A Kinetic Isotope Effect and Isotope Exchange Study of the Nonenzymatic and the Equine Serum Butyrylcholinesterase-Catalyzed Thioester Hydrolysis

Formylthiocholine (FTC) was synthesized and found to be a substrate for nonenzymatic and butyrylcholinesterase (BChE)-catalyzed hydrolysis. Solvent (D2O) and secondary formyl-H kinetic isotope effects (KIEs) were measured by an NMR spectroscopic method. The solvent (D2O) KIEs are D2O k = 0.20 in 200 mM HCl, D2O k = 0.81 in 50 mM HCl, and D2O k = 4.2 in pure water. The formyl-H KIEs are D k = 0.80 in 200 mM HCl, D k = 0.77 in 50 mM HCl, D k = 0.75 in pure water, D k = 0.88 in 50 mM NaOH, and D(V/K) = 0.89 in the BChE-catalyzed hydrolysis in MES buffer at pH 6.8. Positional isotope exchange experiments showed no detectable exchange of 18O into the carbonyl oxygen of FTC or the product, formate, under any of the above conditions. Solvent nucleophile-O KIEs were determined to be 18 k = 0.9917 under neutral conditions, 18 k = 1.0290 (water nucleophile) or 18 k = 0.989 (hydroxide nucleophile) under alkaline conditions, and 18(V/K) = 0.9925 for BChE catalysis. The acidic, neutral, and BChE-catalyzed reactions are explained in terms of a stepwise mechanism with tetrahedral intermediates. Evidence for a change to a direct displacement mechanism under alkaline conditions is presented.