engleski

Acetylcholinesterase mutants possessing enhanced capacity for scavenging organophosphonates

Reaction of organophosphonates with acetylcholinesterase (AChE: EC 3.1.1.7) is characterized by the formation of the serine-conjugated adducts that are only slowly reversible. Since the oximes are the only known antidote to nerve agent poisoning that restores the activity of AChE, mutagenesis of the AChE may lead to means of enhancing the efficiency of antidotal and prophylactic therapy as well. We prepared mouse AChE mutants with substitutions in the choline binding site and the acyl pocket that reflect a transition between AChE and butyrylcholinesterase (BChE ; EC 3.1.1.8) active center geometry due to the known BChE gorge enlargement of BChE. Mutants were phosphonylated with chiral SP- and RP-cycloheptyl, -3, 3-dimethylbutyl and -isopropyl methylphosphonyl thiocholines, and then were subjected to reactivation by oxime, HI-6. Their inhibition and reactivation kinetics were compared with wild-type AChE and BChE. For detection of the phosphonylated enzyme, regenerated enzyme and/or aged enzyme we used additionally MALDI-TOF mass spectrometry. 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 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. Moreover, the mutations described here that enhance oxime reactivation also react rapidly with the methylphosphonates, so the limitations in scavenging capacity depend on efficiency of the oxime to continually regenerate active AChE.

acetylcholinesterase; inhibition; organophosphorus compound; oxime

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