16.2 Reactions of the Citric Acid Cycle in Chapter 16 The Citric Acid Cycle

16.2 Reactions of the Citric Acid Cycle

We are now ready to trace the process by which acetyl-CoA undergoes oxidation. This chemical transformation is carried out by the citric acid cycle, the first cyclic pathway we have encountered (Fig. 16-7). To begin a turn of the cycle, acetyl-CoA donates its acetyl group to the four-carbon compound oxaloacetate to form the six-carbon citrate. Citrate is then transformed into isocitrate, also a six-carbon molecule, which is dehydrogenated with loss of ${CO}_{2}$ $CO Subscript 2$ to yield the five-carbon compound α-ketoglutarate (also called 2-oxoglutarate). α-Ketoglutarate undergoes loss of a second molecule of ${CO}_{2}$ $CO Subscript 2$ and ultimately yields the four-carbon compound succinate. Succinate is enzymatically converted in three steps into the four-carbon oxaloacetate — which is then ready to react with another molecule of acetyl-CoA. In each turn of the cycle, one acetyl group (two carbons) enters as acetyl-CoA and two molecules of ${CO}_{2}$ $CO Subscript 2$ leave; one molecule of oxaloacetate is used to form citrate and one molecule of oxaloacetate is regenerated. No net removal of oxaloacetate occurs; one molecule of oxaloacetate can theoretically bring about oxidation of an infinite number of acetyl groups, effectively acting catalytically; the steady-state concentration of oxaloacetate is very low (micromolar). Four of the eight steps in this process are oxidations, in which the energy of oxidation is very efficiently conserved in the form of the reduced coenzymes NADH and ${FADH}_{2}$ $FADH Subscript 2$ . These two carriers donate their electrons to the respiratory chain where electron flow drives ATP synthesis.

A figure shows the citric acid cycle as a series of 8 reactions. — FIGURE 16-7 **Reactions of the citric acid cycle.** The carbon atoms shaded in pink are those derived from the acetate of acetyl-CoA in the first turn of the cycle; these are *not* the carbons released as ${CO}_{2}$ $CO Subscript 2$ in the first turn. Note that in succinate and fumarate, the two-carbon group derived from acetate can no longer be specifically denoted; because succinate and fumarate are symmetric molecules, C-1 and C-2 are indistinguishable from C-4 and C-3. The red arrows show where energy is conserved by electron transfer to FAD or ${NAD}^{+}$ $NAD Superscript plus$ , forming ${FADH}_{2}$ $FADH Subscript 2$ or $NADH + H^{+}$ $NADH plus upper H Superscript plus$ . Steps , , and are essentially irreversible in the cell; all other steps are reversible. The nucleoside triphosphate product of step may be either ATP or GTP, depending on which succinyl-CoA synthetase isozyme is the catalyst.

FIGURE 16-7 **Reactions of the citric acid cycle.** The carbon atoms shaded in pink are those derived from the acetate of acetyl-CoA in the first turn of the cycle; these are *not* the carbons released as ${CO}_{2}$ $CO Subscript 2$ in the first turn. Note that in succinate and fumarate, the two-carbon group derived from acetate can no longer be specifically denoted; because succinate and fumarate are symmetric molecules, C-1 and C-2 are indistinguishable from C-4 and C-3. The red arrows show where energy is conserved by electron transfer to FAD or ${NAD}^{+}$ $NAD Superscript plus$ , forming ${FADH}_{2}$ $FADH Subscript 2$ or $NADH + H^{+}$ $NADH plus upper H Superscript plus$ . Steps , , and are essentially irreversible in the cell; all other steps are reversible. The nucleoside triphosphate product of step may be either ATP or GTP, depending on which succinyl-CoA synthetase isozyme is the catalyst.

The citric acid cycle is shown as a series of reactions that form a circle that begins with the addition of acetyl-Co A at the twelve o’clock position. Acetyl-Co A is mostly shown in a red box as C H 3 bonded to C double bonded to O above bonded to S outside of the box further bonded to C o A. A blue line extends down to join the cycle at the 12 o’clock position above the words, citrate synthase. Step 1: Aldol condensation: methyl group of acetyl-Co A converted to methylene in citrate. A blue arrow points from the place that acetyl-Co A joins the cycle at the 12 o’clock position to citrate at about the one o’clock position. A blue curved arrow shows that H 2 O is added and Co A – S H is removed. This yields citrate, shown as a three-carbon vertical chain with C 1 and its substituents shown in a red box. C 1 is bonded to 2 H and to C O O minus to the right, C 2 is bonded to O H to the left and C O O minus to the right, C 3 is bonded to 2 H and to C O O minus to the right. Step 2a: Dehydration/rehydration: Bonded O H group of citrate repositioned in isocitrate, which sets up decarboxylation in next step. Blue forward and reverse arrows point from citrate to italicized cis-aconitate in brackets at just before the three o’clock position. The word aconitase is beneath the arrows. The forward-pointing arrow has a branch to show the loss of H 2 O. This yields italicized cis end italics aconitate, which is a three-carbon vertical chain in brackets with the top carbon and its substituents shown in a red box. The top carbon is bonded to 2 H and to C O O minus to the right, the middle carbon is bonded to C O O minus to the right and double bonded to the bottom carbon, and the bottom carbon is bonded to C O O minus to the right and to H below. Step 2b: (Rehydration). Blue forward and reverse arrows next to the word aconitase point from italicized cis end italics aconitate to isocitrate at just past the three o’clock position. The forward arrow is joined by a line showing the addition of H 2 O. This yields isocitrate, shown as a four-carbon chain with the top carbon and its substituents shown in a red box. The top carbon is bonded to 2 H and to C O O minus to the right, the second carbon is bonded to H to the left and C O O minus to the right, the third carbon is bonded to O H to the left and to H to the right, and the bottom carbon forms C O O minus. Step 3: Oxidative decarboxylation: A bonded O H group oxidizes to carbonyl, which in turn facilitates decarboxylation by stabilizing carbanion formed on the adjacent carbon. A blue arrow accompanied by the words isocitrate dehydrogenase splits to show the loss of C O 2 and formation of Greek letter alpha-ketoglutarate at the five o’clock position. A red arrow from the main blue arrow points to an orange circle labeled (3) N A D H in the center of the cycle. Greek letter alpha-ketoglutarate is a four-carbon vertical chain with the top carbon and its substituents shown in a red box. The top carbon is bonded to 2 H and to C O O minus to the right, the second carbon is bonded to 2 H, the third carbon is double bonded to O to the right, and the bottom carbon forms C O O minus. Step 4: Oxidative decarboxylation: pyruvate-dehydrogenase-like mechanism; dependent on the carbonyl on the adjacent carbon. A blue forward arrow points to succinyl-Co A just past the six o’clock position. Accompanying text reads, Greek letter alpha ketoglutarate dehydrogenase complex. A blue curved arrow shows the addition of Co A – S H and loss of C O 2. A red arrow branching from the main blue arrow points to the orange circle labeled (3) N A D H in the center of the cycle. Step 5: Substrate-level phosphorylation: energy of thioester conserved in phosphoanhydride bond of G T P of A T P. Blue forward and reverse arrows point from succinyl-Co A to succinate at the seven o’clock position. Accompanying text reads, succinyl-Co A synthetase. A curved blue arrow that meets the forward arrow shows the addition of G D P (A D P) plus P I and the loss of an orange sphere labeled G T P (A T P). An arrow branches off to show the loss of Co A – S H. This yields succinate, which is a vertical three-carbon chain. The top carbon is bonded to 2 H and to C O O minus, the middle carbon is bonded to 2 H, and the bottom carbon forms C O O minus. Step 6: Dehydrogenation: introduction of double bond initiates methylene oxidation sequence. Blue forward and reverse arrows point from succinate to fumarate just below the nine o’clock position. The arrows are accompanied by text reading, succinate dehydrogenase. A red arrow curves from the forward arrow to show the low of an orange oval labeled F A D H 2. Fumarate is a vertical four-carbon chain. The top carbon forms C O O minus, the second carbon is bonded to H and double bonded to the third carbon, the third carbon is bonded to H, and the fourth carbon forms C O O minus. Step 7: Hydration: addition of water across double bond introduces bonded to H group for next oxidation step. Blue forward and reverse arrows point from fumarate to malate at the ten o’clock position. Accompanying text reads, fumarase. A blue curved line joins the forward arrow to show the addition of H 2 O. Malate is a four-carbon chain. The top carbon forms C O O minus, the second carbon is bonded to H and to O H on the left, the third carbon is bonded to 2 H, and the fourth carbon forms C O O minus. Step 8: Dehydrogenation: oxidation of bonded O H completes oxidation sequence; generates carbonyl positioned to facilitate Claisen condensation in next step. Blue forward and reverse arrows point from malate to oxaloacetate at the eleven o’clock position. Accompanying text reads, malate dehydrogenase. A red arrow points from the forward arrow to the orange oval labeled (3) N A D H in the center of the cycle. Oxaloacetate has C double bonded to O to the left, bonded to C O O minus to the right, and bonded to C H 2 below further bonded to C O O minus. An blue arrow points from oxaloacetate to citrate to continue the cycle. All data are approximate.

