The reactivation of phosphorylated chymotrypsin

Abstract

Oximes and hydroxamic acids will completely reactivate chymotrypsin inhibited with organophosphates, at a rate varying linearly at constant pH with the concentration of the reactivator. Hydroxyiminoacetone, 2-hydroxyiminomethyl-N-methylpyridinium methanesulfonate, picolinohydroxamic acid and salicylohydroxamic acid have about equal potency as reactivators of chymotrypsin inhibited with isopropyl methylphosphonofluoridate (Sarin). This is in line with their roughly equal reactivities with Sarin itself but is in marked contrast with the great difference in their abilities to reactivate inhibited cholinesterase. A bell-shaped pH-activity curve was found for reactivation by hydroxyiminoacetone of chymotrypsin inhibited by Sarin. On storage at 25[degree] for 4 days at various pH values, inhibited chymotrypsin, unlike inhibited cholinesterase, did not lose its ability to be reactivated. Chymotrypsin inhibited with Sarin slowly recovers its activity even in the absence of any specific reactivator. This spontaneous recovery is accelerated by a decrease in pH. A structure is proposed for the phosphorylated or acylated active center of chymotrypsin which will explain (a) the isolation after degradation of phosphorylated or acylated chymotrypsin of peptides in which the phosphoryl or acyl group is attached to a serine hydroxyl group, (b) the increase in the rate of hydrolysis of acylchymotrypsin as the pH is raised, and (c) the increase in the rate of hydrolysis of phosphorylchymotrypsin as the pH is lowered.