Determination of the mechanism of orotidine 5 '-monophosphate decarboxylase by isotope effects

Orotidine 5'-monophosphate shows a N-15 isotope effect of 1.0036 at N-1 for decarboxylation catalyzed by orotidine 5'-monophosphate decarboxylase. Pic olinic acid shows a N-15 isotope effect of 0.9955 for decarboxylation in et hylene glycol at 190 degrees C, while N-methyl picolinic acid shows a N-15 isotope effect of 1.0053 at 120 degrees C. The transition state for enzymat ic decarboxylation of orotidine 5'-monophosphate resembles the transition s tate for N-methyl picolinic acid in that no bond order changes take place a t N-1. This rules out enolization to give a quaternary nitrogen at N-1 in t he enzymatic mechanism and suggests a carbanion intermediate stabilized by simple electrostatic interaction with Lys-93. The driving force for the rea ction appears to be ground-state destabilization resulting from charge repu lsion between the carboxyl of the substrate and Asp-91.