Although the citric acid cycle is central to energy-yielding metabolism, its role is not limited to energy conservation. Four- and five-carbon intermediates of the cycle serve as precursors for a wide variety of products. To replace intermediates removed for this purpose, cells employ anaplerotic (replenishing) reactions, which are described below.

Eugene Kennedy and Albert Lehninger showed in 1948 that, in eukaryotes, the entire set of reactions of the citric acid cycle takes place in mitochondria. Isolated mitochondria were found to contain not only all the enzymes and coenzymes required for the citric acid cycle, but also all the enzymes and proteins necessary for the last stage of respiration — electron transfer and ATP synthesis by oxidative phosphorylation. As we shall see in later chapters, mitochondria also contain the enzymes for the oxidation of fatty acids and some amino acids to acetyl-CoA, and the oxidative degradation of other amino acids to α-ketoglutarate, succinyl-CoA, or oxaloacetate. Thus, in nonphotosynthetic eukaryotes, the mitochondrion is the site of most energy-yielding oxidative reactions and of the coupled synthesis of ATP. In photosynthetic eukaryotes, mitochondria are the major site of ATP production in the dark, but in daylight, chloroplasts produce most of the organism’s ATP. In most bacteria, the enzymes of the citric acid cycle are in the cytosol, and the plasma membrane plays a role analogous to that of the inner mitochondrial membrane in ATP synthesis (Chapter 19).

The Sequence of Reactions in the Citric Acid Cycle Makes Chemical Sense

Acetyl-CoA produced in the breakdown of carbohydrates, fats, and proteins must be completely oxidized to ${CO}_{2}$ $CO Subscript 2$ if the maximum potential energy is to be extracted from these fuels. However, it is not biochemically feasible to directly oxidize acetate (or acetyl-CoA) to ${CO}_{2}$ $CO Subscript 2$ . Decarboxylation of this two-carbon acid would yield ${CO}_{2}$ $CO Subscript 2$ and methane $({CH}_{4})$ $left-parenthesis CH Subscript 4 Baseline right-parenthesis$ . Methane is chemically rather stable, and except for certain methanotrophic bacteria that grow in methane-rich niches, organisms do not have the cofactors and enzymes needed to oxidize methane. Methylene groups $(— {CH}_{2} —)$ $left-parenthesis em-dash CH Subscript 2 Baseline em-dash right-parenthesis$ , however, are readily metabolized by enzyme systems present in most organisms. In typical oxidation sequences, two adjacent methylene groups $(— {CH}_{2} — {CH}_{2} —)$ $left-parenthesis em-dash CH Subscript 2 Baseline em-dash CH Subscript 2 Baseline em-dash right-parenthesis$ are involved, at least one of which is adjacent to a carbonyl group. As we noted in Chapter 13 (p. 473), carbonyl groups are particularly important in the chemical transformations of metabolic pathways. The carbon of the carbonyl group has a partial positive charge due to the electron-withdrawing property of the carbonyl oxygen and is therefore an electrophilic center. A carbonyl group can facilitate the formation of a carbanion on an adjoining carbon by delocalizing the carbanion’s negative charge. We see an example of the oxidation of a methylene group in the citric acid cycle, as succinate is oxidized (steps to in Fig. 16-7) to form a carbonyl (in oxaloacetate) that is more chemically reactive than either a methylene group or methane.

In short, if acetyl-CoA is to be oxidized efficiently, the methyl group of the acetyl-CoA must be attached to something. The first step of the citric acid cycle — the condensation of acetyl-CoA with oxaloacetate — neatly solves the problem of the unreactive methyl group. The carbonyl of oxaloacetate acts as an electrophilic center, which is attacked by the methyl carbon of acetyl-CoA in an aldol condensation (p. 473) to form citrate (step in Fig. 16-7). The methyl group of acetate has been converted into a methylene in citric acid. This tricarboxylic acid then readily undergoes a series of oxidations that eliminate two carbons as ${CO}_{2}$ $CO Subscript 2$ . Note that all steps featuring the breakage or formation of carbon–carbon bonds (steps , , and ) rely on properly positioned carbonyl groups. As in all metabolic pathways, there is a chemical logic to the sequence of steps in the citric acid cycle: each step either involves an energy-conserving oxidation or is a necessary prelude to the oxidation, placing functional groups in position to facilitate oxidation or oxidative decarboxylation. As you learn the steps of the cycle, keep in mind the chemical rationale for each; it will make the process easier to understand and remember.

The Citric Acid Cycle Has Eight Steps

In examining the eight successive reaction steps of the citric acid cycle, we place special emphasis on the chemical transformations taking place as citrate formed from acetyl-CoA and oxaloacetate is oxidized to yield ${CO}_{2}$ $CO Subscript 2$ and the energy of this oxidation is conserved in the reduced coenzymes NADH and ${FADH}_{2}$ $FADH Subscript 2$ .

Formation of Citrate

The first reaction of the cycle is the condensation of acetyl-CoA with oxaloacetate to form citrate, catalyzed by citrate synthase.

A figure shows the reaction of acetyl-Co A and oxaloacetate to form citrate in a reaction catalyzed by citrate synthase and with Greek letter delta G prime degree symbol equals negative 32.2 k J per mole.

Acetyl-Co A is shown mostly in a red box as C H 3 bonded to C double bonded to O to the upper right and bonded to nonhighlighted S to the lower right further bonded to Co A. Oxaloacetate is shown a C double bonded to O to the left, bonded to C O O minus to the right, and bonded to C H 2 below further bonded to C O O minus. An arrow points right above citrate synthase accompanied by a curved arrow above that shows the addition of H 2 O and loss of Co A – S H. This yields citrate, which is shown as a three-carbon vertical chain with the top carbon, C 1, and its substituents shown in a red box. C 1 is bonded to 2 H and to C double bonded to O to the upper right and bonded to nonhighlighted O minus to the lower left; C 2 is bonded to O H to the left and to C O O minus to the right; C 3 is bonded to 2 H and to C O O minus to the right.

In this reaction, the methyl carbon of the acetyl group is joined to the carbonyl group (C-2) of oxaloacetate. Citroyl-CoA is a transient intermediate formed on the active site of the enzyme (see Fig. 16-9). It undergoes hydrolysis to free CoA and citrate, which are released from the active site. The hydrolysis of this high-energy thioester intermediate makes the forward reaction highly exergonic. The large, negative standard free-energy change of the forward citrate synthase reaction is essential to the operation of the cycle because the concentration of oxaloacetate is normally very low (micromolar). The CoA liberated in this reaction is recycled to participate in the oxidative decarboxylation of another molecule of pyruvate by the PDH complex.

Citrate synthase from mitochondria is a homodimer (Fig. 16-8). Each subunit is a single polypeptide with two domains, one large and rigid, the other smaller and more flexible, with the active site between them. Oxaloacetate, the first substrate to bind to the enzyme, induces a large conformational change in the flexible domain, creating a binding site for the second substrate, acetyl-CoA. When citroyl-CoA has formed in the enzyme active site, another conformational change brings about thioester hydrolysis, releasing CoA-SH. This induced fit of the enzyme first to its substrate and then to its reaction intermediate decreases the likelihood of premature and unproductive cleavage of the thioester bond of acetyl-CoA. Kinetic studies of the enzyme are consistent with this ordered bisubstrate mechanism (see Fig. 6-15). The reaction catalyzed by citrate synthase is essentially an aldol condensation (p. 473), involving a thioester (acetyl-CoA) and a ketone (oxaloacetate) (Fig. 16-9).

A figure shows the structure of citrate synthase and how it undergoes a conformational change from an open form in part a to a closed form with bound oxaloacetate in part b. — FIGURE 16-8 Structure of citrate synthase. The flexible domain of each subunit undergoes a large conformational change on binding oxaloacetate, creating a binding site for acetyl-CoA. (a) Open form of the enzyme alone; (b) closed form with bound oxaloacetate and a stable analog of acetyl-CoA (carboxymethyl-CoA). In these representations one subunit is colored tan and one green. [Data from (a) PDB ID 5CSC, D.-I. Liao et al., *Biochemistry* 30:6031, 1991;]

Part a shows citrate synthase without bound oxaloacetate. It has a rounded brown potion on the left that has a hook-like curve extending to its lower left. It has a similar green portion on the right with the hook-like curve extending upward to the upper right. Where the two pieces meet in the center, they overlap so that some green is visible in the middle of the brown portion and vice versa. At the bottom end of the brown lower left hook, there is an arrow showing that a small red molecule of oxaloacetate next to a linear purple acetyl-Co A analog that is thicker in its center can move into the opening. An arrow points from the end of the hook toward the center, showing that it can close the opening. Above the green hook to the upper right, similar oxaloacetate and acetyl-Co A analog molecules are present with an arrow showing movement into the space and an arrow showing that the hook can move inward to close the opening. An arrow points down to part b, which shows a similar structure that is almost rounded because the hooked regions have come together to complete the spherical shape. Where the openings had been, small pieces of red and purple are visible to show where oxaloacetate molecules and the acetyl-Co A analogs are located.

A figure shows the action of citrate synthase in three steps, beginning with the binding of oxaloacetate, followed by the binding of acetyl-Co A, and then the production of citrate. — MECHANISM FIGURE 16-9 Citrate synthase. In the citrate synthase reaction in mammals, oxaloacetate binds first, in a strictly ordered reaction sequence. This binding triggers a conformation change that opens up the binding site for acetyl-CoA. Oxaloacetate is specifically oriented in the active site of citrate synthase by interaction of its two carboxylates with two positively charged Arg residues (not shown here). [Information from S. J. Remington, *Curr. Opin. Struct. Biol.* 2:730, 1992.]

