A MODEL OF THE CONDENSATION STEP IN THE CITRATE SYNTHASE REACTION

Calculations have been performed on the nucleophilic addition of simpl e molecules representing a thioester enolate and enol to acetone, as m odels for the addition step in the Claisen-type condensation reaction of acetyl-CoA and oxaloacetate catalysed by the enzyme citrate synthas e. The structures of the transition state and addition product for the enolate model have been optimized at the semi-empirical (AM1, PM3 and MNDO) and ab initio (RHF/6-31+G(d) and MP2/6-31+G(ci)) levels. The re lative energies of the transition state and addition product given by AM1 are reasonably close to the best ab initio result. The results are compared with AM1 results for addition of the enol form of the thioes ter model, which has been proposed as an alternative possibility for t he nucleophile in the enzyme reaction. The barrier to the addition rea ction for the enol model is considerably larger, due to the expected p oorer nucleophilicity of the neutral enol form. It appears that the ba rrier for enol addition, unless significantly lowered by the enzyme, i s inconsistent with the observed rate of reaction. In agreement with s tudies of the enolization of acetyl-CoA in citrate synthase, the enola te of acetyl-CoA appears to be a more likely nucleophilic intermediate than the enol. This conclusion may also be relevant for other enzymic Claisen condensation reactions in that efficient reaction may require stabilization of the thioester enolate to maintain a high degree of e nolate character in the reactive intermediate. (C) 1998 Elsevier Scien ce B.V.