INVESTIGATION OF THE ENZYMATIC MECHANISM OF YEAST OROTIDINE-5'-MONOPHOSPHATE DECARBOXYLASE USING C-13 KINETIC ISOTOPE EFFECTS

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed C-13 kinetic isotope effect of 1.0247 +/- 0.0008 at 25-degrees-C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 +/- 0.0006 measured at pH 4.0, 25-degrees-C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel [Beak, P. & Siegel, B. (1976) J. Am. Chem. Soc 98, 3601-3606], which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25-degrees-C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. The observed isotope effect does not rise to the intrinsic value above pH 8.5; instead, the observed isotope effects at 25-degrees-C plotted against pH yield an asymmetric curve that at high pH plateaus at about 1.035. These data, in conjunction with the pH profile of V(max)/K(m), fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegel (1976).