Accumulation of tetrahedral intermediates in cholinesterase catalysis: a secondary isotope effect study
| Autor: | Florian Nachon, Jose R. Tormos, Patrick Masson, Yi Wang, Kenneth L. Wiley, Daniel M. Quinn, Didier Fournier |
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| Přispěvatelé: | Department of Chemistry, Iowa State University (ISU), Différenciation Cellulaire et Croissance (DCC), Institut National de la Recherche Agronomique (INRA)-Université Montpellier 2 - Sciences et Techniques (UM2), Centre de Recherche du Service de Santé des Armées |
| Rok vydání: | 2010 |
| Předmět: |
[SDV]Life Sciences [q-bio]
Kinetics 010402 general chemistry 01 natural sciences Biochemistry Medicinal chemistry Catalysis Article Colloid and Surface Chemistry Isotopes Tetrahedral carbonyl addition compound Catalytic Domain Kinetic isotope effect Organic chemistry Animals Cholinesterases [INFO]Computer Science [cs] Butyrylcholinesterase Isotope 010405 organic chemistry Chemistry Substrate (chemistry) General Chemistry Deuterium Recombinant Proteins 0104 chemical sciences INSECTE Acetylthiocholine Drosophila |
| Zdroj: | Journal of the American Chemical Society Journal of the American Chemical Society, American Chemical Society, 2010, 132 (50), pp.17751-17759. ⟨10.1021/ja104496q⟩ |
| ISSN: | 1520-5126 4538-1453 0002-7863 |
| DOI: | 10.1021/ja104496q⟩ |
| Popis: | Contact: daniel-quinn@uiowa.edu; International audience; In a previous communication, kinetic beta-deuterium secondary isotope effects were reported that support a mechanism for substrate-activated turnover of acetylthiocholine by human butyrylcholinesterase (BuChE) wherein the accumulating reactant state is a tetrahedral intermediate (Tormos, J. R.; et al. J. Am. Chem. Soc. 2005, 127, 14538-14539). In this contribution additional isotope effect experiments are described with acetyl-labeled acetylthiocholines (CL(3)COSCH(2)CH(2)N(+)Me(3); L = H or D) that also support accumulation of the tetrahedral intermediate in Drosophila melanogaster acetylcholinesterase (DmAChE) catalysis. In contrast to the aforementioned BuChE-catalyzed reaction, for this reaction the dependence of initial rates on substrate concentration is marked by pronounced substrate inhibition at high substrate concentrations. Moreover, kinetic beta-deuterium secondary isotope effects for turnover of acetylthiocholine depended on substrate concentration, and gave the following: (D3)k(cat)/K(m) = 0.95 +/- 0.03, (D3)k(cat) = 1.12 +/- 0.02 and (D3)beta k(cat) = 0.97 +/- 0.04. The inverse isotope effect on k(cat)/K(m) is consistent with conversion of the sp(2)-hybridized substrate carbonyl in the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis, whereas the markedly normal isotope effect on k(cat) is consistent with hybridization change from sp(3) toward sp(2) as the reactant state for deacylation is converted into the subsequent transition state. Transition states for Drosophila melanogaster AChE-catalyzed hydrolysis of acetylthiocholine were further characterized by measuring solvent isotope effects and determining proton inventories. These experiments indicated that the transition state for rate-determining decomposition of the tetrahedral intermediate is stabilized by multiple protonic interactions. Finally, a simple model is proposed for the contribution that tetrahedral intermediate stabilization provides to the catalytic power of acetylcholinesterase. |
| Databáze: | OpenAIRE |
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