Reinvestigation of the Phenacyl Bromide Modification of α-Chymotrypsin

The modification of α-chymotrypsin with phenacyl bromide has been reinvestigated over a wide pH range. Evidence is presented that indicates that the nature of the phenacyl-modified enzymes prepared by this reaction is dependent upon the pH of the reaction medium. The phenacyl-chymotrypsin produced at low pH is most probably the Met-192 phenacylsulfonium salt, as proposed earlier, since it readily undergoes dealkylation using 2-mercaptoethanol. However, the phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic activity and does not react with 2-mercaptoethanol to regenerate native α-chymotrypsin In addition, 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 enzymalic activity, susceptibility to dealkylation using 2-mercaptoethanol, and ultraviolet difference absorption spectral properties. The stoichiometries of both the low and neutral pH modification reactions have been 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 α-chymotrypsin produced at neutral pH, alkylation reactions of modified achymotrypsins produced by His-57 functionalization with tosylphenylalanine chloromethyl ketone and by Met-192 oxidation to the sulfoxide have been investigated. The combined results of these studies have been 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.