Accumulation of Tetrahedral Intermediates in Cholinesterase Catalysis: A Secondary Isotope Effect Study

In a previous communication, kinetic β-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 (CL3COSCH2CH2N+Me3; 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 β-deuterium secondary isotope effects for turnover of acetylthiocholine depended on substrate concentration, and gave the following: D3kcat/Km = 0.95 ± 0.03, D3kcat = 1...