posted on 2013-12-06, 00:00authored byLori 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.