AMINO-ACID-RESIDUES CONTROLLING REACTIVATION OF ORGANOPHOSPHONYL CONJUGATES OF ACETYLCHOLINESTERASE BY MONOQUATERNARY AND BISQUATERNARY OXIMES

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, CH3PR,S(O)(OC2H5)-AChE(EMP(R,S)-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,