The conversion of pyruvate into acetyl CoA by the pyruvate dehydrogenase complex is the link between glycolysis and cellular respiration because acetyl CoA is the fuel for the citric acid cycle. Indeed, all fuels are ultimately metabolized to acetyl CoA or components of the citric acid cycle.

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The citric acid cycle begins with the condensation of a four-carbon unit, oxaloacetate, and a two-carbon unit, the acetyl group of acetyl CoA. Oxaloacetate reacts with acetyl CoA and H₂O to yield citrate and CoA.

This reaction, which is an aldol condensation followed by a hydrolysis, is catalyzed by citrate synthase. Oxaloacetate first condenses with acetyl CoA to form citryl CoA, a molecule that is energy rich because it contains the thioester bond that originated in acetyl CoA. The hydrolysis of citryl CoA thioester to citrate and CoA drives the overall reaction far in the direction of the synthesis of citrate. In essence, the hydrolysis of the thioester powers the synthesis of a new molecule from two precursors.

Because the condensation of acetyl CoA and oxaloacetate initiates the citric acid cycle, it is very important that side reactions, notably the hydrolysis of acetyl CoA to acetate and CoA, be minimized. Let us briefly consider how the citrate synthase prevents the wasteful hydrolysis of acetyl CoA.

Mammalian citrate synthase is a dimer of identical 49-kDa subunits. Each active site is located in a cleft between the large and the small domains of a subunit, adjacent to the subunit interface. X-ray crystallographic studies of citrate synthase and its complexes with several substrates and inhibitors have revealed that the enzyme undergoes large conformational changes in the course of catalysis. Citrate synthase exhibits sequential, ordered kinetics: oxaloacetate binds first, followed by acetyl CoA. The reason for the ordered binding is that oxaloacetate induces a major structural rearrangement leading to the creation of a binding site for acetyl CoA. The binding of oxaloacetate converts the open form of the enzyme into a closed form (Figure 17.10). In each subunit, the small domain rotates 19 degrees relative to the large domain. Movements as large as 15 Å are produced by the rotation of α helices elicited by quite small shifts of side chains around bound oxaloacetate. These structural changes create a binding site for acetyl CoA.

Citrate synthase catalyzes the condensation reaction by bringing the substrates into close proximity, orienting them, and polarizing certain bonds (Figure 17.11). The donation and removal of protons transforms acetyl CoA into an enol intermediate. The enol attacks oxaloacetate to form a carbon−carbon double bond linking acetyl CoA and oxaloacetate. The newly formed citryl CoA induces additional structural changes in the enzyme, causing the active site to become completely enclosed. The enzyme cleaves the citryl CoA thioester by hydrolysis. CoA leaves the enzyme, followed by citrate, and the enzyme returns to the initial open conformation.

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We can now understand how the wasteful hydrolysis of acetyl CoA is prevented. Citrate synthase is well suited to hydrolyze citryl CoA but not acetyl CoA. How is this discrimination accomplished? First, acetyl CoA does not bind to the enzyme until oxaloacetate is bound and ready for condensation. Second, the catalytic residues crucial for the hydrolysis of the thioester linkage are not appropriately positioned until citryl CoA is formed. As with hexokinase and triose phosphate isomerase (Section 16.1), induced fit prevents an undesirable side reaction.

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The hydroxyl group is not properly located in the citrate molecule for the oxidative decarboxylations that follow (Problem 27). Thus, citrate is isomerized into isocitrate to enable the six-carbon unit to undergo oxidative decarboxylation. The isomerization of citrate is accomplished by a dehydration step followed by a hydration step. The result is an interchange of an H and an OH. The enzyme catalyzing both steps is called aconitase because cis-aconitate is an intermediate.

Aconitase is an iron–sulfur protein, or nonhemeiron protein, in that it contains iron that is not bonded to heme. Rather, its four iron atoms are complexed to four inorganic sulfides and three cysteine sulfur atoms, leaving one iron atom available to bind citrate through one of its COO⁻ groups and an OH group (Figure 17.12). This Fe-S cluster participates in dehydrating and rehydrating the bound substrate.

We come now to the first of four oxidation–reduction reactions in the citric acid cycle. The oxidative decarboxylation of isocitrate is catalyzed by isocitrate dehydrogenase.

The intermediate in this reaction is oxalosuccinate, an unstable β-ketoacid. While bound to the enzyme, it loses CO₂ to form α-ketoglutarate.

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This oxidation generates the first high-transfer-potential electron carrier, NADH, in the cycle.

