Chapter 20
The Tricarboxylic Acid Cycle

A time-lapse photograph of a ferris wheel at night.
Aerobic cells use a metabolic wheel-the tricarboxylic
acid cylce-to gerneate enegry by acetly-CoA oxidation.
(Ferns Whee, DelMar Fair Ó Corbis/Richard Cummins)

The glycolytic pathway converts glucose to pyruvate and produces two molecules of ATP per glucose—only a small fraction of the potential energy available from glucose. Under anaerobic conditions, pyruvate is reduced to lactate in animals and to ethanol in yeast, and much of the potential energy of the glucose molecule remains untapped. In the presence of oxygen, however, a much more interesting and thermodynamically complete story unfolds. Under aerobic conditions, NADH is oxidized in the electron transport chain, rather than becoming oxidized through reduction of pyruvate to lactate or acetaldehyde to ethanol, for example. Further, pyruvate is converted to acetyl-coenzyme A and oxidized to CO₂ in the tricarboxylic acid (TCA) cycle (also called the citric acid cycle). The electrons liberated by this oxidative process are then passed through an elaborate, membrane-associated electron transport pathway to O₂, the final electron acceptor. Electron transfer is coupled to creation of a proton gradient across the membrane. Such a gradient represents an energized state, and the energy stored in this gradient is used to drive the synthesis of many equivalents of ATP.

^{Figure 20.1} · Pyruvate produced in glycolysis is oxidized in the tricarboxylic acid (TCA) cycle. Electrons liberated in this oxidation flow through the electron transport chain and drive the synthesis of ATP in oxidative phosphorylation. In eukaryotic cells, this overall process occurs in mitochondria.

      ATP synthesis as a consequence of electron transport is termed oxidative phosphorylation; the complete process is diagrammed in Figure 20.1. Aerobic pathways permit the production of 30 to 38 molecules of ATP per glucose oxidized. Athough two molecules of ATP come from glycolysis and two more directly out of the TCA cycle, most of the ATP arises from oxidative phosphorylation. Specifically, reducing equivalents released in the oxidative reactions of glycolysis, pyruvate decarboxylation, and the TCA cycle are captured in the form of NADH and enzyme-bound FADH₂, and these reduced coenzymes fuel the electron transport pathway and oxidative phosphorylation. The path to oxidative phosphorylation winds through the TCA cycle, and we will examine this cycle in detail in this chapter.

20.1 · Hans Krebs and the Discovery of the TCA Cycle

Within the orderly and logical confines of a textbook, it is difficult to appreciate the tortuous path of the research scientist through the labyrinth of scientific discovery, the patient sifting and comparing of hypotheses, and the often plodding progress toward new information. The elucidation of the TCA cycle in this century is a typical case, and one worth recounting. Armed with accumulated small contributions—pieces of the puzzle—from many researchers over many years, Hans Krebs, in a single, seminal inspiration, put the pieces together and finally deciphered the cyclic nature of pyruvate oxidation. In his honor, the TCA cycle is often referred to as the Krebs cycle.
      In 1932 Krebs was studying the rates of oxidation of small organic acids by kidney and liver tissue. Only a few substances were active in these experiments—notably succinate, fumarate, acetate, malate, and citrate (Figure 20.2). Later it was found that oxaloacetate could be made from pyruvate in such tissues, and that it could be further oxidized like the other dicarboxylic acids.

Figure 20.2 · The organic acids observed by Krebs to be oxidized in suspensions of liver and kidney tissue. These substances were the pieces in the TCA puzzle that Krebs and others eventually solved.

            In 1935 in Hungary, a crucial discovery was made by Albert Szent-Györgyi, who was studying the oxidation of similar organic substrates by pigeon breast muscle, an active flight muscle with very high rates of oxidation and metabolism. Carefully measuring the amount of oxygen consumed, he observed that addition of any of three four-carbon dicarboxylic acids—fumarate, succinate, or malate—caused the consumption of much more oxygen than was required for the oxidation of the added substance itself. He concluded that these substances were limiting in the cell and, when provided, stimulated oxidation of endogenous glucose and other carbohydrates in the tissues. He also found that malonate, a competitive inhibitor of succinate dehydrogenase (Chapter 14), inhibited these oxidative processes; this finding suggested that succinate oxidation is a crucial step. Szent-Györgyi hypothesized that these dicarboxylic acids were linked by an enzymatic pathway that was important for aerobic metabolism.
      Another important piece of the puzzle came from the work of Carl Martius and Franz Knoop, who showed that citric acid could be converted to isocitrate and then to a-ketoglutarate. This finding was significant because it was already known that a-ketoglutarate could be enzymatically oxidized to succinate. At this juncture, the pathway from citrate to oxaloacetate seemed to be as shown in Figure 20.3. Whereas the pathway made sense, the catalytic effect of succinate and the other dicarboxylic acids from Szent-Györgyi’s studies remained a puzzle.

Figure 20.3 · Martius and Knoop’s observation that citrate could be converted to isocitrate and then a-ketoglutarate provided a complete pathway from citrate to oxaloacetate.

      In 1937 Krebs found that citrate could be formed in muscle suspensions if oxaloacetate and either pyruvate or acetate were added. He saw that he now had a cycle, not a simple pathway, and that addition of any of the intermediates could generate all of the others. The existence of a cycle, together with the entry of pyruvate into the cycle in the synthesis of citrate, provided a clear explanation for the accelerating properties of succinate, fumarate, and malate. If all these intermediates led to oxaloacetate, which combined with pyruvate from glycolysis, they could stimulate the oxidation of many substances besides themselves. (Kreb’s conceptual leap to a cycle was not his first. Together with medical student Kurt Henseleit, he had already elucidated the details of the urea cycle in 1932.) The complete tricarboxylic acid (Krebs) cycle, as it is now understood, is shown in Figure 20.4.

Figure 20.4 · The tricarboxylic acid cycle.

