Interactions of oxime reactivators with diethylphosphoryl adducts of human acetylcholinesterase and its mutant derivatives.
Diethylphosphoryl conjugates of human acetylcholinesterase (AChE) and selected mutants, carrying amino acid replacements at the active center and at the peripheral anionic site, were subjected to reactivation with the monopyridinium oxime 2-hydroxy-iminomethyl-1-methylpyridinium chloride and the bispyridinium oximes 1,3-bis(4'-hydroxyiminomethyl-1'-pyridinium),propane dibromide (TMB-4) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4"-carbamoyl-1"-pyridinium)-2 - oxapropane dichloride (HI-6). The kinetic profiles for all of the reactivation reactions indicate single populations of reactivatable species. Replacement of Trp86, the anionic subsite in the active center, lowered the affinity of the free enzyme toward all three reactivators, but in the corresponding diethylphosphoryl conjugate, only affinity toward TMB-4 was affected. Replacement of other constituents of the hydrophobic subsite (Tyr337, Phe338) had no major effect on either affinity to the free enzymes or rates of reactivation. Substitution of residues of the acyl pocket (Phe295, Phe297) lowered the affinities toward reactivators except for the 20-fold increase in affinity of F295A toward HI-6. Replacement of the acidic residues in the active center (Glu202, Glu450) affected mainly the rates of nucleophilic displacement of the phosphoryl moiety. The effect of substituting residues constituting the peripheral anionic site at the rim of the active site gorge (Tyr72, Asp74, Trp286) was particularly puzzling because for 2-hydroxy-iminomethyl-1-methylpyridinium chloride and HI-6, mainly the nucleophilic reaction rate constants were affected, whereas for TMB-4, the affinities of the phosphorylated enzymes were significantly reduced. The fact that perturbations of the functional architecture of HuAChE active center can account for only some of the observed effects on the reactivation rates suggests that the binding modes of oxime to the phosphorylated and nonphosphorylated enzymes are considerably different and/or that interactions of the reactivators with the phosphoryl moieties play a dominant role in the reactivation process.
Grosfeld
H
H
Department of Biochemistry, Israel Institute for Biological Research, Ness-Ziona, Israel.
Barak
D
D
Ordentlich
A
A
Velan
B
B
Shafferman
A
A
D000110:Acetylcholinesterase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D002801:Cholinesterase Reactivators / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010091:Oximes / Q000737:chemistry / Q000378:metabolism*; D010261:Paraoxon / Q000494:pharmacology*; D010649:Phenylalanine; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014289:Trimedoxime / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan; D014443:Tyrosine
D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D009943:Organophosphorus Compounds; D010091:Oximes; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D014289:Trimedoxime; D014364:Tryptophan; D000110:Acetylcholinesterase; D010261:Paraoxon