Amino acid residues controlling reactivation of organophosphonyl conjugates of acetylcholinesterase by mono- 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(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.
Ashani
Y
Y
Division of Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100.
Radić
Z
Z
Tsigelny
I
I
Vellom
D C
DC
Pickering
N A
NA
Quinn
D M
DM
Doctor
B P
BP
Taylor
P
P
D000110:Acetylcholinesterase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D002091:Butyrylcholinesterase / Q000097:blood; D016466:CHO Cells; D002460:Cell Line; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D002801:Cholinesterase Reactivators / Q000494:pharmacology*; D006224:Cricetinae; D003587:Cytomegalovirus; D005091:Exons; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010091:Oximes; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011726:Pyridinium Compounds / Q000494:pharmacology*; D011806:Quinolinium Compounds / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D014101:Torpedo; D014162:Transfection; D014364:Tryptophan; D014443:Tyrosine
C092538:2-(hydroxyiminomethyl)-1-methylpyridinium methanesulfonate; D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D010091:Oximes; D011726:Pyridinium Compounds; D011806:Quinolinium Compounds; D011994:Recombinant Proteins; D001224:Aspartic Acid; D014443:Tyrosine; D014364:Tryptophan; C044294:7-((methylethoxyphosphinyl)oxy)-1-methylquinolinium; D000110:Acetylcholinesterase; D002091:Butyrylcholinesterase; C022870:asoxime chloride