Correlation of Isotope and Viscosity Effects

Abstract

We have utilized kinetic isotope and viscosity effects to probe the reaction mechanism of acetylcholinesterase-catalyzed hydrolysis of (acetylthio)choline. The bimolecular rate constant kE provides a measure for the acylation stage of catalysis and is assumed to be partially rate limited by diffusional encounter and subsequent chemical transformation. Contributions of these two steps to rate limitation for wild type enzymes from Torpedo californica, mouse, and human, and for various active site mutants of these enzymes have been quantitatively determined by correlation between substrate and solvent isotope effects and viscosity effects on kE. From solvent isotope effect measurements in mixtures of H2O and D2O, called proton inventories, the fraction of rate limitation due to binding and the fraction of rate limitation by the proton transfer step can be calculated. The model of fractional rate determination obtained is consistent with the dependencies of the individual rate constants that contribute to kE on solvent viscosity. In addition, β-deuterium substrate isotope effects were measured in both H2O and D2O to probe changes in transition state geometry. Trends in β-deuterium isotope effects and in solvent effects provide complementary information as to the effect of active site mutation on the rate limiting contributions of the diffusion and catalysis steps. Overall, these independent experimental approaches have proved to be incisive tools for dissecting the rates and characterizing the transition state of an enzyme whose catalytic power is highly evolved.