Reactivation kinetics of diethylphosphoryl acetylcholine esterase

The kinetics of reactivation of diethylphosphorylated acetylcholine esterase by pyridine-2-aldoxime methochloride has been studied using the approach of following the course of the hydrolysis of acetylcholine during the reactivation of the phosphorylated enzyme by the reactivator [Tsou, C.-L. (1965) Acta Biochem. Biophys. Sin. 5, 398-417]. Equations are derived based on the scheme of the formation of a complex between the phosphorylated enzyme and the reactivator and the rate of dissociation of this complex is not necessarily faster than the dephosphorylation and regeneration of the active enzyme. The regenerated enzyme then reacts with the substrate through an acetyl-enzyme intermediate as generally depicted. The equation obtained for product formation during the course of reactivation contains two exponential terms and this is in accord with the experimentally observed biphasic reaction. By making the assumption that the dissociation of the phosphorylated enzyme-reactivator complex is much faster than the dephosphorylation reaction, the above equation can be simplified to a form containing only one exponential term. By following the course of the reactivation reaction with the conventional approach of taking aliquots and assaying for enzyme activity recovery, it would appear likely that one would miss the initial stage of this biphasic reaction.