In the first part of the citric acid cycle, the four-carbon molecule oxaloacetate condenses with a two-carbon acetyl unit to yield citrate, a six-carbon tricarboxylic acid. As citrate moves through the first part of the cycle, two carbon atoms are lost as CO₂ in a process called oxidative decarboxylation. This decarboxylation yields a four-carbon molecule and high-transfer-potential electrons captured as two molecules of NADH.

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.

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This reaction 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 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, side reactions, such as the hydrolysis of acetyl CoA, must be minimized. Let us examine how citrate synthase prevents such wasteful reactions.

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 revealed that the enzyme undergoes large conformational changes in the course of catalysis. Citrate synthase binds substrate in a sequential, ordered fashion: 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 19.3). These structural changes create a binding site for acetyl CoA.

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Citrate synthase first catalyzes the condensation of citrate and acetyl CoA to form citryl CoA. The newly formed citryl CoA induces additional structural changes in the enzyme, causing the active site to become completely enclosed. The enzyme then cleaves the citryl CoA thioester by hydrolysis. CoA leaves the enzyme, followed by citrate, and the enzyme returns to the initial open conformation.

We can now understand how the wasteful hydrolysis of acetyl CoA is prevented. Citrate synthase is well suited to the hydrolysis of 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. Induced fit prevents an undesirable side reaction.

Keep in mind that a major purpose of the citric acid cycle is the oxidation of carbon atoms leading to the capture of high-transfer-potential electrons. The newly formed citrate molecule is not optimally structured for the required oxidation reactions. In particular, the hydroxyl group is not properly located in the citrate molecule for the oxidative decarboxylations that follow. 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 (Figure 19.4).

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

The intermediate in this reaction is oxalosuccinate, an unstable α-ketoacid. While bound to the enzyme, it loses CO₂ to form α-ketoglutarate. This oxidation generates the first high-transfer-potential electron carrier in the cycle, NADH:

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The conversion of isocitrate into α-ketoglutarate is followed by a second oxidative decarboxylation reaction, removing CO₂ from the α-ketoglutarate (five carbon atoms) to form succinyl CoA (four carbon atoms):

This reaction is catalyzed by the α-ketoglutarate dehydrogenase complex, an organized assembly of three kinds of enzymes that is structurally similar 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, employing the same coenzymes and reaction steps. At this point in the citric acid cycle, two carbon atoms have entered the cycle and two carbon atoms have been oxidized to CO₂. The electrons from the oxidations are captured in two molecules of NADH.