Carbon Isotope Effect Study on Orotidine 5'-Monophosphate Decarboxylase: Support for an Anionic Intermediate

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• 作者：Jeremy L. Van Vleet ; Laurie A. Reinhardt ; Brian G. Miller ; Annette Sievers ; W. Wallace Cleland
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：January 15, 2008
• 年：2008
• 卷：47
• 期：2
• 页码：798 - 803
• 全文大小：65K
• 年卷期：v.47,no.2(January 15, 2008)
• ISSN：1520-4995

文摘

Orotidine 5'-monophosphate decarboxylase has been heavily examined in recent years due toits enzymatic proficiency, which provides a catalytic enhancement to a reaction rate ~10¹⁷ times greaterthan that of the nonenzymatic reaction. Several mechanisms proposed to explain this catalytic enhancementhave included covalent addition, ylide or carbene formation, and most recently concerted protonation. Allof these mechanisms have circumvented the formation of a high-energy vinyl anionic intermediate. Toinvestigate the presence of an anionic intermediate, ¹³C isotope effect studies have been performed usingthe alternate substrate 5-fluoro-OMP (OMP = orotidine 5'-monophosphate). Isotope effects obtained forthe wild-type enzyme with OMP and 5-fluoro-OMP are 1.0255 and 1.0106, respectively, correspondingto a decrease of ~1.5% for 5-fluoro-OMP. With the K59A enzyme, the intrinisic isotope effects show asimilar decrease of ~1.9% from 1.0543 with OMP to 1.0356 with 5-fluoro-OMP. This decrease resultsfrom the inductive effect of the fluorine, which stabilizes the carbanion intermediate by electron withdrawaland produces a reaction with an earlier transition state. The isotope effect for the decarboxylation of theslow substrate 2'-deoxy-OMP produced a intrinsic isotope effect of nearly 1.0461.