Studies on the mechanism of action of orotidine 5'-monophosphate decarboxylase.

摘要

The focus of this dissertation is on the mechanism of action of the enzyme orotidine 5'-monophosphate decarboxylase (OMPDC). We are interested in understanding the mechanism for the extraordinarily large rate acceleration achieved by this enzyme. Specifically to understand the mechanism by which binding interactions between the phosphate group of the substrate and the enzyme are "utilized" in the stabilization of a vinyl carbanion intermediate, which forms at a site distant from the phosphate group. We have addressed this problem by determining the effect of several site-directed mutations on the kinetic parameters for the following OMPDC-catalyzed reactions: (1) Decarboxylation of OMP. (2) Decarboxylation of a truncated substrate (EO) that lacks the phosphodianion group, and on the activation of this enzyme-catalyzed decarboxylation reaction by phosphite dianion. This data have been used to evaluate the magnitude of enzymephosphodianion interactions that are utilized to stabilize the ground state Michaelis complex and the transition state for decarboxylation. (3) Decarboxylation of the highly reactive substrate FOMP, for which both kcat and kcat/K m are limited by transport steps. These experiments are providing novel insight into the effect of site-directed mutations on the rate constants for these transport processes.