Transition State Structure and Rate Determination for the Acylation Stage of Acetylcholinesterase Catalyzed Hydrolysis of (Acetylthio)choline
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- 作者：Siobhan Malany ; Monali Sawai ; R. Steven Sikorski ; Javier Seravalli ; Daniel M. Quinn ; Zoran Radi
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- 刊名：Journal of the American Chemical Society
- 出版年：2000
- 出版时间：April 5, 2000
- 年：2000
- 卷：122
- 期：13
- 页码：2981 - 2987
- 全文大小：103K
- 年卷期：v.122,no.13(April 5, 2000)
- ISSN：1520-5126
文摘
Rate-limiting steps and transition state structure for the acylation stage of acetylcholinesterase-catalyzed hydrolysis of (acetylthio)choline have been characterized by measuring substrate and solvent isotopeeffects and viscosity effects on the bimolecular rate constant kE (=kcat/Km). Substrate and solvent isotope effectshave been measured for wild-type enzymes from Torpedo californica, human and mouse, and for variousactive site mutants of these enzymes. Sizable solvent isotope effects, D2OkE ~ 2, are observed when substrate
-deuterium isotope effects are most inverse, DkE = 0.95; conversely, reactions that have D2OkE ~ 1 havesubstrate isotope effects of DkE = 1.00. Proton inventories of kE provide a quantitative measure of thecontributions by the successive steps, diffusional encounter of substrate with the active site and consequentchemical catalysis, to rate limitation of the acylation stage of catalysis. For reactions that have the largestsolvent isotope effects and most inverse substrate isotope effects, proton inventories are linear or nearly so,consistent with prominent rate limitation by a chemical step whose transition state is stabilized by a singleproton bridge. Reactions that have smaller solvent isotope effects and less inverse substrate isotope effectshave nonlinear and upward bulging proton inventories, consistent with partial rate limitations by both diffusionalencounter and chemical catalysis. Curve fitting of such proton inventories provides a measure of the commitmentto catalysis that is in agreement with the effect of solvent viscosity on kE and with the results of a doubleisotope effect measurement, wherein DkE is measured in both H2O and D2O. The results of these variousexperiments not only provide a model for the structure of the acylation transition state but also establish thevalidity of solvent isotope effects as a tool for quantitative characterization of rate limitation for acetylcholinesterase catalysis.
-deuterium isotope effects are most inverse, DkE = 0.95; conversely, reactions that have D2OkE ~ 1 havesubstrate isotope effects of DkE = 1.00. Proton inventories of kE provide a quantitative measure of thecontributions by the successive steps, diffusional encounter of substrate with the active site and consequentchemical catalysis, to rate limitation of the acylation stage of catalysis. For reactions that have the largestsolvent isotope effects and most inverse substrate isotope effects, proton inventories are linear or nearly so,consistent with prominent rate limitation by a chemical step whose transition state is stabilized by a singleproton bridge. Reactions that have smaller solvent isotope effects and less inverse substrate isotope effectshave nonlinear and upward bulging proton inventories, consistent with partial rate limitations by both diffusionalencounter and chemical catalysis. Curve fitting of such proton inventories provides a measure of the commitmentto catalysis that is in agreement with the effect of solvent viscosity on kE and with the results of a doubleisotope effect measurement, wherein DkE is measured in both H2O and D2O. The results of these variousexperiments not only provide a model for the structure of the acylation transition state but also establish thevalidity of solvent isotope effects as a tool for quantitative characterization of rate limitation for acetylcholinesterase catalysis.