20.2 · The TCA Cycle — A Brief Summary

The entry of new carbon units into the cycle is through acetyl-CoA. This entry metabolite can be formed either from pyruvate (from glycolysis) or from oxidation of fatty acids (discussed in Chapter 25). Transfer of the two-carbon acetyl group from acetyl-CoA to the four-carbon oxaloacetate to yield six-carbon citrate is catalyzed by citrate synthase. A dehydration–rehydration rearrangement of citrate yields isocitrate. Two successive decarboxylations produce a-ketoglutarate and then succinyl-CoA, a CoA conjugate of a four-carbon unit. Several steps later, oxaloacetate is regenerated and can combine with another two-carbon unit of acetyl-CoA. Thus, carbon enters the cycle as acetyl-CoA and exits as CO₂. In the process, metabolic energy is captured in the form of ATP, NADH, and enzyme-bound FADH₂ (symbolized as [FADH₂]).

The Chemical Logic of the TCA Cycle
The cycle shown in Figure 20.4 at first appears to be a complicated way to oxidize acetate units to CO₂, but there is a chemical basis for the apparent complexity. Oxidation of an acetyl group to a pair of CO₂ molecules requires CO C cleavage:
                                                                              CH₃COO^- ® CO₂ + CO₂

In many instances, C¾C cleavage reactions in biological systems occur between carbon atoms a- and b- to a carbonyl group:

A good example of such a cleavage is the fructose bisphosphate aldolase reaction (see Chapter 19, Figure 19.14a).

      Another common type of C¾C cleavage is a-cleavage of an a-hydroxy-ketone:

(We see this type of cleavage in the transketolase reaction described in Chapter 23.)
      Neither of these cleavage strategies is suitable for acetate. It has no b-carbon, and the second method would require hydroxylation—not a favorable reaction for acetate. Instead, living things have evolved the clever chemistry of condensing acetate with oxaloacetate and then carrying out a b-cleavage. The TCA cycle combines this b-cleavage reaction with oxidation to form CO₂, regenerate oxaloacetate, and capture the liberated metabolic energy in NADH and ATP.

20.3 · The Bridging Step: Oxidative Decarboxylation of Pyruvate

Pyruvate produced by glycolysis is a significant source of acetyl-CoA for the TCA cycle. Because, in eukaryotic cells, glycolysis occurs in the cytoplasm, whereas the TCA cycle reactions and all subsequent steps of aerobic metabolism take place in the mitochondria, pyruvate must first enter the mitochondria to enter the TCA cycle. The oxidative decarboxylation of pyruvate to acetyl-CoA,
                                    Pyruvate + CoA + NAD⁺ ® acetyl-CoA + CO₂ + NADH + H⁺
is the connecting link between glycolysis and the TCA cycle. The reaction is catalyzed by pyruvate dehydrogenase, a multienzyme complex.
      The pyruvate dehydrogenase complex (PDC) is a noncovalent assembly of three different enzymes operating in concert to catalyze successive steps in the conversion of pyruvate to acetyl-CoA. The active sites of all three enzymes are not far removed from one another, and the product of the first enzyme is passed directly to the second enzyme and so on, without diffusion of substrates and products through the solution. The overall reaction (see A Deeper Look: “Reaction Mechanism of the Pyruvate Dehydrogenase Complex”) involves a total of five coenzymes: thiamine pyrophosphate, coenzyme A, lipoic acid, NAD⁺, and FAD.

20.4 · Entry into the Cycle: The Citrate Synthase Reaction

The first reaction within the TCA cycle, the one by which carbon atoms are introduced, is the citrate synthase reaction (Figure 20.5). Here acetyl-CoA reacts with oxaloacetate in a Perkin condensation (a carbon–carbon condensation between a ketone or aldehyde and an ester). The acyl group is activated in two ways in an acyl-CoA molecule: the carbonyl carbon is activated for attack by nucleophiles, and the C_a carbon is more acidic and can be deprotonated to form a carbanion. The citrate synthase reaction depends upon the latter mode of activation.

Figure 20.5 · Citrate is formed in the citrate synthase reaction from oxaloacetate and acetyl-CoA. The mechanism involves nucleophilic attack by the carbanion of acetyl-CoA on the carbonyl carbon of oxaloacetate, followed by thioester hydrolysis.

As shown in Figure 20.5, a general base on the enzyme accepts a proton from the methyl group of acetyl-CoA, producing a stabilized a-carbanion of acetyl-CoA. This strong nucleophile attacks the a-carbonyl of oxaloacetate, yielding citryl-CoA. This part of the reaction has an equilibrium constant near 1, but the overall reaction is driven to completion by the subsequent hydrolysis of the high-energy thioester to citrate and free CoA. The overall DG°' is -31.4 kJ/mol, and under standard conditions the reaction is essentially irreversible. Although the mitochondrial concentration of oxaloacetate is very low (much less than 1 mM —see example in Section 20.11), the strong, negative DG°' drives the reaction forward.

The Structure of Citrate Synthase
Citrate synthase in mammals is a dimer of 49-kD subunits (Table 20.1).

Table 20.1

The Enzymes and Reactions of the TCA Cycle
Reaction	Enzyme	Subunit Mr	Oligomeric Composition	DG°' (kJ/mol)	Keq at 25° C	DG (kJ/mol)
	Citrate synthase	49,000*	Dimer
	Aconitase	44,500	Dimer
	Isocitrate dehydrogenase		a2bg
	a-Ketoglutarate     dehydrogenase complex	E1 96,000	Dimer
	Succinyl-CoA synthetase	E2 70,000	24-mer
	Succinate dehydrogenase
	Fumarase	48,500	Tetramer
	Malate dehydrogenase	35,000	Dimer

On each subunit, oxaloacetate and acetyl-CoA bind to the active site, which lies in a cleft between two domains and is surrounded mainly by a-helical segments (Figure 20.6). Binding of oxaloacetate induces a conformational change that facilitates the binding of acetyl-CoA and closes the active site, so that the reactive carbanion of acetyl-CoA is protected from protonation by water.