Part of a long oval extends in from the right, representing an opening in the surrounding citrate synthase enzyme. At the left side of citrate synthase, N at the lower right vertex of a five-membered ring has a bond to H within the opening. To the lower left of this N, the ring has a bottom vertex. It also has a double bond between the bottom vertex and lower left vertex, a lower left vertex bonded to H i s superscript 320, an upper left vertex with N substituted for C and bonded to H, and a dashed line between the upper left and lower right vertices with a plus symbol along the line beneath the upper right vertex. At the top center of the oval, there is another N at the lower left vertex of a five-membered ring within citrate synthase that extends an H into the opening below. This ring has a dashed linen connecting the lower left and upper left vertices with a plus symbol at the left side vertex between them, N substituted for C at the upper right vertex and bonded to H, an upper right vertex bonded to H i s superscript 274, and a double bond on its left side. Citrate synthase also has A s p superscript 375 beneath the bottom center of the oval opening with a bond up to C that is double bonded to O to its upper left and bonded to O to its upper right with a red pair of electrons and a negative charge. Oxaloacetate is shown at the left side of the oval opening as C double bonded to O to the left, bonded to C O O minus to the right, and bonded to C H 2 below further bonded to C O O minus to the right. Acetyl-Co A is shown mostly in red in the center right of the opening as C bonded to H above, to the left, and below, and bonded to C to the right double bonded to O above and bonded to nonhighlighted S on the right bonded to C o A. Red curved arrows point from the red pair of electrons on O minus at the boundary of citrate synthase at the bottom center of the oval to H extending down from the left-hand C of acetyl-Co A, from the bond between the same H and the bond between the two C atoms of acetyl-Co A, from the double bond to O in acetyl-Co A to the H extending into the opening from the ring at the upper top of the opening in citrate synthase, and from the bond between the same H and N of the five-membered ring just above the center of the opening in citrate synthase. An arrow points downward. Step 1: The thioester linkage in acetyl-Co A activates the methyl hydrogens. A s p superscript 375 abstracts a proton from the methyl group, forming an enolate intermediate. The intermediate is stabilized by hydrogen bonding to and/or protonation by H i s superscript 274 (full protonation is shown). This yields a similar structure in which A s p superscript 375 beneath the bottom center of the opening is bonded to H within the opening, and acetyl-C o A has become an enol intermediate with red highlighted C bonded to red highlighted H above and to the left connected by a red double bond to red highlighted C connected by a red bond to red highlighted O above and bonded to nonhighlighted S to the right further bonded to C o A. The red highlighted O is further bonded to H connected by three horizontal blue lines to a red pair of electrons beneath N of the five-membered ring below, which no longer has a dashed curved line or positive charge but instead has a double bond at its lower left side. Curved red arrows point from the red pair of electrons on N of the five-membered ring above the opening to H below the three horizontal blue lines, from the bond between this H and the red highlighted O to the bond between the red highlighted O and te C below it, from the double bond between the two C atoms in the enol intermediate and the central C of oxaloacetate that is double bonded to O to its left, and from the double bond between the same C and O and H to the left extending into the ring by its bond from N of the ring just to the left of the boundary of citrate synthase. An arrow points downward. Step 2: The enol(ate) rearranges to attack the carbonyl carbon of oxaloacetate, with H i s superscript 274 positioned to abstract the proton it had previously donated. H i s superscript 320 acts as a general acid. The resulting condensation generates citroyl-Co A. This yields a similar structure in which A s p superscript 375 beneath the center of the opening is no longer bonded to oxygens, the ring to the left of the opening no longer has a curved dashed line from the upper left to lower right vertices with a positive charge on the upper right vertex but instead has a double bond at the upper right side and a red pair of electrons on N at the lower right side adjacent to the boundary with the opening, the ring above the top of the opening has a dashed line between the upper left and lower left vertices again with a positive charge on the left side, and N at the bottom vertex is bonded to H connected by three lines to red highlighted O of citroyl-Co A below. Citroyl-Co A has a three-carbon vertical chain. The top carbon of the chain is highlighted in red, bonded to red highlighted H above and to the left, connected by a red bond to red highlighted C to the right connected by a red double bond to O above connected by three blue lines to H bonded to N of the five-membered ring above and connected to nonhighlighted S to the right further boned to C o A. The middle carbon of the chain is bonded to O H to the left and to C O O minus to the right. The bottom carbon of the chain is bonded to 2 H to the left and to C O O minus to the right. An arrow points downward accompanied by a curved arrow showing the addition of H 2 O with a blue highlighted O and the loss of Co A – S H. Step 3: The thioester is subsequently hydrolyzed, regenerating Co A – S H and producing citrate. This yields a similar oval open region containing citrate, which has a vertical three-carbon chain. The top carbon has red highlighted C bonded to red highlighted H above and to the left and connected by a red bond to red highlighted C O blue highlighted O minus. The middle carbon has C bonded to O H to the left and to C O O minus to the right. The bottom carbon is bonded to 2 H and to C O O minus to the right. In citrate synthase, H i s superscript 274 and further bonded is visible in the upper middle, H i s superscript 320 and further bonded is visible to the left, and A s p superscript 375 and further bonded is visible below.

Formation of Isocitrate via cis-Aconitate

The enzyme aconitase (more formally, aconitate hydratase) catalyzes the reversible transformation of citrate to isocitrate, through the intermediary formation of the tricarboxylic acid cis-aconitate, which normally does not dissociate from the active site. Aconitase can promote the reversible addition of $H_{2} O$ $upper H Subscript 2 Baseline upper O$ to the double bond of enzyme-bound cis-aconitate in two different ways, one leading to citrate and the other to isocitrate:

A figure shows the reaction of citrate to produce isocitrate in a reaction catalyzed by aconitase and with Greek letter delta G prime degree symbol equals 13.3 k J per mole.

Citrate is shown as a three-carbon vertical chain with C 1 bonded to 2 H and to C O O minus, C 2 bonded to O H in a light red box on the left and to C O O minus on the right, and C 3 bonded to H in a red box on the left, to C O O minus on the right, and to H below. Right- and left-pointing arrows are shown above the word aconitase. An arrow branches from the forward arrow to show the loss of H 2 O in a red box. This yields italicized cis end italics-aconitate, shown in brackets. Italicized cis end italics-aconitate is a three-carbon vertical chain with the top C bonded to 2 H and to C O O minus to the right, the middle C bonded to C O O minus to the right and double bonded to the bottom C, and the bottom C bonded to C O O minus to the right and to H below. Right- and left-pointing arrows are shown above the word aconitase. A line joins with the forward arrow to show the addition of H 2 O in a blue box. This yields isocitrate, shown as a four-carbon chain with the top carbon bonded to 2 H and to C O O minus to the right, the second C bonded to blue highlighted H to the left and C O O minus to the right, the middle C bonded to blue highlighted O H to the left and H to the right, and the bottom C forming C O O minus.

Although the equilibrium mixture at pH 7.4 and 25 °C contains less than 10% isocitrate, in the cell the reaction is pulled to the right because isocitrate is immediately consumed in the next step of the cycle, lowering its steady-state concentration. Aconitase contains an iron-sulfur center (Fig. 16-10), which acts both in the binding of the substrate at the active site and in the catalytic addition or removal of $H_{2} O$ $upper H Subscript 2 Baseline upper O$ . In iron-depleted cells, acon-itase loses its iron-sulfur center and acquires a new role in the regulation of iron homeostasis. Aconitase is one of many enzymes known to “moonlight” in a second role (Box 16-1).

A figure shows the structure of the iron-sulfur center in aconitase. — FIGURE 16-10 **Iron-sulfur center in aconitase.** The iron-sulfur center is in red, the citrate molecule in blue. Three Cys residues of the enzyme bind three iron atoms; the fourth iron is bound to one of the carboxyl groups of citrate and also interacts noncovalently with a hydroxyl group of citrate (dashed bond). A basic residue (:B) in the enzyme helps to position the citrate in the active site. The iron-sulfur center acts in both substrate binding and catalysis. The general properties of iron-sulfur proteins are discussed in Chapter 19.

