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

Single and multiple site mutants of recombinant mouse acetylcholineste rase (rMoAChE) were inhibited with racemic 7-(methylethoxyphosphinylox y)-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 methanesul fonate (P2S) and ium)-3-(4''-carbamoyl-1''-pyridinium)-2-oxapropane di chloride (HI-6), Kinetic analysis of the reactivation profiles reveale d biphasic behavior with an approximate 1:1 ratio of two presumed reac tivatable enantiomeric components. Equilibrium dissociation and kineti c rate constants for reactivation of site-specific mutant enzymes were compared with those obtained for wild-type rMoAChE, tissue-derived To rpedo 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 reactiva tion 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 rea ctivation 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 reac tivation potencies of P2S and HI-6, The two kinetic components of oxim e reactivation of MEPQ-inhibited AChEs arise from the chirality of O-e thyl methylphosphonyl moieties conjugated with Ser-203 and may be attr ibutable to the relative stability of the phosphonyl oxygen of the two enantiomers in the oxyanion hole,