The reactant state for substrate-activated turnover of acetylthiocholine by butyrylcholinesterase is a tetrahedral intermediate.

Secondary beta-deuterium kinetic isotope effects have been measured as a function of substrate concentration for recombinant human butyrylcholinesterase-catalyzed hydrolysis of acetyl-L3-thiocholine (L = 1H or 2H). The isotope effect on V/K is inverse, D3V/K = 0.93 +/- 0.03, which is consistent with conversion of the sp2 hybridized carbonyl carbon of the scissile ester bond of the E + A reactant state to a quasi-tetrahedral structure in the acylation transition state. In contrast, the isotope effect on Vmax under conditions of substrate activation is markedly normal, D3(betaVmax) = 1.29 +/- 0.06, an observation that is consistent with accumulation of a tetrahedral intermediate as the reactant state for catalytic turnover. Generally, tetrahedral intermediates for nonenzymatic ester hydrolyses are high-energy steady-state intermediates. Apparently, butyrylcholinesterase displays an unusual ability to stabilize such intermediates. Hence, the catalytic power of cholinesterases can largely be understood in terms of their ability to stabilize tetrahedral intermediates in the multistep reaction mechanism.