Effects of substrate binding determinants in the transition state for orotidine 5 '-monophosphate decarboxylase

To evaluate the effects of individual binding determinants on transition st ate stabilization, the binding properties of substrates and competitive inh ibitors of the OMP decarboxylase activity of human UMP synthase were compar ed with those of fragments obtained by cutting these ligands at various pos itions. The ribofuranosyl group generates little binding affinity las indic ated by comparison of the k(cat)/K-m values of orotidine with that of eroti c acid, and of the K-i value of 6-hydroxyuridine with that of 6-hydroxyurac il), but seems to constrain the relative mobilities of the pyrimidine ring and the phosphoryl group in such a way as to optimize their contributions t o transition state stabilization. The phosphoryl group of OMP appears to co ntribute approximately 10 kcal/mol of binding free energy to transition sta te stabilization, as indicated by comparison of the k(cat)/K-m value of OMP with that of orotidine, and of the K-i value of the transition state analo gue inhibitor 6-hydroxy-UMP with that of the corresponding ribonucleoside. This substituent effect, one of the largest that has been recorded for an e nzyme reaction, is of special interest in view of the phosphoryl group's co nsiderable distance from the site of substrate transformation. (C) 1998 Aca demic Press.