAL-3-P + F6P GLYAL-3-P + S-7-P E-4-P + F6P zole ring’s nitrogen and sulfur atoms on either side of a carbon, allow it to donate a proton and act as an acid, thus forming a carbanion, which gets stabilized by the adjacent tetravalent nitrogen (Figures 6.50 & 6.51)). The stabilized carbanion plays important roles in the reaction mechanism of enzymes, such as transketolase that use TPP as a cofactor. Commonly, the carbanion acts as a nucleophile that attacks the carbonyl carbon of the substrate. Such is the case with transketolase. Attack by the carbanion breaks the carbonyl bond on the substrate and covalently links it to the ionized carbon of TPP, thus allowing it to “carry” the carbonyl group to the other substrate for attachment. In this way, two carbons are moved from Xu- 5-P to E-4-P to make F6P (from E-4-P) and GLYAL-3-P (from Xu-5-P). Similarly, S-7-P and GLYAL-3-P are made from R-5-P and Xu-5-P, respectively. Thiamines Thiamines are a class of compounds involved in catalysis of important respiration-related The Pentose Phosphate Pathway by Kevin Ahern I need erythrose phosphate And don’t know what to do My cells are full of G-6-P And NADP too But I just hit upon a plan As simple as can be I’ll run reactions through the path That’s known as PPP In just two oxidations There’s ribulose-5P Which morphs to other pentoses Each one attached to P The next step it is simple Deserving of some praise The pentose carbons mix and match Thanks to transketolase Glyceraldehyde’s a product Sedoheptulose is too Each with a trailing phosphate But we are not quite through Now three plus seven is the same As adding six and four By swapping carbons back and forth There’s erythrose-P and more At last I’ve got the thing I need From carbons trading places I’m happy that my cells are full Of some transaldolases Figure 6.50 - Thiamine pyrophosphate reactions in the citric acid cycle, pyruvate metabolism, the pentose phosphate pathway, and the Calvin cycle. Thiamine was the first water-soluble vitamin (B1) to be discovered via association with the peripheral nervous system disease known as Beriberi. Thiamine pyrophosphate (TPP) is an enzyme cofactor found in all living systems derived from thiamine by action of the enzyme thiamine diphosphokinase. TPP facilitates catalysis of several biochemical reactions essential for tissue respiration. Deficiency of the vitamin is rare today, though people suffering from Crohn’s disease, anorexia, alcoholism or undergoing kidney dialysis may develop deficiencies. TPP is required for the oxidative decarboxylation of pyruvate to form acetyl-CoA and similar reactions. Transketolase, an important enzyme in the pentose phosphate pathway, also uses it as a coenzyme. Besides these reactions, TPP is also required for oxidative decarboxylation of α-keto acids like α-ketoglutarate and branched-chain α-keto acids arising from metabolism of valine, isoleucine, and leucine. Figure 6.51 - Mechanism of action of thiamine pyrophosphate (TPP) - 1) Carbanion formation; 2) Nucleophilic attack; 3) Covalent attachment of carbonyl; 4) Transfer to second group; 5) Release of product and regeneration of TPP TPP acts in the pyruvate dehydrogenase complex to assist in decarboxylation of pyruvate and “carrying” the activated acetaldehyde molecule to its attachment (and subsequent oxidation) to lipoamide. Central to TPP’s function is the thiazolium ring, which stabilizes carbanion intermediates (through resonance) by acting as an electron sink (Figure 6.51). Such action facilitates breaking of carbon-carbon bonds such as occurs during decarboxylation of pyruvate to produce the activated acetaldehyde. Thiamine deficiency Thiamine is integral to respiration and is needed in every cell. Acute deficiency of thiamine leads to numerous problems - the best known condition is beriberi, whose symptoms include weight loss, weakness, swelling, neurological issues, and irregular heart rhythms. Figure 6.52 - The Calvin cycle - The resynthesis phase has multiple steps and is described below. Image by Aleia Kim Causes of deficiency include poor nutrition, significant intake of foods containing the enzyme known as thiaminase, foods with compounds that counter thiamine action (tea, coffee), and chronic diseases, including diabetes, gastrointestinal diseases, persistent vomiting. People with severe alcoholism often are deficient in thiamine. Calvin cycle The Calvin cycle (Figure 6.52) is a metabolic pathway occurring exclusively in photosynthetic organisms. Commonly referred to as the “Dark Cycle” or the Light-Independent Cycle, the Calvin cycle does not actually occur in the dark. The cell/chloroplast simply is not directly using light energy to drive it. Assimilation It is in the Calvin cycle of photosynthesis that carbon dioxide is taken from the atmosphere and ultimately built into glucose (or other sugars). Reactions of the Calvin cycle take place in regions of the chloroplast known as the stroma, the fluid areas outside of the thylakoid membranes. The cycle can be broken into three phases 1) assimilation of CO2 2) reduction reactions 3) regeneration of the starting material, ribulose 1,5 bisphosphate (Ru1,5BP). Though reduction of carbon dioxide to glucose ultimately requires electrons from twelve molecules of NADPH (and 18 ATPs), it is confusing because one reduction occurs 12 times (1,3 BPG to GLYAL-3P) to input the overall reduction necessary to make one glucose. Carbon dioxide Another reason students find the pathway confusing is because the carbon dioxide molecules are absorbed one at a time into six different molecules of Ru1,5BP. At no point are the six carbons ever together in the same molecule to make a single glucose. Instead, six molecules of Ru1,5BP (30 carbons) gain six more carbons via carbon dioxide and then split into 12 molecules of 3- phosphoglycerate (36 carbons). The gain of six carbons allows two three carbon molecules to be produced in excess for each turn of the cycle. These two molecules molecules are then converted into glucose using the enzymes of gluconeogenesis. The other ten molecules of 3-PG are used to regenerate the six molecules of Ru1,5BP. Figure 6.53 - Rubisco, the most abundant enzyme on Earth Cyclic pathway

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