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 and butyrylcholinesterase (EC 3.1.1.8). Mutations in the choline binding site (Y337A, Y337A/F338A) or combined with acyl pocket mutations (F295L/Y337A, F297I/Y337A, F295L/F297I/Y337A) 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 reactivation rates of the SP-conjugates. The greatest enhancement of 120-fold was achieved with HI-6 for the F295L/Y337A phosphonylated with the most bulky alkoxy moiety, SP-cycloheptyl methylphosphonate. This significant enhancement is likely a direct consequence of simultaneously increasing the dimensions of both the choline binding site and the acyl pocket. The increase in dimensions allows for optimizing the angle of oxime attack in the spatially impacted gorge as suggested from molecular modeling. Rates of reactivation 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.

cholinesterase; reactivation; phosphonates; oximes

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