Mechanistic Insights into Reactivation of Organophosphate-Conjugated Acetylcholinesterase by Nucleophiles as Antidotes

Organophosphates (OP) interacting with acetylcholinesterase (AChE) carry limited therapeutic benefits and are effective biodegradable insecticides when used judiciously. However, certain volatile OP’s have been used insidiously in terrorism. Current drugs that reactivate AChE and are used to combat the inhibitory effects of OP’s are oximes, but the mechanism by which these oximes act in the gorge of the enzyme is not well-understood. General base catalysis dictates that the oximate anion acts as a nucleophile against the organophosphate esters, which is consistent with in-solution pH dependent oximolysis reactions. I employed pH dependent oxime-induced reactivations of OP-AChE and looked at kinetic isotope effects for reactivation in order to deconstruct the mechanism in which these oximes nucleophilic attack within the enzyme. My studies found that oxime-induced reactivation optimizes at pH ~7.5, contradictory to general base catalysis. Solvent isotope effects exhibited a reduction in reactivation rates, with different magnitudes of depression for different oximes and respective oxime concentrations. Therefore, the existence of a hydrogen bonding network that contributed to the formation of the oximate anion is proposed. In addition, I studied fluoride-induced reactivation of OP-AChE, which is predicted to operate differently than oximes. These findings showed that fluoride-induced reactivation was enhanced by low pH and higher ionic strength environments.