Leaving group effects in binding and reaction steps of acetylcholinesterase inhibition by O,O-diethylthiophosphates

Abstract

Affinity and phosphorylation constants for the inhibition of acetylcholinesterase by a series of O,O-diethylthiophosphates, (C 2H 5O) 2P(O)SX, where X = C n H 2 n+1 ( n = 4–8), (CH 2) n SC 2H 5 ( n = 2–6), and (CH 2) n S +(CH 3)C 2H 5 ( n = 3–6), have been measured at 25°C and pH 7.0 in order to quantify the specificity-determining factors in the binding and reaction steps of the process. In contrast to the influence of the leaving group of the acetic ester substrates, where the hydrophobicity of the substituent affects the binding and acetylation steps equally, it has been found that the hydrophobicity of the leaving group of the nonionic organophosphorus inhibitor affects only the binding in the enzyme's active site whereas the rate of the phosphorylation reaction is governed solely by the inductive effect of substituent X. A common phosphorylation rate constant logarithm-σ x ∗ relationship holds for the cationic inhibitors and their nonionic analogs; however, an extra effect of the cationic group in X appears in the dissociation constant for the enzyme-inhibitor complex and points to the putative influence of the enzyme's anionic site in the binding step. The data obtained suggest that the hydrophobic cleft for the binding of the inhibitors' leaving group is not “shut” in the transition state of the phosphorylation of the enzyme's active site, in contrast to the acetylation reaction in which the closed hydrophobic slit in the transition state is shown to stabilize the complex.