An open region is visible between pieces of an enzyme. There are red atoms making up the iron-sulfur center and blue atoms representing citrate. A narrow opening between two pieces of enzyme on the left has C y s shown above and below the opening. The upper C y s has a bond to S at the boundary with the open area. There is a solid wedge bond from this S to red F e at the upper front left side of a roughly cube-shaped structure. A vertical line extends down from this red F e to red S below at the lower front left corner, from which a horizontal line extends to red F e at the right lower front corner. A solid wedge bond connects this right-hand red F e to S to the lower right, outside of the roughly cube-shaped structure and bonded to C y s at the boundary of the bottom center of the enzyme. The same red F e at the lower right front corner has a solid wedge bond up to red S above at the upper right front corner that has a solid wedge bond to red F e at the upper left side and a solid wedge bond to red F e at the right rear corner. The red F e with the solid wedge bond to S bonded to C y s also has a solid wedge bond to red S at the right rear corner that has a bond up to the red F e at the right rear corner. Red F e at the upper right rear corner has a bond to red S at the upper left rear corner that has a solid wedge bond to red F e at the upper front right corner and to red F e at the lower left rear corner. Red F e at the lower left rear corner has a bond to red S at the lower right rear corner, a solid wedge bond to S outside of the cube further bonded to C y s beneath the narrow opening on the left side, and a solid wedge bond to red S at the left bottom front of the cube. Red Fe at the upper right rear of the cube has a bond to O above further bonded to H to the upper left and right, a bond to its upper right to blue O that forms the upper left vertex of a five-membered ring with blue highlighted C double bonded to blue O at the upper right vertex and solid wedge bonded to blue C at its lower right vertex that is solid wedge bonded to blue O at its bottom vertex that is bonded to blue H below and connected by a dashed line to red F e at the lower left vertex. Blue C at the lower right vertex is also solid wedge bonded to blue C H 2 to its upper right further bonded to C O O minus and is bonded to blue C below that is solid wedge bonded to C O O minus to its left, H below, and H to its right next to a red pair of electrons of B in the boundary of the enzyme to the right.

Box 16-1

Moonlighting Enzymes: Proteins with More Than One Job

Before protein and DNA sequencing became routine, two groups of investigators studying two unrelated questions sometimes discovered that “their” proteins had similar properties. Further comparison sometimes showed that they were studying the same protein by following different functions. We now know that many proteins moonlight in a second role, a phenomenon sometimes called gene sharing. Today, researchers using annotated protein and DNA sequence databases can more easily find proteins of the same sequence that have been identified as having different functions, and a protein sequence annotated as having a given function doesn’t necessarily have only that function. When a protein with a known function is made inactive by a mutation, and the resulting mutant organisms show a phenotype with no obvious relation to that function, protein moonlighting can sometimes explain the puzzling result.

One citric acid cycle enzyme is a known moonlighter. Eukaryotic cells have two isozymes of aconitase. The mitochondrial isozyme converts citrate to isocitrate in the citric acid cycle. The cytosolic isozyme has two distinct functions. It catalyzes the conversion of citrate to isocitrate, providing the substrate for a cytosolic isocitrate dehydrogenase that generates NADPH as reducing power for fatty acid synthesis and other anabolic processes in the cytosol. But it also has a role in cellular iron homeostasis.

All cells must obtain iron for the proteins that require it as a cofactor. In humans, severe iron deficiency results in anemia, an insufficient supply of erythrocytes and a reduced oxygen-carrying capacity that can be life-threatening. Too much iron is also harmful: it accumulates in and damages the liver in hemochromatosis and other diseases. Iron obtained in the diet is carried in the blood by the protein transferrin and enters cells via endocytosis mediated by the transferrin receptor. Once inside cells, iron is used in the synthesis of hemes, cytochromes, Fe-S proteins, and other Fe-dependent proteins; excess iron is stored bound to the protein ferritin. The levels of transferrin, transferrin receptor, and ferritin are therefore crucial to cellular iron homeostasis. The synthesis of these three proteins is regulated in response to iron availability — and aconitase, in its moonlighting job, plays a key regulatory role.

Aconitase has an essential Fe-S cluster at its active site (see Fig. 16-10). When a cell is depleted of iron, this Fe-S cluster is disassembled and the enzyme loses its aconitase activity. But the apoenzyme (apoaconitase, lacking its Fe-S cluster) so formed has now acquired its second activity: regulating iron metabolism. Cytosolic apoaconitase is identical to iron regulatory protein 1 (IRP1) and closely related to IRP2. Both IRP1 and IRP2 bind to regions in the mRNAs encoding ferritin and the transferrin receptor, with effects on iron mobilization and iron uptake. These mRNA sequences are part of hairpin structures (see Fig. 8-23) called iron response elements (IREs), located at the $5^{'}$ $5 prime$ and $3^{'}$ $3 prime$ ends of the mRNAs (Fig. 1). When bound to the $5^{'}$ $5 prime$ -untranslated IRE sequence in the ferritin mRNA, IRPs block ferritin synthesis; when bound to the $3^{'}$ $3 prime$ -untranslated IRE sequence in the transferrin receptor mRNA, they stabilize the mRNA, preventing its degradation and thus allowing the synthesis of more copies of the receptor protein per mRNA molecule. So, in iron-deficient cells, iron uptake becomes more efficient and iron storage (bound to ferritin) is reduced. When cellular iron concentrations return to normal levels, IRP1 regains its Fe-S cluster, converting to aconitase, and IRP2 undergoes proteolytic degradation, ending the low-iron response.

FIGURE 1 Effect of IRP1 and IRP2 on the mRNAs for ferritin and the transferrin receptor. [Information from R. S. Eisenstein, Annu. Rev. Nutr. 20:627, 2000, Fig. 1.]

The figure shows two major horizontal rows and four columns. The first column shows the m R N A structure; the second column specifies changes in translation, stability, and synthesis; the third column shows the effects of low concentrations of iron; and the fourth column shows the effects of high concentrations of iron. The top row shows a green piece of ferritin m R N A with its 5 prime end to the left, then a stem loop extending upward labeled I R E, then a short piece, then a thicker rectangular piece, then another thin piece before ending with a short black line and then A A A (A) subscript italicized n end italics 3 prime. The bottom row shows a green piece of transferring receptor (T f R) m R N A with its 5 prime end to the left, then a short thin region, then a long, thick rectangular piece, then five stem loops extending upward labeled I R E s, then another short thin piece before ending with a short black line and then A A A (A) subscript italicized n end italics 3 prime. The third and fourth columns show low iron concentration and high iron concentrations, respectively. A small blue oval labeled I R P 1 undergoes a reversible reaction to become a large blue oval labeled aconitase when there is a high iron concentration. A small blue oval labeled I R P 2 is converted to many small fragments when there is a high iron concentration. I R P is bound to the iron response element (I R E) under the low iron condition but not under the low iron condition. For the top major row, ferritin m R N A, the low iron concentration condition leads to the presence of I R P 1 and I R P 2, I R P is bound to the iron response element (I R E), ferritin m R N A translation is repressed, and ferritin synthesis is decreased. The high iron condition leads to the presence of aconitase and no I R P 2, I R P is not bound to the iron response element (I R E), ferritin m R N A translation is activated, and ferritin synthesis is increased. For the bottom major row, transferring receptor (T f R) m R N A, the low iron concentration condition leads to the presence of I R P 1 and I R P 2, I R P is bound to the iron response element (I R E), T f R m R N A stability is increased, and T f R synthesis is increased. The high iron condition leads to the presence of aconitase and no I R P 2, I R P is not bound to the iron response element (I R E), T f R m R N A stability is decreased, and T f R synthesis is decreased.

The enzymatically active aconitase and the moonlighting, regulatory apoaconitase have different structures. As the active aconitase, the protein has two lobes that close around the Fe-S cluster; as IRP1, the two lobes open, exposing the mRNA-binding site (Fig. 2).

It makes evolutionary sense that the enzymes of central metabolism have had a long time to evolve additional functions. The glycolytic enzyme pyruvate kinase acts in the nucleus to regulate the transcription of genes that respond to thyroid hormone. Glyceraldehyde 3-phosphate dehydrogenase moonlights both as uracil DNA glycosylase, effecting the repair of damaged DNA, and as a regulator of histone H2B transcription. Several glycolytic enzymes, including phosphoglycerate kinase, triose phosphate isomerase, and lactate dehydrogenase, moonlight as crystallins in the lens of the vertebrate eye.

FIGURE 2 Two forms of cytosolic aconitase/IRP1 with two distinct functions. (a) In aconitase, the two major lobes are closed and the Fe-S cluster is buried; the protein has been made transparent here to show the Fe-S cluster. (b) In IRP1, the lobes open, exposing a binding site for the mRNA hairpin. [Data from (a) PDB ID 2B3Y, J. Dupuy et al., Structure 14:129, 2006; (b) PDB ID 2IPY, W. E. Walden et al., Science 314:1903, 2006.]

Oxidation of Isocitrate to α-Ketoglutarate and $C O_{2}$ $bold upper C bold upper O Subscript bold 2 Baseline$

In the next step, isocitrate dehydrogenase catalyzes oxidative decarboxylation of isocitrate to form α-ketoglutarate (Fig. 16-11). ${Mn}^{2 +}$ $Mn Superscript 2 plus$ in the active site interacts with the carbonyl group of the intermediate oxalosuccinate, which is formed transiently but does not leave the binding site until decarboxylation converts it to α-ketoglutarate. ${Mn}^{2 +}$ $Mn Superscript 2 plus$ also stabilizes the enol formed transiently by decarboxylation.

A figure shows the conversion of isocitrate to Greek letter alpha-ketoglutarate in three steps. — MECHANISM FIGURE 16-11 Isocitrate dehydrogenase. In this reaction, the substrate, isocitrate, loses one carbon by oxidative decarboxylation.

