engleski

Oxime reactivation of enantiomeric organophosphonyl conjugates of cholinesterase mutants

Single and multiple site mutants of mouse acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), were inhibited with enantiomeric SP and RP cycloheptyl (CHMP), isopropyl (iPrMP), and dimethylbutyl (DMBMP) methylphosphonyl thiocholines and resulting conjugates subjected to reactivation with oximes 2-PAM or HI-6. Mutations of AChE were in the two active site domains important for specificity of substrates and inhibitors: the acyl pocket (F295L, F297I) and the choline binding site (Y337A). The F295L mutation enhanced the HI-6 induced reactivation rates of the SP conjugates up to 10-20 fold, whereas the F297I mutation preferentially enhanced 2-PAM reactivation by an equivalent or greater magnitude. The RP conjugates were far more resistant to reactivation, and little enhancement of reactivation rates was seen with the acyl pocketmutants. By contrast, the Y337A mutation accelerated reactivation rate of all RP conjugates up to 6000-fold. Y337A showed a 5-25 fold faster HI-6 reactivation rate for SP -DMBMP and SP -CHMP, and a 12-fold slower rate for SP -iPrMP compared to wildtype AChE. On other hand, complete removal of the aromatic residues as in BChE, which has L, I and A at positions corresponding to 295, 297 and 337, did not enhance reactivation of many SP and RP conjugates. Hence, both access of the oxime nucleophile to the phosphorus and the configuration of the conjugated phosphonate in the active center appeared critical to reactivation. (Supported by GM18360, DAMD1718014 and a Croatian Ministry of Science Fellowship)

ACETYLCHOLINESTERASE; OXIME REACTIVATION; ENANTIOMERIC ORGANOPHOSPHONATES

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