Enzymology
Amino Acid Residues Controlling Reactivation of Organophosphonyl Conjugates of Acetylcholinesterase by Mono- and Bisquaternary Oximes (∗)

https://doi.org/10.1074/jbc.270.11.6370Get rights and content
Under a Creative Commons license
open access

Single and multiple site mutants of recombinant mouse acetylcholinesterase (rMoAChE) were inhibited with racemic 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide (MEPQ) and the resulting mixture of two enantiomers, CH3PRiS(O)(OC2H5)-AChE(EMPRiS-AChE), were subjected to reactivation with 2-(hydroxyiminomethyl)-1-methylpyridinium methanesulfonate (P2S) and 1-(2′-hydroxyiminomethyl-1′-pyridinium)-3-(4″-carbamoyl-1″-pyridinium)-2-oxapropane dichloride (HI-6). Kinetic analysis of the reactivation profiles revealed biphasic behavior with an approximate 1:1 ratio of two presumed reactivatable enantiomeric components. Equilibrium dissociation and kinetic rate constants for reactivation of site-specific mutant enzymes were compared with those obtained for wild-type rMoAChE, tissue-derived Torpedo AChE and human plasma butyrylcholinesterase. Substitution of key amino acid residues at the entrance to the active-site gorge (Trp-286, Tyr-124, Tyr-72, and Asp-74) had a greater influence on the reactivation kinetics of the bisquaternary reactivator HI-6 compared with the monoquaternary reactivator P2S. Replacement of Phe-295 by Leu enhanced reactivation by HI-6 but not by P2S. Of residues forming the choline-binding subsite, the E202Q mutation had a dominant influence where reactivation by both oximes was decreased 16- to 33-fold. Residues Trp-86 and Tyr-337 in this subsite showed little involvement. These kinetic findings, together with energy minimization of the oxime complex with the phosphonylated enzyme, provide a model for differences in the reactivation potencies of P2S and HI-6. The two kinetic components of oxime reactivation of MEPQ-inhibited AChEs arise from the chirality of O-ethyl methylphosphonyl moieties conjugated with Ser-203 and may be attributable to the relative stability of the phosphonyl oxygen of the two enantiomers in the oxyanion hole.

Cited by (0)

This work was supported in part by U. S. Army Research and Material Command Grant DAMD17-91-C-1056 (to P. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Visiting scientist from the Israel Institute for Biological Research, Ness-Ziona, Israel.

Visiting fellow from the Institute for Medical Research and Occupational Health, University of Zagreb, Croatia.

∗∗

Visiting scientist from the Department of Chemistry, University of Iowa, Iowa City, IA.