Figure 20.6 · Citrate synthase. In the monomer shown here, citrate is shown in green, and CoA is pink.

Regulation of Citrate Synthase
Citrate synthase is the first step in this metabolic pathway, and as stated the reaction has a large negative DG°'. As might be expected, it is a highly regulated enzyme. NADH, a product of the TCA cycle, is an allosteric inhibitor of citrate synthase, as is succinyl-CoA, the product of the fifth step in the cycle (and an acetyl-CoA analog).

20.5 · The Isomerization of Citrate by Aconitase

Citrate itself poses a problem: it is a poor candidate for further oxidation because it contains a tertiary alcohol, which could be oxidized only by breaking a carbon–carbon bond. An obvious solution to this problem is to isomerize the tertiary alcohol to a secondary alcohol, which the cycle proceeds to do in the next step.
      Citrate is isomerized to isocitrate by aconitase in a two-step process involving aconitate as an intermediate (Figure 20.7). In this reaction, the elements of water are first abstracted from citrate to yield aconitate, which is then rehydrated with H¾ and HO¾ adding back in opposite positions to produce isocitrate. The net effect is the conversion of a tertiary alcohol (citrate) to a secondary alcohol (isocitrate). Oxidation of the secondary alcohol of isocitrate involves breakage of a C¾H bond, a simpler matter than the C¾C cleavage required for the direct oxidation of citrate.

Figure 20.7 · (a) The aconitase reaction converts citrate to cis-aconitate and then to isocitrate. Aconitase is stereospecific and removes the pro-R hydrogen from the pro-R arm of citrate. (b) The active site of aconitase. The iron-sulfur cluster (red) is coordinated by cysteines (yellow) and isocitrate (white).

      Inspection of the citrate structure shows a total of four chemically equivalent hydrogens, but only one of these—the pro-R H atom of the pro-R arm of citrate—is abstracted by aconitase, which is quite stereospecific. Formation of the double bond of aconitate following proton abstraction requires departure of hydroxide ion from the C-3 position. Hydroxide is a relatively poor leaving group, and its departure is facilitated in the aconitase reaction by coordination with an iron atom in an iron–sulfur cluster.

Aconitase Utilizes an Iron – Sulfur Cluster
Aconitase contains an iron–sulfur cluster consisting of three iron atoms and four sulfur atoms in a near-cubic arrangement (Figure 20.8). This cluster is bound to the enzyme via three cysteine groups from the protein. One corner of the cube is vacant and binds Fe²⁺, which activates aconitase. The iron atom in this position can coordinate the C-3 carboxyl and hydroxyl groups of citrate. This iron atom thus acts as a Lewis acid, accepting an unshared pair of electrons from the hydroxyl, making it a better leaving group. The equilibrium for the aconitase reaction favors citrate, and an equilibrium mixture typically contains about 90% citrate, 4% cis-aconitate, and 6% isocitrate. The DG°' is +6.7 kJ/mol.

Figure 20.8 · The iron–sulfur cluster of aconitase. Binding of Fe²⁺ to the vacant position of the cluster activates aconitase. The added iron atom coordinates the C-3 carboxyl and hydroxyl groups of citrate and acts as a Lewis acid, accepting an electron pair from the hydroxyl group and making it a better leaving group.

Fluoroacetate Blocks the TCA Cycle
Fluoroacetate is an extremely poisonous agent that blocks the TCA cycle in vivo, although it has no apparent effect on any of the isolated enzymes. Its LD₅₀, the lethal dose for 50% of animals consuming it, is 0.2 mg per kilogram of body weight; it has been used as a rodent poison. The action of fluoroacetate has been traced to aconitase, which is inhibited in vivo by fluorocitrate, which is formed from fluoroacetate in two steps (Figure 20.9). Fluoroacetate readily crosses both the cellular and mitochondrial membranes, and in mitochondria it is converted to fluoroacetyl-CoA by acetyl-CoA synthetase. Fluoroacetyl-CoA is a substrate for citrate synthase, which condenses it with oxaloacetate to form fluorocitrate. Fluoroacetate may thus be viewed as a trojan horse inhibitor. Analogous to the giant Trojan Horse of legend—which the soldiers of Troy took into their city, not knowing that Greek soldiers were hidden inside it and waiting to attack—fluoroacetate enters the TCA cycle innocently enough, in the citrate synthase reaction. Citrate synthase converts fluoroacetate to inhibitory fluorocitrate for its TCA cycle partner, aconitase, blocking the cycle.

Figure 20.9 · The conversion of fluoroacetate to fluorocitrate.

20.6 · Isocitrate Dehydrogenase — The First Oxidation in the Cycle

In the next step of the TCA cycle, isocitrate is oxidatively decarboxylated to yield a-ketoglutarate, with concomitant reduction of NAD⁺ to NADH in the isocitrate dehydrogenase reaction (Figure 20.10). The reaction has a net DG°' of -8.4 kJ/mol, and it is sufficiently exergonic to pull the aconitase reaction forward. This two-step reaction involves (1) oxidation of the C-2 alcohol of iso­citrate to form oxalosuccinate, followed by (2) a b-decarboxylation reaction that expels the central carboxyl group as CO₂, leaving the product a-ketoglutarate. Oxalosuccinate, the b-keto acid produced by the initial dehydrogenation reaction, is unstable and thus is readily decarboxylated. Isocitrate Dehydrogenase Links the TCA Cycle and Electron Transport Isocitrate dehydrogenase provides the first connection between the TCA cycle and the electron transport pathway and oxidative phosphorylation, via its production of NADH. As a connecting point between two metabolic pathways, isocitrate dehydrogenase is a regulated reaction. NADH and ATP are allosteric inhibitors, whereas ADP acts as an allosteric activator, lowering the K_m for isocitrate by a factor of 10. The enzyme is virtually inactive in the absence of ADP. Also, the product, a-ketoglutarate, is a crucial a-keto acid for aminotransferase reactions (see Chapters 14 and 27), connecting the TCA cycle (that is, carbon metabolism) with nitrogen metabolism.