The conversion of isocitrate into α-ketoglutarate is followed by a second oxidative decarboxylation reaction, the formation of succinyl CoA from α-ketoglutarate.

This reaction is catalyzed by the α-ketoglutarate dehydrogenase complex, an organized assembly of three kinds of enzymes that is homologous to the pyruvate dehydrogenase complex. In fact, the E₃ component is identical in both enzymes. The oxidative decarboxylation of α-ketoglutarate closely resembles that of pyruvate, also an α-ketoacid.

Both reactions include the decarboxylation of an α-ketoacid and the subsequent formation of a thioester linkage with CoA that has a high transfer potential. The reaction mechanisms are entirely analogous.

Succinyl CoA is an energy-rich thioester compound. The ΔG°′ for the hydrolysis of succinyl CoA is about −33.5 kJ mol⁻¹ (−8.0 kcal mol⁻¹), which is comparable to that of ATP (−30.5 kJ mol⁻¹, or −7.3 kcal mol⁻¹). In the citrate synthase reaction, the cleavage of the thioester bond powers the synthesis of the six-carbon citrate from the four-carbon oxaloacetate and the two-carbon fragment. The cleavage of the thioester bond of succinyl CoA is coupled to the phosphorylation of a purine nucleoside diphosphate, usually ADP. This reaction, which is readily reversible, is catalyzed by succinyl CoA synthetase (succinate thiokinase).

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This reaction is the only step in the citric acid cycle that directly yields a compound with high phosphoryl-transfer potential. In mammals, there are two isozymic forms of the enzyme, one specific for ADP and one for GDP. In tissues that perform large amounts of cellular respiration, such as skeletal and heart muscle, the ADP-requiring isozyme predominates. In tissues that perform many anabolic reactions, such as the liver, the GDP-requiring enzyme is common. The GDP-requiring enzyme is believed to work in reverse of the direction observed in the TCA cycle; that is, GTP is used to power the synthesis of succinyl CoA, which is a precursor for heme synthesis. The E. coli enzyme uses either GDP or ADP as the phosphoryl-group acceptor.

Note that the enzyme nucleoside diphosphokinase, which catalyzes the following reaction,

allows the γ phosphoryl group to be readily transferred from GTP to form ATP, thereby allowing the adjustment of the concentration of GTP or ATP to meet the cell’s need.

The mechanism of this reaction is a clear example of an energy transformation: energy inherent in the thioester molecule is transformed into phosphoryl-group-transfer potential (Figure 17.13). The first step is the displacement of coenzyme A by orthophosphate, which generates another energy-rich compound, succinyl phosphate. A histidine residue plays a key role as a moving arm that detaches the phosphoryl group, then swings over to a bound ADP and transfers the group to form ATP. The participation of high-energy compounds in all the steps is attested to by the fact that the reaction is readily reversible: ΔG°′ = −3.4 kJ mol⁻¹ (−0.8 kcal mol⁻¹). The formation of ATP at the expense of succinyl CoA is an example of substrate-level phosphorylation (Section 16.1).

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Succinyl CoA synthetase is an α₂β₂ heterodimer; the functional unit is one αβ pair. The enzyme mechanism shows that a phosphoryl group is transferred first to succinyl CoA bound in the α subunit and then to a nucleoside diphosphate bound in the β subunit. Examination of the three-dimensional structure of succinyl CoA synthetase reveals that each subunit comprises two domains (Figure 17.14). The amino-terminal domains of the two subunits have different structures, each characteristic of its role in the mechanism. The amino-terminal domain of the α subunit forms a Rossmann fold (Section 16.1), which binds the ADP substrate of succinyl CoA synthetase. The amino-terminal domain of the β subunit is an ATP-grasp domain, found in many enzymes, which here binds and activates ADP. Succinyl CoA synthetase has evolved by adopting these domains and harnessing them to capture the energy associated with succinyl CoA cleavage, which is used to drive the generation of a nucleoside triphosphate.

Reactions of four-carbon compounds constitute the final stage of the citric acid cycle: the regeneration of oxaloacetate.

The reactions constitute a metabolic motif that we will see again in fatty acid synthesis and degradation as well as in the degradation of some amino acids. A methylene group (CH₂) is converted into a carbonyl group CPO in three steps: an oxidation, a hydration, and a second oxidation reaction. Oxaloacetate is thereby regenerated for another round of the cycle, and more energy is extracted in the form of FADH₂ and NADH.

