26.12: How do Enzymes Work? Citrate Synthase

As we saw in the previous section, enzymes work by bringing substrate and other reactant molecules together, holding them in the orientation necessary for reaction, providing any necessary acidic or basic sites to catalyze specific steps, and stabilizing the transition state for reaction. As an example, let’s look at citrate synthase, an enzyme that catalyzes the aldol-like addition of acetyl CoA to oxaloacetate to give citrate. This reaction is the first step in the citric acid cycle, in which acetyl groups produced by degradation of food molecules are metabolized to yield CO₂ and H₂O. We’ll look at the details of the citric acid cycle in Section 29.7.

Citrate synthase is a globular protein of 433 amino acids with a deep cleft lined by an array of functional groups that can bind to the substrate, oxaloacetate. On binding oxaloacetate, the original cleft closes and another opens up nearby to bind acetyl CoA. This second cleft is also lined by appropriate functional groups, including a histidine at position 274 and an aspartic acid at position 375. The two reactants are now held by the enzyme in close proximity and with a suitable orientation for reaction. Figure 26.10 shows the structure of citrate synthase as determined by X-ray crystallography, along with a close-up of the active site.

Table 26.3 Structures and Functions of Some Common Coenzymes

Figure 26.10: X-ray crystal structure of citrate synthase. Part (a) is a space-filling model and part (b) is a ribbon model, which emphasizes the α-helical segments of the protein chain and indicates that the enzyme is dimeric; that is, it consists of two identical chains held together by hydrogen bonds and other intermolecular attractions. Part (c) is a close-up of the active site in which oxaloacetate and an unreactive acetyl CoA mimic are bound.

As shown in Figure 26.11, the first step in the aldol reaction of acetyl CoA and oxaloacetate is generation of the enol of acetyl CoA. The side-chain carboxyl of an aspartate residue acts as base to abstract an acidic α proton, while at the same time the side-chain imidazole ring of a histidine donates H⁺ to the carbonyl oxygen. The enol thus produced then performs a nucleophilic addition to the ketone carbonyl group of oxaloacetate. The first histidine acts as a base to remove the –OH hydrogen from the enol, while a second histidine residue simultaneously donates a proton to the oxaloacetate carbonyl group, giving citryl CoA. Water then hydrolyzes the thiol ester group in citryl CoA in a nucleophilic acyl substitution reaction, releasing citrate and coenzyme A as the final products.

Figure 26.11 MECHANISM Mechanism of the addition of acetyl CoA to oxaloacetate to give (S)-citryl CoA, catalyzed by citrate synthase.