Transition state structure and rate determination for the acylation stage of acetylcholinesterase catalyzed hydrolysis of (acetylthio)choline

Rate-limiting steps and transition state structure for the acylation stage of acetylcholinesterase-catalyzed hydrolysis of (acetylthio)choline have be ewith the effect of solvent viscosity on k(E) and with the results of a dou ble isotope effect measurement, wherein (beta D)k(E) is measured in both H2 O and D2O. The results of these various experiments not only provide a mode ln characterized by measuring substrate and solvent isotope effects and vis cosity effects on the bimolecular rate constant k(E) (= k(cat)/K-m). Substr ate and solvent isotope effects have been measured for wild-type enzymes fr om Torpedo californica, human and mouse, and for various active site mutant s of these enzymes. Sizable solvent isotope effects, (D2O)k(E) similar to 2 , are observed when substrate beta-deuterium isotope effects are most inver se, (beta D)k(E) = 0.95; conversely, reactions that have (D2O)k(E) similar to 1 have substrate isotope effects of (beta D)k(E) = 1.00. Proton inventor ies of k(E) provide a quantitative measure of the contributions by the succ essive steps, diffusional encounter of substrate with the active site and c onsequent chemical, catalysis, to rate limitation of the acylation stage of catalysis; For reactions that have the largest solvent isotope effects and most inverse substrate isotope effects; proton inventories are linear or n early so, consistent with prominent rate limitation by a chemical step whos e transition state is stabilized by a single proton bridge. Reactions that have smaller solvent isotope effects and less inverse substrate isotope eff ects have nonlinear and upward bulging proton inventories, consistent with partial rate limitations by both diffusional encounter and chemical catalys is. Curve fitting of such proton inventories provides a measure of the comm itment to catalysis that is in agreement with the effect of solvent viscosi ty on k(E) and with the results of a double isotope effect measurement, whe rein (beta D)k(E) is measured in both H2O and D2O. The results of these var ious experiments not only provide a model for the structure of the acylatio n transition state but also establish the validity of solvent isotope effec ts as a tool for quantitative characterization of rate limitation for acety lcholinesterase catalysis.