Isocitrate is shown as a five-carbon chain with the top C forming C O O minus; the second carbon bonded to 2 H; the third carbon bonded to H on the left and to C on the right in a light red box and further double bonded to O to the upper left and bonded to O minus to the lower right, all within a light red box; the further carbon bonded to O on the left further bonded to H in a light blue box and to H on the right in a light blue box; and the bottom carbon double bonded to O to the lower left and bonded to O minus to the lower right. Step 1: Isocitrate is oxidized by hydride transfer to N A D plus or N A D P plus (depending on the isocitrate dehydrogenase isozyme). An arrow points right above isocitrate dehydrogenase accompanied by a curved arrow showing the addition of N A D (P) plus and the loss of N A D (P) H, with H in a light blue box, plus H plus in a blue box. This yields oxalosuccinate, shown within brackets as a similar molecule in which the fourth carbon is double bonded to O on the right. Additionally, there are curved red arrows from O minus to the bond between it and C within the red box bonded to the third carbon, between C in the red box bonded to the third carbon and the bond between the third and fourth carbons in the vertical chain, and between the double bond between the fourth carbon in the vertical chain and O and the same O. There are also red arrows from this double bonded O and from the O minus on the bottom carbon to M n 2 plus between them. Step 2: Decarboxylation is facilitated by electron withdrawal by the adjacent carbonyl and coordinated M n 2 plus. An arrow points right accompanied by a branched arrow showing the loss of C O 2 in a red box. This yields an intermediate shown in brackets. It is similar except that the third carbon in the vertical chain is bonded to H on the left and double bonded to the fourth carbon below and the further carbon has a single bond to O minus to its right. Curved red arrows point from the double bond between the third and fourth carbons in the vertical chain to H plus to the right and from the negative charge on O bonded to the further carbon in the chain to the bond between this O and carbon. Step 3: Rearrangement of the enol intermediate generates Greek letter alpha-ketoglutarate. An arrow points right. This yields Greek letter alpha-ketoglutarate, shown as a vertical five-carbon chain with the top carbon forming C O O minus, the second carbon bonded to 2 H, the third carbon bonded to H to the left and H to the right, the fourth carbon double bonded to O, and the fifth carbon double bonded to O to the left and bonded to O minus to the right.

There are two different forms of isocitrate dehydrogenase in all cells, one requiring ${NAD}^{+}$ $NAD Superscript plus$ as electron acceptor and the other requiring ${NADP}^{+}$ $NADP Superscript plus$ . The overall reactions are otherwise identical. In eukaryotic cells, the NAD-dependent enzyme occurs in the mitochondrial matrix and serves in the citric acid cycle. The main function of the NADP-dependent enzyme, found in both the mitochondrial matrix and the cytosol, is the generation of NADPH, which is essential for reductive anabolic pathways such as fatty acid and sterol synthesis.

Oxidation of α-Ketoglutarate to Succinyl-CoA and $C O_{2}$ $bold upper C bold upper O Subscript bold 2 Baseline$

The next step is another oxidative decarboxylation, in which α-ketoglutarate is converted to succinyl-CoA and ${CO}_{2}$ $CO Subscript 2$ by the action of the α-ketoglutarate dehydrogenase complex; ${NAD}^{+}$ $NAD Superscript plus$ serves as electron acceptor and CoA as the carrier of the succinyl group. The energy of oxidation of α-ketoglutarate is conserved in the formation of the thioester bond of succinyl-CoA:

A figure shows the reaction of Greek letter alpha-ketoglutarate to form succinyl-Co A in a reaction catalyzed by Greek letter alpha-ketoglutarate dehydrogenase complex and with Greek letter delta G prime degree symbol equals negative 33.5 k J per mole.

This reaction is virtually identical to the pyruvate dehydrogenase reaction discussed above and to the reaction sequence responsible for the breakdown of branched-chain amino acids (Fig. 16-12). The α-ketoglutarate dehydrogenase complex closely resembles the PDH complex and the complex that degrades branched-chain α-keto acids in both structure and function. All three have homologous $E_{1}$ $upper E Subscript 1$ and $E_{2}$ $upper E Subscript 2$ components, identical $E_{3}$ $upper E Subscript 3$ components, enzyme-bound TPP and lipoate, coenzyme A, FAD, and NAD. These related enzymes can employ the same $E_{3}$ $upper E Subscript 3$ subunit because the substrate for $E_{3}$ $upper E Subscript 3$ — a reduced lipoate — is the same for both complexes. They are certainly derived from a common evolutionary ancestor by gene duplication and subsequent divergent evolution, as described in Figure 1-32.

A figure shows a conserved mechanism for oxidative decarboxylation in three pathways: decarboxylations by the pyruvate dehydrogenase complex, in the citric acid cycle, and in the oxidation of isoleucine. — FIGURE 16-12 **A conserved mechanism for oxidative decarboxylation.** The pathways shown employ the same five cofactors (thiamine pyrophosphate, coenzyme A, lipoate, FAD, and ${NAD}^{+}$ $NAD Superscript plus$ ), closely similar multienzyme complexes, and the same enzymatic mechanism to carry out oxidative decarboxylations of pyruvate (by the pyruvate dehydrogenase complex), α-ketoglutarate (in the citric acid cycle), and the carbon skeletons of the three branched-chain amino acids, isoleucine (shown here), leucine, and valine.

FIGURE 16-12 **A conserved mechanism for oxidative decarboxylation.** The pathways shown employ the same five cofactors (thiamine pyrophosphate, coenzyme A, lipoate, FAD, and ${NAD}^{+}$ $NAD Superscript plus$ ), closely similar multienzyme complexes, and the same enzymatic mechanism to carry out oxidative decarboxylations of pyruvate (by the pyruvate dehydrogenase complex), α-ketoglutarate (in the citric acid cycle), and the carbon skeletons of the three branched-chain amino acids, isoleucine (shown here), leucine, and valine.

Three examples are shown. Pyruvate dehydrogenase complex: Pyruvate is converted to acetyl-Co A. Pyruvate is shown in a red box as C H 3 bonded to C double bonded to O above and bonded to C O O minus to the right. An arrow points down accompanied by a branched arrow showing the loss of C O 2 next to a curved line showing the addition of H S – Co A and a curved arrow showing the addition of N A D plus and loss of an orange oval labeled N A D H. This yields acetyl Co A, shown as C H 3 bonded to C double bonded to O to the upper right and bonded to S to the lower right further bonded to Co A. Citric acid cycle: Greek letter alpha-ketoglutarate is converted to succinyl Co A. Greek letter alpha-ketoglutarate is shown as C O O minus bonded to C H 2 bonded to C H 2 in a red box that is bonded to C double bonded to O above and to C O O minus to the right within the red box. An arrow points down accompanied by a branched arrow showing the loss of C O 2 next to a curved line showing the addition of H S – Co A and a curved arrow showing the addition of N A D plus and loss of an orange oval labeled N A D H. This yields succinyl Co A, shown as C O O minus bonded to C H 2 bonded to C H 2 bonded to C double bonded to O to the upper right and bonded to S to the lower right further bonded to C o A. Oxidation of isoleucine (leucine, valine): A Greek letter alpha-keto acid from isoleucine is converted to Greek letter alpha-methylbutyryl-Co A. Greek letter alpha-keto acid from isoleucine is shown as C H 3 bonded to C H 2 bonded to C H 2 bonded to C H in a red box bonded to C H 3 above outside of the box and to C double bonded to O above and to C O O minus to the right within the light red box. An arrow points down accompanied by a branched arrow showing the loss of C O 2 next to a curved line showing the addition of H S – Co A and a curved arrow showing the addition of N A D plus and loss of an orange oval labeled N A D H. This yields Greek letter alpha-methylbutyryl-Co A, shown as C H 3 C H 2 C H bonded to C H 3 above and bonded to C on the right further double bonded to O to the upper right and to S to the lower right further bonded to Co A.

Conversion of Succinyl-CoA to Succinate

Succinyl-CoA, like acetyl-CoA, has a thioester bond with a strongly negative standard free energy of hydrolysis $(Δ G^{'} ° \approx - 36 kJ/mol)$ $left-parenthesis upper Delta upper G Superscript prime Baseline degree almost-equals negative 36 kJ slash mol right-parenthesis$ . In the next step of the citric acid cycle, energy released in the breakage of this bond is used to drive the synthesis of a phosphoanhydride bond in either GTP or ATP, with a net $Δ G^{'} °$ $upper Delta upper G prime degree$ of only –2.9 kJ/mol. Succinate is formed in the process:

A figure shows the reaction of succinyl-Co A to form succinate in a reaction catalyzed by succinyl-Co A synthetase and with Greek letter delta G prime degree symbol equals negative 2.9 k J per mole.

The enzyme that catalyzes this reversible reaction is called succinyl-CoA synthetase or succinic thiokinase; both names indicate the participation of a nucleoside triphosphate in the reaction.

This energy-conserving reaction involves an intermediate step in which the enzyme molecule itself becomes phosphorylated at a His residue in the active site (Fig. 16-13a). This phosphoryl group, which has a high group transfer potential, is transferred to ADP (or GDP) to form ATP (or GTP). Animal cells have two isozymes of succinyl-CoA synthetase, one specific for ADP and the other for GDP. The enzyme has two subunits, $α (M_{r} 32, 000)$ $alpha left-parenthesis upper M Subscript r Baseline 32 comma 000 right-parenthesis$ , which has the –His residue $({His}^{246})$ $left-parenthesis His Superscript 246 Baseline right-parenthesis$ and the binding site for CoA, and $β (M_{r} 42, 000)$ $beta left-parenthesis upper M Subscript r Baseline 42 comma 000 right-parenthesis$ , which confers specificity for either ADP or GDP. The active site is at the interface between subunits. The crystal structure of succinyl-CoA synthetase reveals two “power helices” (one from each subunit), oriented so that their electric dipoles situate partial positive charges close to the negatively charged –His (Fig. 16-13b), stabilizing the phosphoenzyme intermediate. (Recall the similar role of helix dipoles in stabilizing $K^{+}$ $upper K Superscript plus$ ions in the $K^{+}$ $upper K Superscript plus$ channel; see Fig. 11-45.)

