Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5鈥?Monophosphate Decarboxylase

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• 作者：Bogdana Goryanova ; Lawrence M. Goldman ; Shonoi Ming ; Tina L. Amyes ; John A. Gerlt ; John P. Richard
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：July 28, 2015
• 年：2015
• 卷：54
• 期：29
• 页码：4555-4564
• 全文大小：457K
• ISSN：1520-4995

文摘

The caged complex between orotidine 5鈥?monophosphate decarboxylase (ScOMPDC) and 5-fluoroorotidine 5鈥?monophosphate (FOMP) undergoes decarboxylation 鈭?00 times faster than the caged complex between ScOMPDC and the physiological substrate, orotidine 5鈥?monophosphate (OMP). Consequently, the enzyme conformational changes required to lock FOMP at a protein cage and release product 5-fluorouridine 5鈥?monophosphate (FUMP) are kinetically significant steps. The caged form of ScOMPDC is stabilized by interactions between the side chains from Gln215, Tyr217, and Arg235 and the substrate phosphodianion. The control of these interactions over the barrier to the binding of FOMP and the release of FUMP was probed by determining the effect of all combinations of single, double, and triple Q215A, Y217F, and R235A mutations on k_cat/K_m and k_cat for turnover of FOMP by wild-type ScOMPDC; its values are limited by the rates of substrate binding and product release, respectively. The Q215A and Y217F mutations each result in an increase in k_cat and a decrease in k_cat/K_m, due to a weakening of the protein鈥損hosphodianion interactions that favor fast product release and slow substrate binding. The Q215A/R235A mutation causes a large decrease in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP, which are limited by the rate of the decarboxylation step, but much smaller decreases in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of FOMP, which are limited by the rate of enzyme conformational changes. By contrast, the Y217A mutation results in large decreases in k_cat/K_m for ScOMPDC-catalyzed decarboxylation of both OMP and FOMP, because of the comparable effects of this mutation on rate-determining decarboxylation of enzyme-bound OMP and on the rate-determining enzyme conformational change for decarboxylation of FOMP. We propose that k_cat = 8.2 s^鈥? for decarboxylation of FOMP by the Y217A mutant is equal to the rate constant for cage formation from the complex between FOMP and the open enzyme, that the tyrosyl phenol group stabilizes the closed form of ScOMPDC by hydrogen bonding to the substrate phosphodianion, and that the phenyl group of Y217 and F217 facilitates formation of the transition state for the rate-limiting conformational change. An analysis of kinetic data for mutant enzyme-catalyzed decarboxylation of OMP and FOMP provides estimates for the rate and equilibrium constants for the conformational change that traps FOMP at the enzyme active site.