Amino acid residues controlling reactivation of organophosphonyl conjugates of acetylcholinesterase by mono- and bisquaternary oximes

J Biol Chem. 1995 Mar 17;270(11):6370-80. doi: 10.1074/jbc.270.11.6370.


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(EMPR,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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acetylcholinesterase / biosynthesis
  • Acetylcholinesterase / chemistry*
  • Acetylcholinesterase / metabolism*
  • Amino Acid Sequence
  • Animals
  • Aspartic Acid
  • Binding Sites
  • Butyrylcholinesterase / blood
  • CHO Cells
  • Cell Line
  • Cholinesterase Inhibitors / pharmacology*
  • Cholinesterase Reactivators / pharmacology*
  • Cricetinae
  • Cytomegalovirus
  • Exons
  • Humans
  • Kidney
  • Kinetics
  • Models, Molecular
  • Molecular Structure
  • Mutagenesis, Site-Directed
  • Oximes
  • Promoter Regions, Genetic
  • Protein Conformation
  • Pyridinium Compounds / pharmacology*
  • Quinolinium Compounds / pharmacology*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Stereoisomerism
  • Structure-Activity Relationship
  • Torpedo
  • Transfection
  • Tryptophan
  • Tyrosine


  • 2-(hydroxyiminomethyl)-1-methylpyridinium methanesulfonate
  • Cholinesterase Inhibitors
  • Cholinesterase Reactivators
  • Oximes
  • Pyridinium Compounds
  • Quinolinium Compounds
  • Recombinant Proteins
  • Aspartic Acid
  • Tyrosine
  • Tryptophan
  • 7-((methylethoxyphosphinyl)oxy)-1-methylquinolinium
  • Acetylcholinesterase
  • Butyrylcholinesterase
  • asoxime chloride