RATE CONSTANTS OF CARBAMYLATION AND DECARBAMYLATION OF ACETYLCHOLINESTERASE FOR PHYSOSTIGMINE AND CARBARYL IN THE PRESENCE OF AN OXIME

Membrane-bound bovine erythrocyte acetylcholinesterase was inhibited with physostigmine or carbaryl, and the rate constants of carbamylation and decarbonylation were determined from the proportion of inhibited acetylcholinesterase in the steady state, and the rate of approach to the steady state. The oximes 2-PAM, HI-6, HS-6, TMB-4 and toxogonin, at 0.1 mM, all decreased the rate of carbamylation by physostigmine, but increased the rate of carbamylation by carbaryl. TMB-4 and toxogonin were the most effective oximes in potentiating carbamylation by carbaryl, with an enhancement of the second-order rate constant of 54- and 17-fold respectively. The greatest reduction in the rate constant for carbamylation by physostigmine (3.7-fold) was caused by HI-6. HS-6 and HI-6 increased the rate of decarbamylation, while 2-PAM reduced the rate of decarbamylation if physostigmine was the carbamate. 2-PAM and HI-6 were also studied with soluble bovine erythrocyte acetylcholinesterase, and similar results were obtained. The results extend those in a recent report by other authors who studied the half-life of carbamylation for acetylcholinesterase and butyrylcholinesterase in an attempt to understand the mechanism by which oximes increase the toxicity of carbaryl in vivo. These authors proposed binding of the oximes to an allosteric site on the enzyme. While not discounting this possibility, the present results, taken with other reports in the literature, suggest that binding of the oximes to the anionic subsite of the active site of the enzyme is also feasible. The present results also offer an explanation for another recent report, in which anomalous results were presented for decarbamylation of physostigmine-inhibited and carbaryl-inhibited erythrocyte acetylcholinesterase in the presence of 2-PAM or HI-6.