Carbon isotope effect study on orotidine 5'-monophosphate decarboxylase: support for an anionic intermediate.
Orotidine 5'-monophosphate decarboxylase has been heavily examined in recent years due to its enzymatic proficiency, which provides a catalytic enhancement to a reaction rate approximately 1017 times greater than that of the nonenzymatic reaction. Several mechanisms proposed to explain this catalytic enhancement have included covalent addition, ylide or carbene formation, and most recently concerted protonation. All of these mechanisms have circumvented the formation of a high-energy vinyl anionic intermediate. To investigate the presence of an anionic intermediate, 13C isotope effect studies have been performed using the alternate substrate 5-fluoro-OMP (OMP = orotidine 5'-monophosphate). Isotope effects obtained for the wild-type enzyme with OMP and 5-fluoro-OMP are 1.0255 and 1.0106, respectively, corresponding to a decrease of approximately 1.5% for 5-fluoro-OMP. With the K59A enzyme, the intrinisic isotope effects show a similar decrease of approximately 1.9% from 1.0543 with OMP to 1.0356 with 5-fluoro-OMP. This decrease results from the inductive effect of the fluorine, which stabilizes the carbanion intermediate by electron withdrawal and produces a reaction with an earlier transition state. The isotope effect for the decarboxylation of the slow substrate 2'-deoxy-OMP produced a intrinsic isotope effect of nearly 1.0461.
Van Vleet
Jeremy L
JL
Institute for Enzyme Research and Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53726, USA.
Reinhardt
Laurie A
LA
Miller
Brian G
BG
Sievers
Annette
A
Cleland
W Wallace
WW
D001665:Binding Sites; D002247:Carbon Isotopes; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation
D002247:Carbon Isotopes; D050505:Mutant Proteins; D009964:Orotidine-5'-Phosphate Decarboxylase