Succinate is oxidized to fumarate by succinate dehydrogenase. The hydrogen acceptor is FAD rather than NAD⁺, which is used in the other three oxidation reactions in the cycle. FAD is the hydrogen acceptor in this reaction because the free-energy change is insufficient to reduce NAD⁺. FAD is nearly always the electron acceptor in oxidations that remove two hydrogen atoms from a substrate. In succinate dehydrogenase, the isoalloxazine ring of FAD is covalently attached to a histidine side chain of the enzyme (denoted E-FAD).

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Succinate dehydrogenase, like aconitase, is an iron–sulfur protein. Indeed, succinate dehydrogenase contains three different kinds of iron– sulfur clusters: 2Fe-2S (two iron atoms bonded to two inorganic sulfides), 3Fe-4S, and 4Fe-4S. Succinate dehydrogenase—which consists of a 70-kDa and a 27-kDa subunit—differs from other enzymes in the citric acid cycle in being embedded in the inner mitochondrial membrane. In fact, succinate dehydrogenase is directly associated with the electron-transport chain, the link between the citric acid cycle and ATP formation. FADH₂ produced by the oxidation of succinate does not dissociate from the enzyme, in contrast with NADH produced in other oxidation–reduction reactions. Rather, two electrons are transferred from FADH₂ directly to iron–sulfur clusters of the enzyme, which in turn passes the electrons to coenzyme Q (CoQ). Coenzyme Q, an important member of the electron-transport chain, passes electrons to the ultimate acceptor, molecular oxygen, as we shall see in Chapter 18.

The next step is the hydration of fumarate to form l-malate. Fumarase catalyzes a stereospecific trans addition of H⁺ and OH⁻. The OH⁻ group adds to only one side of the double bond of fumarate; hence, only the L isomer of malate is formed.

Finally, malate is oxidized to form oxaloacetate. This reaction is catalyzed by malate dehydrogenase, and NAD⁺ is again the hydrogen acceptor.

The standard free energy for this reaction, unlike that for the other steps in the citric acid cycle, is significantly positive (ΔG°′ = +29.7 kJ mol⁻¹, or +7.1 kcal mol⁻¹). The oxidation of malate is driven by the use of the products—oxaloacetate by citrate synthase and NADH by the electron-transport chain.

The net reaction of the citric acid cycle is

Let us recapitulate the reactions that give this stoichiometry (Figure 17.15 and Table 17.2):

Isotope-labeling studies have revealed that the two carbon atoms that enter each cycle are not the ones that leave. The two carbon atoms that enter the cycle as the acetyl group are retained during the initial two decarboxylation reactions (Figure 17.15) and then remain incorporated in the four-carbon acids of the cycle. Note that succinate is a symmetric molecule. Consequently, the two carbon atoms that enter the cycle can occupy any of the carbon positions in the subsequent metabolism of the four-carbon acids. The two carbons that enter the cycle as the acetyl group will be released as CO₂ in subsequent trips through the cycle. To understand why citrate is not processed as a symmetric molecule, see Problems 34 and 35.

Evidence is accumulating that the enzymes of the citric acid cycle are physically associated with one another. The close arrangement of enzymes enhances the efficiency of the citric acid cycle because a reaction product can pass directly from one active site to the next through connecting channels, a process called substrate channeling.

As will be considered in Chapter 18, the electron-transport chain oxidizes the NADH and FADH₂ formed in the citric acid cycle. The transfer leads of electrons from these carriers to O₂, the ultimate electron acceptor, to the generation of a proton gradient across the inner mitochondrial membrane. This proton-motive force then powers the generation of ATP; the net stoichiometry is about 2.5 ATP per NADH, and 1.5 ATP per FADH₂. Consequently, nine high-transfer-potential phosphoryl groups are generated when the electron-transport chain oxidizes 3 NADH molecules and 1 FADH₂ molecule, and one ATP is directly formed in one round of the citric acid cycle. Thus, one acetyl unit generates approximately 10 molecules of ATP. In dramatic contrast, the anaerobic glycolysis of an entire glucose molecule generates only 2 molecules of ATP (and 2 molecules of lactate).

Recall that molecular oxygen does not participate directly in the citric acid cycle. However, the cycle operates only under aerobic conditions because NAD⁺ and FAD can be regenerated in the mitochondrion only by the transfer of electrons to molecular oxygen. Glycolysis has both an aerobic and an anaerobic mode, whereas the citric acid cycle is strictly aerobic. Glycolysis can proceed under anaerobic conditions because NAD⁺ is regenerated in the conversion of pyruvate into lactate or ethanol.