A Mechanistic Study of Thioester Hydrolysis with Heavy Atom Kinetic Isotope Effects

The carbonyl-C, carbonyl-O, and leaving-S kinetic isotope effects (KIEs) were determined for the hydrolysis of formylthiocholine. Under acidic conditions, ¹³<i>k</i>_obs = 1.0312, ¹⁸<i>k</i>_obs = 0.997, and ³⁴<i>k</i>_obs = 0.995; for neutral conditions, ¹³<i>k</i>_obs = 1.022, ¹⁸<i>k</i>_obs = 1.010, and ³⁴<i>k</i>_obs = 0.996; and for alkaline conditions, ¹³<i>k</i>_obs = 1.0263, ¹⁸<i>k</i>_obs = 0.992, and ³⁴<i>k</i>_obs = 1.000. The observed KIEs provided helpful insights into a qualitative description of the bond orders in the transition state structure

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<i>k</i> = 0.20 in 200 mM HCl, ^D₂O<i>k</i> = 0.81 in 50 mM HCl, and ^D₂O<i>k</i> = 4.2 in pure water. The formyl-H KIEs are ^D<i>k</i> = 0.80 in 200 mM HCl, ^D<i>k</i> = 0.77 in 50 mM HCl, ^D<i>k</i> = 0.75 in pure water, ^D<i>k</i> = 0.88 in 50 mM NaOH, and ^D(<i>V</i>/<i>K</i>) = 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<i>k</i> = 0.9917 under neutral conditions, ¹⁸<i>k</i> = 1.0290 (water nucleophile) or ¹⁸<i>k</i> = 0.989 (hydroxide nucleophile) under alkaline conditions, and ¹⁸(<i>V</i>/<i>K</i>) = 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