Citrate synthase complexes with the transition-state analog inhibitor, carboxymethyl-CoA(CM-CoA), are believed to mimic those with the activated form of acetyl-CoA. The X-ray structure [Karpusas, M., Branchaud, B., & Remington, S. J. (1990) Biochemistry 29, 2213] of the ternary complex of the enzyme, oxaloacetate, and CMCoA has been used as the basis for a proposal that a neutral enol of acetyl-CoA is that activated form. Since the inhibitor carboxyl has a pK(a) of 3.90, analogy with an enolic acetyl-CoA intermediate leads to the prediction that a proton should be taken up from solution upon formation of the analog complex so that the transition-state analog carboxyl is protonated when bound. We have obtained evidence in solution for this proposal by comparing the isoelectric points and the pH dependence of the dissociation constants of the ternary complexes of the pig heart enzyme with the neutral ground-state analog inhibitor, acetonyl-CoA (KCoA), and the anionic transition-state analog inhibitor (CMCoA) and by studying the NMR spectra of the transition-state analog complexes of allosteric (Escherichia coli) and nonallosteric (pig heart) enzymes. The pH dependence of the dissociation constant of the ground-state analog indicates no proton uptake, while that for the transition-state analog indicates that 0.55 +/- 0.04 proton is taken up when the analog binds to the citrate synthase-oxaloacetate binary complex. The overall charges of ternary complexes of the pig heart enzyme with the transition-state and ground-state analog inhibitors are the same, as monitored by their isoelectric points. In [1-C-13]carboxymethyl-CoA binary complexes and in the oxaloacetate ternary complex of the allosteric enzyme in the absence of KCl, the chemical shift of the bound CMCoA carboxylate indicates that the inhibitor is bound as the anion. In ternary complexes with OAA (or with OAA plus KCl for the allosteric enzyme), the chemical shift of the bound CMCoA carboxyl is consistent with inhibitor carboxyl protonation in the active site. The chemical shift of the bound carboxyl of CMCoA is unaffected by pH (pH 6.4-10.0), indicating that the potential ionization of the bound carboxyl is suppressed as long as the enzyme ternary complex remains intact. The allosteric enzyme from E. coli requires KCl to achieve protonation of CMCoA, implying that one effect of KCl is to facilitate the activation, as well as the binding, of acetyl-CoA.