Figure 20.10 · (a) The isocitrate dehydrogenase reaction. (b) The active site of isocitrate dehydrogenase. Isocitrate is shown in green, NADP⁺ is shown in gold, with Ca²⁺ in red.

20.7 · a-Ketoglutarate Dehydrogenase — A Second Decarboxylation

A second oxidative decarboxylation occurs in the a-ketoglutarate dehydrogenase reaction (Figure 20.11).

Figure 20.11 · The a-ketoglutarate dehydrogenase reaction.

Like the pyruvate dehydrogenase complex, a-ketoglutarate dehydrogenase is a multienzyme complex—consisting of a-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and dihydrolipoyl dehydrogenase —that employs five different coenzymes (Table 20.2). The dihydrolipoyl dehydrogenase in this reaction is identical to that in the pyruvate dehydrogenase reaction. The mechanism is analogous to that of pyruvate dehydrogenase, and the free energy changes for these reactions are -29 to -33.5 kJ/mol. As with the pyruvate dehydrogenase reaction, this reaction produces NADH and a thioester product—in this case, succinyl-CoA. Succinyl-CoA and NADH products are energy-rich species that are important sources of metabolic energy in subsequent cellular processes.

Table 20.2

20.8 · Succinyl-CoA Synthetase — A Substrate-Level Phosphorylation

The NADH produced in the foregoing steps can be routed through the electron transport pathway to make high-energy phosphates via oxidative phosphorylation. However, succinyl-CoA is itself a high-energy intermediate and is utilized in the next step of the TCA cycle to drive the phosphorylation of GDP to GTP (in mammals) or ADP to ATP (in plants and bacteria). The reaction (Figure 20.12) is catalyzed by succinyl-CoA synthetase, sometimes called succinate thiokinase.

Figure 20.12 · The succinyl-CoA synthetase reaction.

The free energies of hydrolysis of succinyl-CoA and GTP or ATP are similar, and the net reaction has a DG°' of -3.3 kJ/mol. Succinyl-CoA synthetase provides another example of a substrate-level phosphorylation (Chapter 19), in which a substrate, rather than an electron transport chain or proton gradient, provides the energy for phosphorylation. It is the only such reaction in the TCA cycle. The GTP produced by mammals in this reaction can exchange its terminal phosphoryl group with ADP via the nucleoside diphosphate kinase reaction:
                                                                                  Nucleoside diphosphate
                                                                                                kinase
                                                                                GTP + ADP ATP + GDP

The Mechanism of Succinyl-CoA Synthetase
The mechanism of succinyl-CoA synthetase is postulated to involve displacement of CoA by phosphate, forming succinyl phosphate at the active site, followed by transfer of the phosphoryl group to an active-site histidine (making a phosphohistidine intermediate) and release of succinate. The phosphoryl moiety is then transferred to GDP to form GTP (Figure 20.13). This sequence of steps “preserves” the energy of the thioester bond of succinyl-CoA in a series of high-energy intermediates that lead to a molecule of ATP: 
                                                        Thioester ® [succinyl-P] ® [phosphohistidine] ® GTP ® ATP

Figure 20.13 · The mechanism of the succinyl-CoA synthetase reaction.

The First Five Steps of the TCA Cycle Produce NADH, CO₂, GTP (ATP), and Succinate
This is a good point to pause in our trip through the TCA cycle and see what has happened. A two-carbon acetyl group has been introduced as acetyl-CoA and linked to oxaloacetate, and two CO₂ molecules have been liberated. The cycle has produced two molecules of NADH and one of GTP or ATP, and has left a molecule of succinate.
      The TCA cycle can now be completed by converting succinate to oxaloace-tate. This latter process represents a net oxidation. The TCA cycle breaks it down into (consecutively) an oxidation step, a hydration reaction, and a second oxidation step. The oxidation steps are accompanied by the reduction of an [FAD] and an NAD⁺. The reduced coenzymes, [FADH₂] and NADH, subsequently provide reducing power in the electron transport chain. (We see in Chapter 24 that virtually the same chemical strategy is used in b-oxidation of fatty acids.)

20.9 · Succinate Dehydrogenase — An Oxidation Involving FAD

The oxidation of succinate to fumarate (Figure 20.14) is carried out by succinate dehydrogenase, a membrane-bound enzyme that is actually part of the electron transport chain. As will be seen in Chapter 21, succinate dehydrogenase is part of the succinate–coenzyme Q reductase of the electron transport chain. In contrast with all of the other enzymes of the TCA cycle, which are soluble proteins found in the mitochondrial matrix, succinate dehydrogenase is an integral membrane protein tightly associated with the inner mitochondrial membrane. Succinate oxidation involves removal of H atoms across a C¾C bond, rather than a C¾O or C¾N bond, and produces the trans-unsaturated fumarate. This reaction (the oxidation of an alkane to an alkene) is not sufficiently exergonic to reduce NAD⁺, but it does yield enough energy to reduce [FAD]. (By contrast, oxidations of alcohols to ketones or aldehydes are more energetically favorable and provide sufficient energy to reduce NAD⁺.) This important point is illustrated and clarified in an example in Chapter 21.

Figure 20.14 · The succinate dehydrogenase reaction. Oxidation of succinate occurs with reduction of [FAD]. Reoxidation of [FADH₂] transfers electrons to coenzyme Q.

      Succinate dehydrogenase is a dimeric protein, with subunits of molecular masses 70 kD and 27 kD (see Table 20.1). FAD is covalently bound to the larger subunit; the bond involves a methylene group of C-8a of FAD and N-3 of a histidine on the protein (Figure 20.15).

Figure 20.15 · The covalent bond between FAD and succinate dehydrogenase involves the C-8a methylene group of FAD and the N-3 of a histidine residue on the enzyme.

Succinate dehydrogenase also contains three different iron–sulfur clusters (Figure 20.16).

Figure 20.16 · The Fe₂S₂ cluster of succinate dehydrogenase.