A two-part figure shows the succinyl-Co A synthetase reaction in three steps in part a and an illustration of the active site of succinyl-Co A synthetase in part b. — FIGURE 16-13 The succinyl-CoA synthetase reaction. (a) In step a phosphoryl group replaces the CoA of succinyl-CoA bound to the enzyme, forming a high-energy acyl phosphate. In step the succinyl phosphate donates its phosphoryl group to a His residue of the enzyme, forming a high-energy phosphohistidyl enzyme. In step the phosphoryl group is transferred from the His residue to the terminal phosphate of GDP (or ADP), forming GTP (or ATP). (b) Active site of succinyl-CoA synthetase of *Escherichia coli*. The active site includes part of both the α (blue) and the β (brown) subunits. The power helices (blue, brown) place the partial positive charges of the helix dipole near the phosphate group of ${-His}^{246}$ $hyphen His Superscript 246$ in the α chain, stabilizing the phosphohistidyl enzyme. The bacterial and mammalian enzymes have similar amino acid sequences and three-dimensional structures. [Data from PDB ID 1SCU, W. T. Wolodko et al., J. *Biol. Chem.* 269:10,883, 1994.]

FIGURE 16-13 The succinyl-CoA synthetase reaction. (a) In step a phosphoryl group replaces the CoA of succinyl-CoA bound to the enzyme, forming a high-energy acyl phosphate. In step the succinyl phosphate donates its phosphoryl group to a His residue of the enzyme, forming a high-energy phosphohistidyl enzyme. In step the phosphoryl group is transferred from the His residue to the terminal phosphate of GDP (or ADP), forming GTP (or ATP). (b) Active site of succinyl-CoA synthetase of *Escherichia coli*. The active site includes part of both the α (blue) and the β (brown) subunits. The power helices (blue, brown) place the partial positive charges of the helix dipole near the phosphate group of ${-His}^{246}$ $hyphen His Superscript 246$ in the α chain, stabilizing the phosphohistidyl enzyme. The bacterial and mammalian enzymes have similar amino acid sequences and three-dimensional structures. [Data from PDB ID 1SCU, W. T. Wolodko et al., J. *Biol. Chem.* 269:10,883, 1994.]

Part a shows a clockwise cycle of reactions. Step 1 is shown at the three o’clock position. Succinyl Co A is shown as a four-carbon chain with the top carbon double bonded to O to the upper left and bonded to O minus to the upper right, the second and third carbons bonded to 2 H, and the bottom carbon double bonded to O to the lower left and bonded to S to the lower left further bonded to S to the lower right further bonded to C o A. Succinyl Co A is added at the two o’clock position, then a new arrow starts from the three o’clock to five o’clock positions accompanied by a curved arrow labeled 1 showing the addition of red highlighted P subscript I and the loss of Co A – S H. This yields enzyme-bound succinyl phosphate, which is similar to succinyl Co A except that the third carbon is enclosed in a blue oval with H i s at the far right with a bond to the right, and the bottom carbon has a bond to O at the lower right that is further bonded to a red circle labeled P. An arrow points from enzyme-bound succinyl phosphate to phosphohistidyl enzyme at the ten o’clock position. At the seven o’clock position, an arrow labeled 2 curves away and points to succinate, shown as C double bonded to O to the upper left, bonded to O minus to the lower left, and bonded to C H 2 to the right further bonded to C H 2 bonded to C double bonded to O to the upper right and bonded to O minus to the lower right. All data are approximate. The arrow within the circle continues up to a structure labeled phosphohidtidyl enzyme at the ten o’clock position. This is a blue oval with H i s at the right side with a bond to the right and a bond to the lower right to a red circle labeled P outside of the blue oval. An arrow points from this point to a similar blue oval labeled succinyl-Co A synthetase at the twelve o’clock position with H i s at the right side with a bond to the right but with no bond to a red circle labeled P. A curved arrow labeled 3 at the eleven o’clock position shows the addition of G D P and loss of G T P with P highlighted in red. An arrow points from succinyl-Co A synthetase to the three o’clock position to continue the cycle. Part b shows a brown enzyme labeled Greek letter beta subunit in the background with helices and strands. It has a vertical purple piece labeled coenzyme A containing a ball-and-stick model that bends toward the left near its top. At the bottom of coenzyme A, several structures come together and the brown Greek letter beta subunit above meets the blue Greek letter alpha subunit below. A ball-and-stick model of phosphate is visible between the structures with a red structure behind. There are circles containing minus signs next to the two lower O atoms of the phosphate. To the left, a yellow structure contains a ball-and-stick model and is labeled H i s superscript 246 bonded to a red circle labeled P. A purple helix labeled Greek letter alpha subunit power helix has a circle with a minus symbol near its bottom and extends up from just to the right of the center bottom to the phosphate group. It has an arrow running through its center that ends with a blue point containing a circle containing a plus sign near one of the negatively charged O atoms. A brown helix labeled Greek letter beta subunit power helix extends from the upper center right to the phosphate and has a circle with a minus symbol near its left end. It also contains an arrow with a blue point containing a circle with a plus sign next to a negatively charged O atom.

The formation of ATP (or GTP) at the expense of the energy released by the oxidative decarboxylation of α-ketoglutarate is a substrate-level phosphorylation, like the synthesis of ATP in the glycolytic reactions catalyzed by phosphoglycerate kinase and pyruvate kinase (see Fig. 14-2). The GTP formed by succinyl-CoA synthetase can donate its terminal phosphoryl group to ADP to form ATP, in a reversible reaction catalyzed by nucleoside diphosphate kinase (p. 487):

\begin{matrix} GTP + ADP ⇌ GDP + ATP & Δ G^{'} ° = 0 kJ/mol \end{matrix}

$StartLayout 1st Row 1st Column GTP plus ADP right harpoon over left harpoon GDP plus ATP 2nd Column Blank 3rd Column upper Delta upper G Superscript prime Baseline degree equals 0 kJ slash mol EndLayout$

Thus the net result of the activity of either isozyme of succinyl-CoA synthetase is the conservation of energy as ATP. There is no change in free energy for the nucleoside diphosphate kinase reaction; ATP and GTP are energetically equivalent.

Oxidation of Succinate to Fumarate

The succinate formed from succinyl-CoA is oxidized to fumarate by the flavoprotein succinate dehydrogenase:

A figure shows the reaction of succinate to form fumarate in a reaction catalyzed by succinate dehydrogenase and with Greek letter delta G prime degree symbol equals 0 k J per mole.

In eukaryotes, succinate dehydrogenase is an integral protein of the mitochondrial inner membrane; in bacteria, of the plasma membrane. The enzyme contains three different iron-sulfur clusters and one molecule of covalently bound FAD (see Fig. 19-9). Electrons pass from succinate through the FAD and iron-sulfur centers before entering the chain of electron carriers in the mitochondrial inner membrane (the plasma membrane in bacteria). Electron flow from succinate through these carriers to the final electron acceptor, $O_{2}$ $upper O Subscript 2$ , is coupled to the synthesis of about 1.5 ATP molecules per pair of electrons (respiration-linked phosphorylation). Malonate, an analog of succinate not normally present in cells, is a strong competitive inhibitor of succinate dehydrogenase, and its addition to mitochondria in the laboratory blocks the activity of the citric acid cycle.

A figure shows the structures of malonate and succinate.

Hydration of Fumarate to Malate

The reversible hydration of fumarate to l-malate is catalyzed by fumarase (formally, fumarate hydratase). The transition state in this reaction is a carbanion:

A figure shows the series of reactions to convert fumarate to L-malate catalyzed by fumarase and with a Greek letter delta G prime degree symbol of negative 3.8 k J per mole.

Fumarate is shown as C bonded to H to the upper left, bonded to C O O minus to the upper right, and double bonded to C below bonded to C O O minus to the lower left and to H to the lower right. Right- and left-pointing arrows are shown above the word fumarase. A line shows the addition of O H minus to the right-pointing arrow. This yields a carbanion transition state, shown as C with a negative charge bonded to H to the upper left, bonded to C O O minus to the upper right, and bonded to C below that is bonded to C O O minus to the lower left, solid wedge bonded to the lower right, and hashed wedge bonded to O H and slightly down. Upward- and downward- pointing arrows are shown below accompanied by the word fumarase. A curved line shows the addition of H plus to the downward arrow. This yields L-malate, shown as C with a solid wedge bond to H to the left, a hashed wedge bond to H to the upper left, a bond to C O O minus to the upper right, and a bond to C below bonded to C O O minus to the lower left, solid wedge bonded to H to the lower right, and hashed bonded to O H to the right and slightly down.

This enzyme is highly stereospecific; it catalyzes hydration of the trans double bond of fumarate but not the cis double bond of maleate. In the reverse direction (from l-malate to fumarate), fumarase is equally stereospecific: d-malate is not a substrate.

