Reinvestigation of the phenacyl bromide modification of α-chymotrypsin

Abstract

The modification of [bovine] .alpha.-chymotrypsin with phenacyl bromide was reinvestigated over a wide pH range. The nature of the phenacyl-modified enzymes prepared by this reaction apparently depends upon the pH of the reaction medium. The phenacyl .alpha.-chymotrypsin produced at low pH is most probably the Met-192 phenacylsulfonium salt, as proposed earlier, since it readily undergoes dealkylation using 2-mercaptoethanol. The phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic acitivity and does not react with 2-mercaptoethanol to regenerate native .alpha.-chymotrypsin. Incubation of the Met-192 phenacyl sulfonium enzyme at neutral pH causes a smooth irreversible change to the new phenacyl-enzyme as monitored by changes in enzymatic activity, susceptibility to dealkylation using 2-mercaptoethanol, and UV difference absorption spectral properties. The stoichiometries of both the low and neutral pH modification reactions were determined, using [carbonyl-14C]phenacyl bromide, to be 1 phenacyl group/enzyme molecule. In efforts to obtain information about the nature and mechanism of formation of the phenacyl .alpha.-chymotrypsin produced at neutral pH, alkylation reactions of modified .alpha.-chymotrypsins produced by His-57 functionalization with tosylphenylalainine chloromethyl ketone and by Met-192 oxidation to the sulfoxide were investigated. The combined results of these studies were initially interpreted in terms of a neutral pH, phenacyl bromide modification resulting in formation of a new modified enzyme via the Met-192 sulfonium salt.