Viewed from either end of the succinate molecule, the reaction involves dehydrogenation a,b to a carbonyl (actually, a carboxyl) group. The dehydrogenation is stereospecific (Figure 20.14), with the pro-S hydrogen removed from one carbon atom and the pro-R hydrogen removed from the other. The electrons captured by [FAD] in this reaction are passed directly into the iron–sulfur clusters of the enzyme and on to coenzyme Q(UQ). The covalently bound FAD is first reduced to [FADH₂] and then reoxidized to form [FAD] and the reduced form of coenzyme Q, UQH₂. Electrons captured by UQH₂ then flow through the rest of the electron transport chain in a series of events that is discussed in detail in Chapter 21.
      Note that flavin coenzymes can carry out either one-electron or two-electron transfers. The succinate dehydrogenase reaction represents a net two-electron reduction of FAD.

20.10 · Fumarase Catalyzes Trans-Hydration of Fumarate

Fumarate is hydrated in a stereospecific reaction by fumarase to give L-malate (Figure 20.17).

Figure 20.17 · The fumarase reaction.

The reaction involves trans-addition of the elements of water across the double bond. Recall that aconitase carries out a similar reaction, and that trans-addition of ¾H and ¾OH occurs across the double bond of cis-aconitate. Though the exact mechanism is uncertain, it may involve protonation of the double bond to form an intermediate carbonium ion (Figure 20.18) or possibly attack by water or OH^- anion to produce a carbanion, followed by protonation.

Figure 20.18 · Two possible mechanisms for the fumarase reaction.

20.11 · Malate Dehydrogenase — Completing the Cycle

In the last step of the TCA cycle, L-malate is oxidized to oxaloacetate by malate dehydrogenase (Figure 20.19). This reaction is very endergonic, with a DG°' of +30 kJ/mol. Consequently, the concentration of oxaloacetate in the mitochondrial matrix is usually quite low (see the following example). The reaction, however, is pulled forward by the favorable citrate synthase reaction. Oxidation of malate is coupled to reduction of yet another molecule of NAD⁺, the third one of the cycle. Counting the [FAD] reduced by succinate dehydrogenase, this makes the fourth coenzyme reduced through oxidation of a single acetate unit.

Figure 20.19 · The malate dehydrogenase reaction.

A Deeper Look
Steric Preferences in NAD+ Dependent Dehydrogenases
As noted in Chapter 18, the enzymes tha trequire nicotinamide coenzymes are seterospecific and transer hydride to either the pro-R or the pro-S positions selectively. The table (facing page) lists the preferences of serveral dehydrogenases.         What accounts for this stereospecificity? It arises from the fact that the enzymes (and especially the active sites of enzymes) are inherently asymmetric structures. The nicotinamide coenzyme (and the substrate) fit the active site in only one way. Malate dehydrogenase, the citric acid cycle enzyme, transfers hydride to the H R position of NADH, but glyceraldehyde-3-P dehydrogenase in the glycolytic pathway transfers hydride to the HS position, as shown in the accompanying table. Dehydrogenases are stereo-specific with respect to the substrates as well. Note that alcohol dehydrogenase removes hydrogen from the pro-R position of ethanol and transfers it to the pro-R position of NADH.	Steric Specificity for NAD of Various Pyridine Nucleotide-Linked Enzymes Dehydrogenase Source Steric Specificity Alcohol (with ethanol) Yeast, Pseudomonas, liver, wheat germ   Alcohol (with isopropyl alcohol) Yeast   Acetaldehyde Liver   L-Lactate Heart muscle, Lactobacillus HR L-Malate  Pig heart, wheat germ   D-Glycerate Spinach   Dihydroorotate Zymobacterium oroticum   a-Glycerophosphate Muscle   Glyceraldehyde-3-P  Yeast, muscle   L-Glutamate Liver   D-Glucose Liver b-Hydroxysteroid Pseudomonas HS NADH cytochrome c reductase Rat liver mitochondria, pig heart   NADPH transhydrogenase Pseudomonas   NADH diaphorase Pig heart   L-b-Hydroxybutyryl-CoA Heart muscle
NAD(P) +-dependent enzymes are stereospecific. Malate dehydrogenase, for example, transfers a hydride to the pro-R position of NADH, whereas glyceraldehyde-3-phosphate dehydrogenase transfers a hydride to the pro-S position of the nicotinamide. Alcohol dehydrogenase removes a hydride from the pro-R position of ethanol and transfers it to the pro-R position of NADH.		The stereospecificity of hydride transfer in dehydrogenases is a consequence of the asymmetric nature of the active site.
Adapted from Kaplan, N. O., 1960. In The Enzymes, vol. 3, p. 115, edited by Boyer, Lardy, and Myrbäck. New York: Academic Press.

Example
A typical intramitochondrial concentration of malate is 0.22 mM. If the [NAD⁺]/[NADH] ratio in mitochondria is 20 and if the malate dehydrogenase reaction is at equilibrium, calculate the intramitochondrial concentration of oxaloacetate at 25°C.
SOLUTION
For the malate dehydrogenase reaction, 
                   
with the value of DG°' being +30 kJ/mol. Then 
                   
                                                                                                                                  

Malate dehydrogenase is structurally and functionally similar to other dehydrogenases, notably lactate dehydrogenase (Figure 20.20). Both consist of alternating b-sheet and a-helical segments. Binding of NAD⁺ causes a conformational change in the 20-residue segment that connects the D and E strands of the b-sheet. The change is triggered by an interaction between the adenosine phosphate moiety of NAD⁺ and an arginine residue in this loop region. Such a conformational change is consistent with an ordered single-displacement mechanism for NAD⁺-dependent dehydrogenases (Chapter 14).

Figure 20.20 · (a) The structure of malate dehydrogenase. (b) The active site of malate dehydrogenase. Malate is shown in red; NAD⁺ is blue.