A figure shows the structures of fumarate, maleate, L-malate, and D-malate.

Oxidation of Malate to Oxaloacetate

In the last reaction of the citric acid cycle, l-malate dehydrogenase catalyzes the oxidation of l-malate to oxaloacetate, coupled to the reduction of ${NAD}^{+}$ $NAD Superscript plus$ to NADH:

A figure shows the reaction of L-malate to form oxaloacetate in a reaction catalyzed by L-malate dehydrogenase and with Greek letter delta G prime degree symbol equals 29.7 k J per mole.

The equilibrium of this reaction lies far to the left under standard thermodynamic conditions, but in intact cells oxaloacetate is continually removed by the highly exergonic citrate synthase reaction (step of Fig. 16-7). This keeps the concentration of oxaloacetate in the cell extremely low $(< 10^{- 6} M)$ $left-parenthesis less-than 10 Superscript negative 6 Baseline upper M right-parenthesis$ , pulling the malate dehydrogenase reaction toward the formation of oxaloacetate.

Although the individual reactions of the citric acid cycle were initially worked out in vitro, using minced muscle tissue, the pathway and its regulation have also been studied extensively in vivo. By using precursors isotopically labeled with $^{14} C$ $Superscript 14 Baseline upper C$ , researchers have traced the fate of individual carbon atoms through the citric acid cycle. Some of the earliest experiments with $^{14} C$ $Superscript 14 Baseline upper C$ produced an unexpected result, however, which aroused considerable controversy about the pathway and mechanism of the citric acid cycle. In fact, these experiments at first seemed to show that citrate was not the first tricarboxylic acid to be formed. Box 16-2 gives some details of this episode in the history of citric acid cycle research. Today biochemists use $^{13} C$ $Superscript 13 Baseline upper C$ -labeled precursors and whole-tissue NMR spectroscopy to monitor metabolic flux through the cycle in living tissue. Because the NMR signal is unique to the compound containing the $^{13} C$ $Superscript 13 Baseline upper C$ , precursor carbons can be traced into each cycle intermediate and into compounds derived from the intermediates. This technique makes it possible to study regulation of the citric acid cycle and its interconnections with other metabolic pathways.

Box 16-2

Citrate: A Symmetric Molecule That Reacts Asymmetrically

When compounds enriched in the heavy-carbon isotope $^{13} C$ $Superscript 13 Baseline zero width space upper C$ and the radioactive carbon isotopes $^{11} C$ $Superscript 11 Baseline zero width space upper C$ and $^{14} C$ $Superscript 14 Baseline zero width space upper C$ became available in the mid-1940s, they were soon put to use in tracing the pathway of carbon atoms through the citric acid cycle. One such experiment initiated the controversy over the role of citrate. Acetate labeled in the carboxyl group (designated $[1 -^{14} C]$ $left-bracket 1 hyphen Superscript 14 Baseline upper C right-bracket$ acetate) was incubated aerobically with an animal tissue preparation. Acetate is enzymatically converted to acetyl-CoA in animal tissues, and the pathway of the labeled carboxyl carbon of the acetyl group in the cycle reactions could thus be traced. α-Ketoglutarate was isolated from the tissue after incubation, then degraded by known chemical reactions to establish the position(s) of the isotopic carbon.

Condensation of unlabeled oxaloacetate with carboxyl-labeled acetate would be expected to produce citrate labeled in one of the two primary carboxyl groups. Citrate is a symmetric molecule, its two terminal carboxyl groups being chemically indistinguishable. Therefore, half the labeled citrate molecules were expected to yield α-ketoglutarate labeled in the α-carboxyl group and the other half to yield α-ketoglutarate labeled in the γ-carboxyl group; that is, the α-ketoglutarate isolated was expected to be a mixture of the two types of labeled molecules (Fig. 1, pathways and ). Contrary to this expectation, the labeled α-ketoglutarate isolated from the tissue suspension contained $^{14} C$ $Superscript 14 Baseline zero width space upper C$ only in the γ-carboxyl group (Fig. 1, pathway ). The investigators concluded that citrate (or any other symmetric molecule) could not be an intermediate in the pathway from acetate to α-ketoglutarate. Rather, an asymmetric tricarboxylic acid, presumably cis-aconitate or isocitrate, must be the first product formed from condensation of acetate and oxaloacetate.

FIGURE 1 Incorporation of the isotopic carbon $(^{14} C)$ $left-parenthesis Superscript 14 Baseline upper C right-parenthesis$ of the labeled acetyl group into α-ketoglutarate by the citric acid cycle. The carbon atoms of the entering acetyl group are shown in red.

Labeled acetate is shown as red highlighted C bonded to nonhighlighted 3 H bonded to red highlighted superscript 14 end superscript C nonhighlighted O O minus. Oxaloacetate is shown as C double bonded to O to the left, bonded to C O O minus to the right, and bonded to C H 2 below further bonded to C O O minus. Labeled acetate and oxaloacetate are added and a right-pointing arrow shows that this yields labeled citrate, shown as a vertical three-carbon chain with the top carbon highlighted in red, bonded to 2 H, and further bonded to red highlighted superscript 14 end superscript C nonhighlighted O O minus, the middle carbon bonded to O H to the left and C O O minus to the right, and the bottom carbon bonded to 2 H and to C O O minus. An arrow pointing up and to the right is numbered 1, and an arrow pointing down and to the right is numbered 2. Both reactions yield isocitrate, but the labeled carbon atoms are in different positions. Arrow 1 points to a product shown as a vertical three-carbon chain with the top carbon highlighted in red, bonded to 2 H, and further bonded to red highlighted superscript 14 end superscript C nonhighlighted O O minus, the middle carbon bonded to H and to C O O minus to the right, and the bottom carbon bonded to O H to the left and to C O O minus to the right. A right-pointing arrow shows that this yields a vertical three-carbon chain shown with the top superscript Greek letter gamma end superscript red highlighted carbon bonded to 2 H bonded to red highlighted superscript 14 end superscript C nonhighlighted O O minus, the middle superscript Greek letter beta end superscript carbon bonded to 2 H, and the bottom superscript Greek letter alpha end superscript C bonded to H, double bonded to O to the left, and bonded to C O O minus to the right. Accompanying text reads, Only this product was formed. Arrow 2 points to a product shown as a vertical three-carbon chain with the top carbon bonded to 2 H and to C O O minus, the middle carbon bonded to H and to C O O minus to the right, and the bottom carbon highlighted in red, bonded to O H to the left, and bonded to red highlighted superscript 14 end superscript C nonhighlighted O O minus on the right. A right-pointing arrow shows that this yields a vertical three-carbon chain shown with the top superscript Greek letter gamma end superscript carbon bonded to 2 H bonded to C O O minus to the right, the middle superscript Greek letter beta end superscript carbon bonded to H and to C O O minus to the right, and the bottom superscript Greek letter alpha end superscript red highlighted C double bonded to O to the left and bonded to red highlighted superscript 14 end superscript C nonhighlighted O O minus to the right. Accompanying text reads, This second form of labeled Greek letter alpha-ketoglutarate was also expected, but was not formed.

In 1948, however, Alexander Ogston pointed out that although citrate has no chiral center (see Fig. 1-19), it has the potential to react asymmetrically if an enzyme with which it interacts has an active site that is asymmetric. He suggested that the active site of aconitase may have three points to which the citrate must be bound and that the citrate must undergo a specific three-point attachment to these binding points. As seen in Figure 2, the binding of citrate to three such points could happen in only one way, and this would account for the formation of only one type of labeled α-ketoglutarate. Organic molecules such as citrate that have no chiral center but are potentially capable of reacting asymmetrically with an asymmetric active site are now called prochiral molecules.

FIGURE 2 The prochiral nature of citrate. (a) Structure of citrate; (b) schematic representation of citrate: $upper X equals em-dash OH$ $upper X equals em-dash OH$ ; $Y = — {COO}^{-}$ $upper Y equals em-dash COO Superscript minus$ ; $Z = — {CH}_{2} {COO}^{-}$ $upper Z equals em-dash CH Subscript 2 Baseline COO Superscript minus$ . (c) Correct complementary fit of citrate to the binding site of aconitase. There is only one way in which the three specified groups of citrate can fit on the three points of the binding site. Thus only one of the two $— {CH}_{2} {COO}^{-}$ $em-dash CH Subscript 2 Baseline COO Superscript minus$ groups is bound by aconitase.

Part a shows citrate with a central C bonded to C H 2 C O O minus above, bonded to C H 2 C O O minus to the lower right, solid wedge bonded to C O O minus below to the front, and bonded to O H to the lower left. The bond between C and C H 2 C O O minus to the lower right is labeled susceptible bond. Part b shows C bonded to Z above, bonded to Z to the lower right, solid wedge bonded to Y below to the front, and bonded to X to the lower left. Part c shows the schematic illustration in part b above a blue oval with three labeled positioned. The bond between C and Z to the lower right is accompanied by text reading, This bond can be positioned correctly and attached. A dashed vertical line extends down from this Z to red highlighted Z prime at the rear right of the blue oval. The solid wedge bond between C and Y below to the front has a dashed vertical line extending down to red highlighted Y prime at the lower front of the blue oval. The bond between C and X to the lower left has a dashed vertical line extending down to red highlighted X prime at the rear left of the blue oval. The bond between C and Z above is accompanied by text reading, This bond cannot be positioned correctly and is not attached.

