Investigation of the enzymic mechanism of yeast orotidine-5'-monophosphate decarboxylase using carbon-13 kinetic isotope effects

Abstract

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13C 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 Vmax/km, 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).

The crystal structure and mechanism of orotidine 5′-monophosphate decarboxylase

“…One possibility is that the enzymatic synthesis of 2-thioOMP did not give the anticipated product. Isotope effect studies suggest that the protonation and the decarboxylation are not concerted (35,36). This result does not contradict our mechanistic proposal if we assume that a substantial component of the observed solvent isotope effect is attributable to substrate binding or to a protein conformational change.…”

“…This is consistent with the use of substrate binding energy to hold the two carboxylate groups in close proximity. OMP decarboxylase shows a V͞K vs. pH profile with a maximum at pH ϭ 7 (35). We propose that Lys62 is protonated (pKa ϭ 7.5) and Asp60 is deprotonated (pKa ϭ 6.5) under these conditions.…”

Anatomy of a proficient enzyme: The structure of orotidine 5′-monophosphate decarboxylase in the presence and absence of a potential transition state analog

“…This reaction shows a 13 C kinetic isotope effect on k cat ͞K m of sufficient magnitude to indicate that cleavage of the scissile COC bond is almost fully rate determining (16). The potency of BMP as an inhibitor may be explained (4) by its resemblance to a carbanionic intermediate proposed by Beak and Siegel (17) and would be expected to contact the same enzyme residues that participate in OMP decarboxylation.…”

QM/MM Metadynamics Study of the Direct Decarboxylation Mechanism for Orotidine-5‘-monophosphate Decarboxylase Using Two Different QM Regions: Acceleration Too Small To Explain Rate of Enzyme Catalysis

“…5 Additionally, the rate constant for the reaction at low substrate concentration, measured as the ratio of the maximum speed of the enzyme reaction, V max , to the Michaelis dissociation constant for enzyme-substrate complex, K m , has been shown to peak at pH 7, indicating a catalytic group with a pK a near 7. 6 There have been several informative reviews on ODCase. 7,8,9 The direct decarboxylation mechanism for OMP involves stretching of the C6-CO 2 bond, and leads to formation of carbon dioxide and a deprotonated uridine with an unstabilized carbanion at C6 (Figure 1, b).…”