20.12 · A Summary of the Cycle

The net reaction accomplished by the TCA cycle, as follows, shows two molecules of CO₂, one ATP, and four reduced coenzymes produced per acetate group oxidized. The cycle is exergonic, with a net DG°' for one pass around the cycle of approximately -40kJ/mol. Table 20.1 compares the DG°' values for the individual reactions with the overall DG°' for the net reaction.Acetyl-CoA+1
Glucose metabolized via glycolysis produces two molecules of pyruvate and thus two molecules of acetyl-CoA, which can enter the TCA cycle. Combining glycolysis and the TCA cycle gives the net reaction shown:

All six carbons of glucose are liberated as CO₂, and a total of four molecules of ATP are formed thus far in substrate-level phosphorylations. The 12 reduced coenzymes produced up to this point can eventually produce a maximum of 34 molecules of ATP in the electron transport and oxidative phosphorylation pathways. A stoichiometric relationship for these subsequent processes is

Thus, a total of 3 ATP per NADH and 2 ATP per FADH₂ may be produced through the processes of electron transport and oxidative phosphorylation.

The Fate of the Carbon Atoms of Acetyl-CoA in the TCA Cycle
It is instructive to consider how the carbon atoms of a given acetate group are routed through several turns of the TCA cycle. As shown in Figure 20.21, neither of the carbon atoms of a labeled acetate unit is lost as CO₂ in the first turn of the cycle. The CO₂ evolved in any turn of the cycle derives from the carboxyl groups of the oxaloacetate acceptor (from the previous turn), not from incoming acetyl-CoA. On the other hand, succinate labeled on one end from the original labeled acetate forms two different labeled oxaloacetates. The carbonyl carbon of acetyl-CoA is evenly distributed between the two carboxyl carbons of oxaloacetate, and the labeled methyl carbon of incoming acetyl-CoA ends up evenly distributed between the methylene and carbonyl carbons of oxaloacetate.

Figure 20.21 · The fate of the carbon atoms of acetate in successive TCA cycles. (a) The carbonyl carbon of acetyl-CoA is fully retained through one turn of the cycle but is lost completely in a second turn of the cycle. (b) The methyl carbon of a labeled acetyl-CoA survives two full turns of the cycle but becomes equally distributed among the four carbons of oxaloacetate by the end of the second turn. In each subsequent turn of the cycle, one-half of this carbon (the original labeled methyl group) is lost.

      When these labeled oxaloacetates enter a second turn of the cycle, both of the carboxyl carbons are lost as CO₂, but the methylene and carbonyl carbons survive through the second turn. Thus, the methyl carbon of a labeled acetyl-CoA survives two full turns of the cycle. In the third turn of the cycle, one-half of the carbon from the original methyl group of acetyl-CoA has become one of the carboxyl carbons of oxaloacetate and is thus lost as CO₂. In the fourth turn of the cycle, further “scrambling” results in loss of half of the remaining labeled carbon (one-fourth of the original methyl carbon label of acetyl-CoA), and so on.
      It can be seen that the carbonyl and methyl carbons of labeled acetyl-CoA have very different fates in the TCA cycle. The carbonyl carbon survives the first turn intact but is completely lost in the second turn. The methyl carbon survives two full turns, then undergoes a 50% loss through each succeeding turn of the cycle.
      It is worth noting that the carbon–carbon bond cleaved in the TCA pathway entered as an acetate unit in the previous turn of the cycle. Thus, the oxidative decarboxylations that cleave this bond are just a cleverly disguised acetate C¾C cleavage and oxidation.

20.13 · The TCA Cycle Provides Intermediates for Biosynthetic Pathways

Until now we have viewed the TCA cycle as a catabolic process because it oxidizes acetate units to CO₂ and converts the liberated energy to ATP and reduced coenzymes. The TCA cycle is, after all, the end point for breakdown of food materials, at least in terms of carbon turnover. However, as shown in Figure 20.22, four-, five-, and six-carbon species produced in the TCA cycle also fuel a variety of biosynthetic processes.

Figure 20.22 · The TCA cycle provides intermediates for numerous biosynthetic processes in the cell.

a-Ketoglutarate, succinyl-CoA, fumarate, and oxaloacetate are all precursors of important cellular species. (In order to participate in eukaryotic biosynthetic processes, however, they must first be transported out of the mitochondria.) A transamination reaction converts a-keto­glutarate directly to glutamate, which can then serve as a versatile precursor for proline, arginine, and glutamine (as described in Chapter 26). Succinyl-CoA provides most of the carbon atoms of the porphyrins. Oxaloacetate can be transaminated to produce aspartate. Aspartic acid itself is a precursor of the pyrimidine nucleotides and, in addition, is a key precursor for the synthesis of asparagine, methionine, lysine, threonine, and isoleucine. Oxaloacetate can also be decarboxylated to yield PEP, which is a key element of several pathways, namely (1) synthesis (in plants and microorganisms) of the aromatic amino acids phenylalanine, tyrosine, and tryptophan; (2) formation of 3-phosphoglycerate and conversion to the amino acids serine, glycine, and cysteine; and (3) gluconeogenesis, which, as we will see in Chapter 23, is the pathway that synthesizes new glucose and many other carbohydrates.
      Finally, citrate can be exported from the mitochondria and then broken down by ATP-citrate lyase to yield oxaloacetate and acetyl-CoA, a precursor of fatty acids (Figure 20.23). Oxaloacetate produced in this reaction is rapidly reduced to malate, which can then be processed in either of two ways: it may be transported into mitochondria, where it is reoxidized to oxaloacetate, or it may be oxidatively decarboxylated to pyruvate by malic enzyme, with subsequent mitochondrial uptake of pyruvate. This cycle permits citrate to provide acetyl-CoA for biosynthetic processes, with return of the malate and pyruvate by-products to the mitochondria.

Figure 20.23 · Export of citrate from mitochondria and cytosolic breakdown produces oxaloacetate and acetyl-CoA. Oxaloacetate is recycled to malate or pyruvate, which re-enters the mitochondria. This cycle provides acetyl-CoA for fatty acid synthesis in the cytosol.

