CATALYTIC STRATEGY OF CITRATE SYNTHASE - EFFECTS OF AMINO-ACID CHANGES IN THE ACETYL-COA BINDING-SITE ON TRANSITION-STATE ANALOG INHIBITOR COMPLEXES

Acetyl-CoA enol has been proposed as an intermediate in the citrate synthase (CS) reaction with Asp375 acting as a base, removing a proton from the methyl carbon of acetyl-CoA, and His274 acting as an acid, donating a proton to the carbonyl [Karpusas, M., Branchaud, B., & Remington, S. J. (1990) Biochemistry 29, 2213]. CS-oxaloacetate (OAA) complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CMCoA), mimic those with acetyl-CoA enol. Asp375 and His274 interact intimately with the carboxyl of the bound inhibitor. While enzymes in which these residues have been changed to other amino acids have very low catalytic activity, we find that they retain their ability to form complexes with substrates and the transition-state analog inhibitor. In comparison with the value of the chemical shift of the protonated CMCoA carboxyl in acidic aqueous solutions or its value in the wild-type ternary complex, the values in the Asp375 mutants are unusually low. Model studies suggest that these low values result from complete absence of one hydrogen bond partner for the Gly mutant and distortions in the active site hydrogen bond systems for the Glu mutant. The high affinity of Asp375Gly-OAA for CMCoA suggests that the unfavorable proton uptake required to stabilize the CMCoA-OAA ternary complex of the wild-type enzyme [Kurz, L. C., Shah, S., Crane, B. R., Donald, L. J., Duckworth, H. W., & Drysdale, G. R. (1992) Biochemistry (preceding paper in this issue)] is not required by this mutant; the needed proton is most likely provided by His274. This supports the proposed role of His274 as a general acid. The surprising low catalytic activity of the Asp375Glu mutant is discussed in terms of steric hindrance to the functioning of the carboxylate as general acid-base and/or in terms of an impairment of the mutant enzyme's ability to access the closed conformational form of the enzyme in which the active site is isolated from the bulk solvent. The Asp mutants are able to achieve full activation of OAA through carbonyl polarization as assessed by the [2-C-13]OAA chemical shift of ternary complexes. In contrast, the His274Gly mutant is unable to polarize the OAA carbonyl in binary or ternary complexes; it may be impaired in its access to the closed conformational form, and the C-13 chemical shift of the bound CMCoA is consistent with a carboxylate anion in an hydrophobic environment in the active site of this mutant protein.