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

Abstract

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 (18)O 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.

Mechanistic investigations of the hydrolysis of amides, oxoesters and thioesters via kinetic isotope effects and positional isotope exchange

“…In the PIX experiment FTC displayed no detectable 18 O exchange within experimental limits [24]. In this particular experiment only exchange into FTC (not formic acid) can be used because of the known 18 O exchange into formic acid.…”

“…The PIX experiment did not detect 18 O exchange from solvent into the carbonyl-O of FTC [24]. Once again this is not surprising because the chiral environment of the enzyme renders the two oxygen atoms diastereotopic and thus of intrinsically different reactivity.…”

“…The major drawback is that the experimental protocols are extremely difficult to develop. The procedure used in our laboratories [20,[22][23][24] for the amides, oxoesters and thioesters of formic acid is highlighted in this review to give the reader a feeling for both the complexity of measuring this type of heavy-atom KIE and the rewards from obtaining such data.…”

“…Alkaline conditions are difficult to investigate because the reaction rate is so much faster than under acidic and neutral conditions. Nevertheless we have developed techniques in our laboratories to obtain results from PIX and KIE experiments [24]. The substrate used was formylthiocholine (FTC), which is shown in Fig.…”

An investigation into the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine using heavy atom kinetic isotope effects

“…Quinn attributed this normal KIE as arising from an sp 3 Several KIEs for BChE-catalyzed hydrolysis of FTC have been measured previously in our labs using the competitive method, which measures only k cat /K m isotope effects (Table 1). 11 In these studies FTC was used as the substrate instead of ATC. The reason for choosing FTC was the existence of analytical procedures for formate which allowed measurement of multiple KIEs.…”