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

Siobhan Malany, Monali Sawai, R. Steven Sikorski, Javier Seravalli, Daniel M. Quinn, Zoran Radić, Palmer Taylor, Chanoch Kronman, Baruch Velan, Avigdor Shafferman

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35 Scopus citations


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 isotope effects and viscosity effects on the bimolecular rate constant k(E) (=k(cat)/K(m)). Substrate and solvent isotope effects have been measured for wild-type enzymes from Torpedo californica, human and mouse, and for various active site mutants of these enzymes. Sizable solvent isotope effects, (D2O)k(E) ~ 2, are observed when substrate β-deuterium isotope effects are most inverse, (βD)k(E) = 0.95; conversely, reactions that have (D2O)k(E) ~ 1 have substrate isotope effects of (βD)k(E) = 1.00. Proton inventories of k(E) provide a quantitative measure of the contributions by the successive steps, diffusional encounter of substrate with the active site and consequent 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 nearly so, consistent with prominent rate limitation by a chemical step whose transition state is stabilized by a single proton bridge. Reactions that have smaller solvent isotope effects and less inverse substrate isotope effects have nonlinear and upward bulging proton inventories, consistent with partial rate limitations by both diffusional encounter and chemical catalysis. Curve fitting of such proton inventories provides a measure of the commitment to catalysis that is in agreement with the effect of solvent viscosity on k(E) and with the results of a double isotope effect measurement, wherein (βD)k(E) is measured in both H2O and D2O. The results of these various experiments not only provide a model for the structure of the acylation transition state but also establish the validity of solvent isotope effects as a tool for quantitative characterization of rate limitation for acetylcholinesterase catalysis.

Original languageEnglish (US)
Pages (from-to)2981-2987
Number of pages7
JournalJournal of the American Chemical Society
Issue number13
StatePublished - Apr 5 2000

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


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