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

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journal contribution
posted on 06.12.2013, 00:00 by Lori I. Robins, Kristen M. Meisenheimer, Emily J. Fogle, Cory A. Chaplan, Richard L. Redman, Joseph T. Vacca, Michelle R. Tellier, Brittney R. Collins, Dorothea H. Duong, Kathrin Schulz, John F. Marlier
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 D2Ok = 0.20 in 200 mM HCl, D2Ok = 0.81 in 50 mM HCl, and D2Ok = 4.2 in pure water. The formyl-H KIEs are Dk = 0.80 in 200 mM HCl, Dk = 0.77 in 50 mM HCl, Dk = 0.75 in pure water, Dk = 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 18k = 0.9917 under neutral conditions, 18k = 1.0290 (water nucleophile) or 18k = 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.