20.14 · The Anaplerotic, or “Filling Up,” Reactions

In a sort of reciprocal arrangement, the cell also feeds many intermediates back into the TCA cycle from other reactions. Since such reactions replenish the TCA cycle intermediates, Hans Kornberg proposed that they be called anaplerotic reactions (literally, the “filling up” reactions). Thus, PEP carboxylase and pyruvate carboxylase synthesize oxaloacetate from pyruvate (Figure 20.24).

Figure 20.24 · Phosphoenolpyruvate (PEP) carboxylase, pyruvate carboxylase, and malic enzyme catalyze anaplerotic reactions, replenishing TCA cycle intermediates.

      Pyruvate carboxylase is the most important of the anaplerotic reactions. It exists in the mitochondria of animal cells but not in plants, and it provides a direct link between glycolysis and the TCA cycle. The enzyme is tetrameric and contains covalently bound biotin and an Mg²⁺ site on each subunit. (It is examined in greater detail in our discussion of gluconeogenesis in Chapter 23.) Pyruvate carboxylase has an absolute allosteric requirement for acetyl-CoA. Thus, when acetyl-CoA levels exceed the oxaloacetate supply, allosteric activation of pyruvate carboxylase by acetyl-CoA raises oxaloacetate levels, so that the excess acetyl-CoA can enter the TCA cycle.
      PEP carboxylase occurs in yeast, bacteria, and higher plants, but not in animals. The enzyme is specifically inhibited by aspartate, which is produced by transamination of oxaloacetate. Thus, organisms utilizing this enzyme control aspartate production by regulation of PEP carboxylase. Malic enzyme is found in the cytosol or mitochondria of many animal and plant cells and is an NADPH-dependent enzyme.
      It is worth noting that the reaction catalyzed by PEP carboxykinase (Figure 20.25) could also function as an anaplerotic reaction, were it not for the particular properties of the enzyme. CO₂ binds weakly to PEP carboxykinase, whereas oxaloacetate binds very tightly ( K_D = 2 x 10^-6 M), and, as a result, the enzyme favors formation of PEP from oxaloacetate.

Figure 20.25 · The phosphoenolpyruvate carboxykinase reaction.

      The catabolism of amino acids provides pyruvate, acetyl-CoA, oxaloacetate, fumarate, a-ketoglutarate, and succinate, all of which may be oxidized by the TCA cycle. In this way, proteins may serve as excellent sources of nutrient energy, as seen in Chapter 26.

20.15 · Regulation of the TCA Cycle

Situated as it is between glycolysis and the electron transport chain, the TCA cycle must be carefully controlled by the cell. If the cycle were permitted to run unchecked, large amounts of metabolic energy could be wasted in over-production of reduced coenzymes and ATP; conversely, if it ran too slowly, ATP would not be produced rapidly enough to satisfy the needs of the cell. Also, as just seen, the TCA cycle is an important source of precursors for biosynthetic processes and must be able to provide them as needed.
      What are the sites of regulation in the TCA cycle? Based upon our experience with glycolysis (Figure 19.31), we might anticipate that some of the reactions of the TCA cycle would operate near equilibrium under cellular conditions (with DG » 0), whereas others—the sites of regulation—would be characterized by large, negative DG values. Estimates for the values of DG in mitochondria, based on mitochondrial concentrations of metabolites, are summarized in Table 20.1. Three reactions of the cycle—citrate synthase, isocitrate dehydrogenase, and a-keto­glutarate dehydrogenase—operate with large, negative DG values under mitochondrial conditions and are thus the primary sites of regulation in the cycle.
      The regulatory actions that control the TCA cycle are shown in Figure 20.26. As one might expect, the principal regulatory “signals” are the concentrations of acetyl-CoA, ATP, NAD⁺, and NADH, with additional effects provided by several other metabolites. The main sites of regulation are pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase. All of these enzymes are inhibited by NADH, so that when the cell has produced all the NADH that can conveniently be turned into ATP, the cycle shuts down. For similar reasons, ATP is an inhibitor of pyruvate dehydrogenase and isocitrate dehydrogenase. The TCA cycle is turned on, however, when either the ADP/ATP or NAD⁺/NADH ratio is high, an indication that the cell has run low on ATP or NADH. Regulation of the TCA cycle by NADH, NAD⁺, ATP, and ADP thus reflects the energy status of the cell. On the other hand, succinyl-CoA is an intracycle regulator, inhibiting citrate synthase and a-ketoglutarate dehydrogenase. Acetyl-CoA acts as a signal to the TCA cycle that glycolysis or fatty acid breakdown is producing two-carbon units. Acetyl-CoA activates pyruvate carboxylase, the anaplerotic reaction that provides oxaloacetate, the acceptor for increased flux of acetyl-CoA into the TCA cycle.

Figure 20.26 · Regulation of the TCA cycle.

Regulation of Pyruvate Dehydrogenase
As we shall see in Chapter 23, most organisms can synthesize sugars such as glucose from pyruvate. However, animals cannot synthesize glucose from acetyl-CoA. For this reason, the pyruvate dehydrogenase complex, which converts pyruvate to acetyl-CoA, plays a pivotal role in metabolism. Conversion to acetyl-CoA commits nutrient carbon atoms either to oxidation in the TCA cycle or to fatty acid synthesis (see Chapter 25). Because this choice is so crucial to the organism, pyruvate dehydrogenase is a carefully regulated enzyme. It is subject to product inhibition and is further regulated by nucleotides. Finally, activity of pyruvate dehydrogenase is regulated by phosphorylation and dephosphorylation of the enzyme complex itself.
      High levels of either product, acetyl-CoA or NADH, allosterically inhibit the pyruvate dehydrogenase complex. Acetyl-CoA specifically blocks dihydrolipoyl transacetylase, and NADH acts on dihydrolipoyl dehydrogenase. The mammalian pyruvate dehydrogenase is also regulated by covalent modifications. As shown in Figure 20.27, a Mg²⁺-dependent pyruvate dehydrogenase kinase is associated with the enzyme in mammals. This kinase is allosterically activated by NADH and acetyl-CoA, and when levels of these metabolites rise in the mitochondrion, they stimulate phosphorylation of a serine residue on the pyruvate dehydrogenase subunit, blocking the first step of the pyruvate dehydrogenase reaction, the decarboxylation of pyruvate. Inhibition of the dehydrogenase in this manner eventually lowers the levels of NADH and acetyl-CoA in the matrix of the mitochondrion. Reactivation of the enzyme is carried out by pyruvate dehydrogenase phosphatase, a Ca²⁺-activated enzyme that binds to the dehydrogenase complex and hydrolyzes the phosphoserine moiety on the dehydrogenase subunit. At low ratios of NADH to NAD⁺ and low acetyl-CoA levels, the phosphatase maintains the dehydrogenase in an activated state, but a high level of acetyl-CoA or NADH once again activates the kinase and leads to the inhibition of the dehydrogenase. Insulin and Ca²⁺ ions activate dephosphorylation, and pyruvate inhibits the phosphorylation reaction.