The Energy of Oxidations in the Cycle Is Efficiently Conserved

We have now covered one complete turn of the citric acid cycle (Fig. 16-14). A two-carbon acetyl group entered the cycle by combining with oxaloacetate. Two carbon atoms emerged from the cycle as ${CO}_{2}$ $CO Subscript 2$ from the oxidation of isocitrate and α-ketoglutarate. The energy released by these oxidations was conserved in the reduction of three ${NAD}^{+}$ $NAD Superscript plus$ and one FAD and the production of one ATP or GTP. At the end of the cycle a molecule of oxaloacetate was regenerated. Note that the two carbon atoms appearing as ${CO}_{2}$ $CO Subscript 2$ are not the same two carbons that entered in the form of the acetyl group; additional turns around the cycle are required to release these carbons as ${CO}_{2}$ $CO Subscript 2$ (Fig. 16-7).

A figure shows the citric acid cycle, emphasizing the products produced by one turn: three N A D H, one F A D H 2, one G T P (or A T P), and two C O 2. — FIGURE 16-14 **Products of one turn of the citric acid cycle.** At each turn of the cycle, three NADH, one ${FADH}_{2}$ $FADH Subscript 2$ , one GTP (or ATP), and two ${CO}_{2}$ $CO Subscript 2$ are released in oxidative decarboxylation reactions. Here and in several of the following figures, all cycle reactions are shown as proceeding in one direction only, but keep in mind that most of the reactions are reversible.

FIGURE 16-14 **Products of one turn of the citric acid cycle.** At each turn of the cycle, three NADH, one ${FADH}_{2}$ $FADH Subscript 2$ , one GTP (or ATP), and two ${CO}_{2}$ $CO Subscript 2$ are released in oxidative decarboxylation reactions. Here and in several of the following figures, all cycle reactions are shown as proceeding in one direction only, but keep in mind that most of the reactions are reversible.

The cycle consists of clockwise blue arrows. The first blue arrow begins at oxaloacetate at the eleven o’clock position and points to citrate at the one o’clock position. A curved blue line shows the addition of acetyl Co A to this line at the twelve o’clock position. A short blue arrow points from citrate to isocitrate at the two o’clock position. A blue arrow points from isocitrate to Greek letter alpha-ketoglutarate at the three o’clock position. This arrow has a blue arrow that branches off to show the loss of a red box labeled C O 2 and is accompanied by a curved red arrow showing that something is added and an orange oval of N A D H is produced. A blue arrow points from Greek letter alpha-ketoglutarate to succinyl-Co A at the five o’clock position. This arrow has a blue arrow that branches off to show the loss of a red box labeled C O 2 and is accompanied by a curved red arrow showing that something is added and an orange oval of N A D H is produced. An arrow points from succinyl-Co A to succinate at just past the six o’clock position. This arrow is accompanied by a curved blue arrow showing the production of an orange oval labeled G T P (A T P). A blue arrow points from succinate to fumarate at the seven o’clock position and is accompanied by a red arrow showing that something is added and that an orange oval labeled F A D H 2 is produced. A short blue arrow points from fumarate to malate at the nine o’clock position. An arrow points from malae to oxaloacetate at the eleven o’clock position and is accompanied by a red arrow showing that something is added and that an orange oval labeled F A D H 2 is produced. All data are approximate.

Although the citric acid cycle directly generates only one ATP per turn (in the conversion of succinyl-CoA to succinate), the four oxidation steps in the cycle provide a large flow of electrons into the respiratory chain via NADH and ${FADH}_{2}$ $FADH Subscript 2$ and thus lead to formation of almost 10 times more ATP during oxidative phosphorylation.

We saw in Chapter 14 that the production of two molecules of pyruvate from one molecule of glucose in glycolysis yields 2 ATP and 2 NADH. In oxidative phosphorylation (Chapter 19), passage of two electrons from NADH to $O_{2}$ $upper O Subscript 2$ drives the formation of about 2.5 ATP, and passage of two electrons from ${FADH}_{2}$ $FADH Subscript 2$ to $O_{2}$ $upper O Subscript 2$ yields about 1.5 ATP. This stoichiometry allows us to calculate the overall yield of ATP from the complete oxidation of glucose. When both pyruvate molecules are oxidized to 6 ${CO}_{2}$ $CO Subscript 2$ via the pyruvate dehydrogenase complex and the citric acid cycle, and the electrons are transferred to $O_{2}$ $upper O Subscript 2$ via oxidative phosphorylation, as many as 32 ATP are obtained per glucose (Table 16-1). In round numbers, this represents the conservation of $32 \times 30.5 kJ/mol = 976 kJ/mol$ $32 times 30.5 kJ slash mol equals 976 kJ slash mol$ , or 34% of the theoretical maximum of about 2,840 kJ/mol available from the complete oxidation of glucose. These calculations employ the standard free-energy changes; when corrected for the actual free energy required to form ATP within cells (see Worked Example 13-2, p. 480), the calculated efficiency of the process is closer to 65%. When cells under anaerobic conditions depend on glycolysis for ATP, one glucose yields just 2 ATP. Aerobic metabolism is far more effective in capturing the energy in glucose as a fuel.

TABLE 16-1 Stoichiometry of Coenzyme Reduction and ATP Formation in the Aerobic Oxidation of Glucose via Glycolysis, the Pyruvate Dehydrogenase Complex Reaction, the Citric Acid Cycle, and Oxidative Phosphorylation
Reaction	Number of ATP or reduced coenzyme directly formed	Number of ATP ultimately formed^a
$Glucose \to glucose 6 -phosphate$ $Glucose right-arrow glucose 6 hyphen phosphate$	$− 1 ATP$ $negative 1 ATP$	$− 1$ $negative 1$
$Fructose 6 -phosphate \to fructose 1,6-bisphosphate$ $Fructose 6 hyphen phosphate right-arrow fructose 1 comma 6 hyphen bisphosphate$	$− 1 ATP$ $negative 1 ATP$	$− 1$ $negative 1$
$2 Glyceraldehyde 3 -phosphate \to 2 1, 3 -bisphosphoglycerate$ $2 Glyceraldehyde 3 hyphen phosphate right-arrow 2 1 comma 3 hyphen bisphosphoglycerate$	2 NADH	3 or 5^b
$2 1, 3 -Bisphosphoglycerate \to 2 3 -phosphoglycerate$ $2 1 comma 3 hyphen Bisphosphoglycerate right-arrow 2 3 hyphen phosphoglycerate$	2 ATP	2
$2 Phosphoenolpyruvate \to 2 pyruvate$ $2 Phosphoenolpyruvate right-arrow 2 pyruvate$	2 ATP	2
$2 Pyruvate \to 2 acetyl-CoA$ $2 Pyruvate right-arrow 2 acetyl hyphen CoA$	2 NADH	5
$2 Isocitrate \to 2 α -ketoglutarate$ $2 Isocitrate right-arrow 2 alpha hyphen ketoglutarate$	2 NADH	5
$2 α -Ketoglutarate \to 2 succinyl-CoA$ $2 alpha hyphen Ketoglutarate right-arrow 2 succinyl hyphen CoA$	2 NADH	5
$2 Succinyl-CoA \to 2 succinate$ $2 Succinyl hyphen CoA right-arrow 2 succinate$	2 ATP (or 2 GTP)	2
$2 Succinate \to 2 fumarate$ $2 Succinate right-arrow 2 fumarate$	$2 {FADH}_{2}$ $2 FADH Subscript 2 Baseline$	3
$2 Malate \to 2 oxaloacetate$ $2 Malate right-arrow 2 oxaloacetate$	2 NADH	5
Total		30–32
^aThis is calculated as 2.5 ATP per NADH and 1.5 ATP per ${FADH}_{2}$ $FADH Subscript 2$ . A negative value indicates consumption. ^bThis number is either 3 or 5, depending on the mechanism used to shuttle NADH equivalents from the cytosol to the mitochondrial matrix; see Figures 19-31 and 19-32.

SUMMARY 16.2 Reactions of the Citric Acid Cycle

The citric acid cycle (Krebs cycle, TCA cycle) is a nearly universal central catabolic pathway in which compounds derived from the breakdown of carbohydrates, fats, and proteins are oxidized to ${CO}_{2}$ $CO Subscript 2$ , with most of the energy of oxidation temporarily held in the electron carriers ${FADH}_{2}$ $FADH Subscript 2$ and NADH. During aerobic metabolism, these electrons are transferred to $O_{2}$ $upper O Subscript 2$ and the energy of electron flow is conserved as ATP.
Acetyl-CoA enters the citric acid cycle as citrate synthase catalyzes its condensation with oxaloacetate to form citrate. In seven sequential reactions, including two decarboxylations, the citric acid cycle converts citrate to oxaloacetate and releases two ${CO}_{2}$ $CO Subscript 2$ . The pathway is cyclic in that the intermediates of the cycle are not used up; for each oxaloacetate consumed in the path, one is produced.
For each acetyl-CoA oxidized by the citric acid cycle, the energy gain consists of three molecules of NADH, one ${FADH}_{2}$ $FADH Subscript 2$ , and one nucleoside triphosphate (either ATP or GTP).