Evidence from Inhibitor Studies for Conformational Changes of Citrate Synthase

Abstract

Substrate analog CoA derivatives were applied as inhibitors of [pig heart] citrate synthase. Substitution of the acyl-CoA oxygen next to sulfur by hydrogen was without marked influence on the affinity. Carboxymethyl-CoA, a structural analog of enolic acetyl-CoA, was characterized as a transition state analog by an affinity 100-fold higher than that of acetyl-CoA. Ks of the binary inhibitor-enzyme complex was high (230 .mu.M) but that of the ternary inhibitor-oxaloacetate-enzyme complex was 0.07 .mu.M. Both enzyme subunits bound the inhibitor independently, also in the presence of oxaloacetate. (3R,S)-3,4-Dicarboxy-3-hydroxybutyl-CoA, an analog of citryl-CoA, inhibited the overall reaction noncompetitively against acetyl-CoA and against oxaloacetate; it was a competitive inhibitor against the hydrolysis and cleavage reactions of (3S)-citryl-CoA. Kinetic data suggest that this inhibitor represents an intermediate analog. Conformational changes of the synthase probably occur during the catalytic cycle. In the proposed mechanism the free enzyme represents a hydrolase which in the presence of oxaloacetate, by a well-known conformational change, is converted into a ligase. If both substrates are present, the ligase is reconverted into the hydrolase upon formation of the intermediate, (3S)-citryl-CoA.