Formation of active site thiol esters of CoA transferase and the dependence of catalysis on specific binding interactions.

The enzyme-CoA intermediate of succinyl-CoA:3-ketoacid coenzyme A transferase reacts with methyl mercaptopropionate, a short chain analog of coenzyme A, to release coenzyme A and give a new thiol ester at the active site, enzyme-SR (E-SR). The reaction is reversible, with Keq = 2.4 X 10(-4). Other thiols react similarly, acetyl-CoA. On the other hand, the value of kcat/km for the reaction of succinyl-SR with the enzyme to form E-SR is smaller than that for the reaction of succinyl-CoA to form enzyme-CoA by a factor of 3 X 10(12). Thus, interactions of the enzyme with the nonreacting portions of coenzyme A are responsible for a rate increase of 3 X 10(12) compared with the short chain analog. This increase is close to the total rate acceleration of 5 X 10(13) that is brought about by the enzyme. The rate increase is interpreted as representing 17 kcal mol-1 of intrinsic is interpreted as representing 17 kcal mol-1 of intrinsic binding energy of coenzyme A that is utilized to stabilize the transition state of the reaction; the observed binding of coenzyme A to the enzyme is relatively weak. The thiol ester group of enzyme-CoA is activated for reaction with the nonspecific reagents water, borohydride anion, and thiol anions by approximately 10(2)-10(3) compared with E-SR and thiol esters of acetate. The formation of E-SR at equilibrium is unfavorable by 5.7 kcal mol-2 compared with thiol esters of acetate, in spite of its normal chemical reactivity. This is consistent with a thermodynamically unfavorable conformation change upon thiol ester formation. The observed more favorable formation of enzyme-CoA compared to E-SR by 4.9 kcal mol-1 can be accounted for by utilization of the binding energy of coenzyme A to overcome the unfavorable Gibbs energy of this conformation change, for which there is independent evidence. The reaction of succinate with E-SR is 10(5) faster than with a thiol ester of acetate. This rate increase represents utilization of the binding energy of the specific substrate succinate for catalysis; however, no binding of succinate to E-SR was observed. A rate acceleration of 8 X 10(5) was observed for the nonphysiological reaction of coenzyme A with E-SR to give enzyme-CoA compared to the analogous nonenzymic reaction. This represents utilization of the binding energy of coenzyme A, in large part to overcome the entropy barrier for reaction. The combined effects of several modest contributions, especially the entropy loss of bound substrates, appear sufficient to account for most of the observed rate increase of the enzymic reaction without invoking ordinary mechanisms of chemical catalysis.