EFFECTS OF CHANGES IN 3 CATALYTIC RESIDUES ON THE RELATIVE STABILITIES OF SOME OF THE INTERMEDIATES AND TRANSITION-STATES IN THE CITRATE SYNTHASE REACTION

This work reports the relative importance of the interactions provided by three catalytic residues to individual steps in the mechanism of c itrate synthase. When the side chains of any of the residues (H320, D3 75, and H274) are mutated, the data indicate that they are involved in the stabilization of one or more of the transition/intermediate state s in the multistep citrate synthase reaction. H320 forms a hydrogen bo nd with the carbonyl of oxaloacetate and the alcohols of the citryl-co enzyme A and citrate products. Enzymes substituted at H320 (Q, G, N, a nd R) have reaction profiles for which the condensation reaction is cl eanly rate determining. None of these mutants can activate the carbony l of oxaloacetate by polarization. All these mutants catalyze the nece ssary proton transfer from the methyl group of acetyl-coenzyme A only poorly, a process which occurs in a structurally separate site. Furthe rmore, all H320 mutants hydrolyze the citryl-coenzyme A intermediate s ignificantly more slowly than does the wild-type. D375 is the base rem oving the proton of acetyl-coenzyme A. D375E and D375G have greatly di minished ability to catalyze proton transfer from acetyl-CoA. The D375 mutants polarize the oxaloacetate carbonyl as well as wild-type. For D375E, the hydrolysis of citryl-CoA is rate determining. D375G, having no side chain capable of acid-base chemistry in either the condensati on or hydrolysis reactions is nearly completely devoid of activity in any of the reactions catalyzed by the wild-type. H274 hydrogen bonds t o the carbonyl of acetyl-coenzyme A but also forms the back wall of th e oxaloacetate-binding site. H274G cannot properly activate either oxa loacetate or acetyl-coenzyme A, and the condensation reaction is overw helmingly rate determining. Nonetheless, hydrolysis of the intermediat e is impaired. All the enzymes except H320R and H274G show kinetic coo perativity with CitCoA as substrate, indicating changes in the subunit interactions with these latter two mutants. The energetics of citrate synthase are surprisingly tightly coupled. All changes affect more th an one step in the catalytic cycle. Within the condensation reaction, the intermediate of proton transfer must occupy a shallow well between transition states close in free energy so that perturbations of one h ave substantial effects on that of the other.