engleski

Mutant cholinesterases possessing enhanced capacity for reactivation of their phosphonylated conjugates

Selective mutants of mouse acetylcholinesterase (AChE ; EC 3.1.1.7) phosphonylated with chiral SP- and RP-cycloheptyl, -3, 3-dimethylbutyl and -isopropyl, methylphosphonyl thiocholines were subjected to reactivation by the oximes HI-6 and 2-PAM and their reactivation kinetics compared with wild-type AChE. Mutations in the choline binding site (Y337A, F338A) or combined with acyl pocket mutations (F295L, F297I) were employed to enlarge active center gorge dimensions. HI-6 was more efficient than 2-PAM (up to 29, 000 times) as a reactivator of SP-phosphonates (kr ranged from 50 to 13, 000 min-1M-1), while RP-conjugates were reactivated by both oximes at similar, but far slower rates (kr<10 min-1M-1). The Y337A substitution accelerated all reactivation rates over the wild-type AChE, and enabled reactivation even of RP-cycloheptyl and RP-3, 3-dimethylbutyl conjugates that when formed in wild-type AChE are resistant to reactivation. When combined with the F295L or F297I mutations in the acyl pocket, the Y337A mutation showed substantial enhancements of HI-6 reactivation rates of the SP-conjugates. Molecular modeling analysis was performed in order to ascertain probable HI-6 orientations inside the active center gorges. The model of SP-cycloheptyl methylphosphonylated wild-type AChE indicates that HI-6 can assume only one distinct productive orientation, while within the F295L/Y337A mutant additional orientations appear likely. Obtained reactivation rates reach values sufficient for consideration of mixtures of a mutant enzyme and an oxime as a scavenging strategy in protection and treatment of organophosphate exposure. The enhanced scavenging capacity is manifested to the greatest extent in those bulky SP enantiomers that are most intractable to reactivation. Methylphosphonates, cyclosarin and soman, would fall into that category.

acetylcholinesterase; inhibition; organophosphorus compound; oxime

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