Evidence from Inhibitor Studies for Conformational Changes of Citrate Synthase

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Abstract
Substrate analogue CoA derivatives were applied as inhibitors of citrate synthase. Substitution of the acyl‐CoA oxygen next to sulfur by hydrogen was without marked influence on the affinity. Carboxymethyl‐CoA, a structural analogue of enolic acetyl‐CoA. Ks of the binary inhibitor‐enzyme comples was high (230 μM) but that of the ternary inhibitor‐oxaloacetate‐enzyme complex was 0.07 μM. Both enzyme sub‐units bond the inhibitor independently, also in the presence of oxaloacetate. (3R,S)‐3,4‐Dicarboxy‐3hydroxybutyl‐CoA, an analogue 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 and intermediate analogue. The results given above indicate conformational changes of the synthase during the catalytic cycle. In the proposed mechanism the free enzyme represents a hydrolase which in the presence of oxaloacetate, by a wellknown conformational change, is converted into a ligase. If both substrates are presents, the ligase is reconverted into the hydrolase upon formation of the intermediate, (3S)‐citryl‐CoA.