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 cente r and at the peripheral anionic site, were subjected to reactivation w ith the monopyridinium oxime 2-hydroxy-iminomethyl-1-methylpyridinium chloride and the bispyridinium oximes 1,3-bis(4'-hydroxyiminomethyl-1' -pyri dinium),propane dibromide (TMB-4) and 1-(2'-hydroxyiminomethyl-1 '-pyridinium)-3-(4 ''-carbamoyl-1 ''-pyridinium)-2-oxapropane dichlori de (HI-6). The kinetic profiles for all of the reactivation reactions indicate single populations of reactivatable species. Replacement of T rp86, the anionic subsite in the active center, lowered the affinity o f the free enzyme toward all three reactivators, but in the correspond ing diethylphosphoryl conjugate, only affinity toward TMB-4 was affect ed. Replacement of other constituents of the hydrophobic subsite (Tyr3 37, 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 fo r the 20-fold increase in affinity of F295A toward HI-6. Replacement o f the acidic residues in the active center (Glu202, Glu450) affected m ainly the rates of nucleophilic displacement of the phosphoryl moiety, The effect of substituting residues constituting the peripheral anion ic site at the rim of the active site gorge (Tyr72, Asp74, Trp286) was particularly puzzling because for 2-hydroxy-iminomethyl-1-methylpyrid inium chloride and HI-6, mainly the nucleophilic reaction rate constan ts were affected, whereas for TMB-4, the affinities of the phosphoryla ted enzymes were significantly reduced. The fact that perturbations of the functional architecture of HuAChE active center can account for o nly some of the observed effects on the reactivation rates suggests th at the binding modes of oxime to the phosphorylated and nonphosphoryla ted enzymes are considerably different and/or that interactions of the reactivators with the phosphoryl moieties play a dominant role in the reactivation process.