Effects of Substrate Binding Determinants in the Transition State for Orotidine 5′-Monophosphate Decarboxylase

Abstract

To evaluate the effects of individual binding determinants on transition state stabilization, the binding properties of substrates and competitive inhibitors of the OMP decarboxylase activity of human UMP synthase were compared with those of fragments obtained by cutting these ligands at various positions. The ribofuranosyl group generates little binding affinity (as indicated by comparison of thekcat/Kmvalues of orotidine with that of orotic acid, and of theKivalue of 6-hydroxyuridine with that of 6-hydroxyuracil), but seems to constrain the relative mobilities of the pyrimidine ring and the phosphoryl group in such a way as to optimize their contributions to transition state stabilization. The phosphoryl group of OMP appears to contribute approximately 10 kcal/mol of binding free energy to transition state stabilization, as indicated by comparison of thekcat/Kmvalue of OMP with that of orotidine, and of theKivalue of the transition state analogue inhibitor 6-hydroxy-UMP with that of the corresponding ribonucleoside. This substituent effect, one of the largest that has been recorded for an enzyme reaction, is of special interest in view of the phosphoryl group's considerable distance from the site of substrate transformation.