A secondary isotope effect study of equine serum butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine.

Abstract β-Secondary deuterium isotope effects have been measured for equine serum butyrylcholinesterase-catalyzed hydrolysis of acetyl-L 3 -thiocholine (L = H or 2 H). The dependencies of initial rates on isotopic substrate concentrations show close adherence to Michaelis–Menten kinetics, and yield the following isotope effects: D3 k cat / K m  = 0.98 ± 0.02 and D3 k cat  = 1.10 ± 0.02. The modestly inverse isotope effect on k cat / K m is consistent with partial rate limitation by a step that converts the sp 2 -hybridized ester carbonyl of the E  +  A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis. On the other hand, the markedly normal isotope effect on k cat indicates that the Michaelis complex that accumulates at substrate saturation of the active site during catalytic turnover is a tetrahedral intermediate, whose decomposition is the rate-limiting step. These results compliment a previous report [J.R. Tormos et al., J. Am. Chem. Soc. 127 (2005) 14538–14539] that showed that substrate-activated hydrolysis of acetylthiocholine (ATCh), catalyzed by recombinant human butyrylcholinesterase, is also rate limited by decomposition of an accumulating tetrahedral intermediate.