Figure 20.27 · Regulation of the pyruvate dehydrogenase reaction.

      Pyruvate dehydrogenase is also sensitive to the energy status of the cell. AMP activates pyruvate dehydrogenase, whereas GTP inhibits it. High levels of AMP are a sign that the cell may become energy-poor. Activation of pyruvate dehydrogenase under such conditions commits pyruvate to energy production.

Regulation of Isocitrate Dehydrogenase
The mechanism of regulation of isocitrate dehydrogenase is in some respects the reverse of pyruvate dehydrogenase. The mammalian isocitrate dehydrogenase is subject only to allosteric activation by ADP and NAD⁺ and to inhibition by ATP and NADH. Thus, high NAD⁺/NADH and ADP/ATP ratios stimulate isocitrate dehydrogenase and TCA cycle activity. The Escherichia coli enzyme, on the other hand, is regulated by covalent modification. Serine residues on each subunit of the dimeric enzyme are phosphorylated by a protein kinase, causing inhibition of the isocitrate dehydrogenase activity. Activity is restored by the action of a specific phosphatase. When TCA cycle and glycolytic intermediates—such as isocitrate, 3-phosphoglycerate, pyruvate, PEP, and oxaloacetate—are high, the kinase is inhibited, the phosphatase is activated, and the TCA cycle operates normally. When levels of these intermediates fall, the kinase is activated, isocitrate dehydrogenase is inhibited, and iso­citrate is diverted to the glyoxylate pathway, as explained in the next section.
      It may seem surprising that isocitrate dehydrogenase is strongly regulated, because it is not an apparent branch point within the TCA cycle. However, the citrate/isocitrate ratio controls the rate of production of cytosolic acetyl-CoA, because acetyl-CoA in the cytosol is derived from citrate exported from the mitochondrion. (Breakdown of cytosolic citrate produces oxaloacetate and acetyl-CoA, which can be used in a variety of biosynthetic processes.) Thus, isocitrate dehydrogenase activity in the mitochondrion favors catabolic TCA cycle activity over anabolic utilization of acetyl-CoA in the cytosol.

20.16 · The Glyoxylate Cycle of Plants and Bacteria

Plants (particularly seedlings, which cannot yet accomplish efficient photosynthesis), as well as some bacteria and algae, can use acetate as the only source of carbon for all the carbon compounds they produce. Although we saw that the TCA cycle can supply intermediates for some biosynthetic processes, the cycle gives off 2 CO₂ for every two-carbon acetate group that enters and cannot effect the net synthesis of TCA cycle intermediates. Thus, it would not be possible for the cycle to produce the massive amounts of biosynthetic intermediates needed for acetate-based growth unless alternative reactions were available. In essence, the TCA cycle is geared primarily to energy production, and it “wastes” carbon units by giving off CO₂. Modification of the cycle to support acetate-based growth would require eliminating the CO₂-producing reactions and enhancing the net production of four-carbon units (i.e., oxaloacetate). Plants and bacteria employ a modification of the TCA cycle called the glyoxylate cycle to produce four-carbon dicarboxylic acids (and eventually even sugars) from two-carbon acetate units. The glyoxylate cycle bypasses the two oxidative decarboxylations of the TCA cycle, and instead routes isocitrate through the isocitrate lyase and malate synthase reactions (Figure 20.28). Glyoxylate produced by isocitrate lyase reacts with a second molecule of acetyl-CoA to form L-malate. The net effect is to conserve carbon units, using two acetyl-CoA molecules per cycle to generate oxaloacetate. Some of this is converted to PEP and then to glucose by pathways discussed in Chapter 23.

Figure 20.28 · The glyoxylate cycle. The first two steps are identical to TCA cycle reactions. The third step bypasses the CO₂-evolving steps of the TCA cycle to produce succinate and glyoxylate. The malate synthase reaction forms malate from glyoxylate and another acetyl-CoA. The result is that one turn of the cycle consumes one oxaloacetate and two acetyl-CoA molecules but produces two molecules of oxaloacetate. The net for this cycle is one oxaloacetate from two acetyl-CoA molecules.

The Glyoxylate Cycle Operates in Specialized Organelles

The enzymes of the glyoxylate cycle in plants are contained in glyoxysomes, organelles devoted to this cycle. Yeast and algae carry out the glyoxylate cycle in the cytoplasm. The enzymes common to both the TCA and glyoxylate pathways exist as isozymes, with spatially and functionally distinct enzymes operating independently in the two cycles.

Isocitrate Lyase Short-Circuits the TCA Cycle
by Producing Glyoxylate and Succinate
The isocitrate lyase reaction (Figure 20.29) produces succinate, a four-carbon product of the cycle, as well as glyoxylate, which can then combine with a second molecule of acetyl-CoA.

Figure 20.29 · The isocitrate lyase reaction.

Isocitrate lyase catalyzes an aldol cleavage and is similar to the reaction mediated by aldolase in glycolysis. The malate synthase reaction (Fi