Reinvestigation of the phenacyl bromide modification of α-chymotrypsin

Abstract

The modification of alpha-chymotrysin 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 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. However, the phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic activity and does not react with 2-mercaptoethanol to regenerate native alpha-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 enzymatic 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]phyenacyl 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 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.

Evidence for a pH-dependent irreversible formation of a stable conformation of phenacyl-α-chymotrypsin

“…The high-and low-pH modified a-chymotrypsins were -prepared by reactions of the native enzyme with phenacyl bromide and purified by procedures reported previously (Glover et al, 1976). Thermolytic cleavage reactions of the modified phenacyl-achymotrypsins and the model compound, N-acetyl-S-phenacyl-L-methionylglycine ethyl ester, were carried out by the method of Lawson et al (1962).…”

“…In our previous studies (Glover et al, 1976) several alternative explanations for the pH-dependent nature ofthe phenacyl-a-chymotrypsins had been considered. In one of these, the inertness of the high-pH enzyme under 2-mercaptoethanol dealkylation conditions was temporarily rationalized on the basis of irreversible formation of the sulphonium ylide at methionine-192.…”

“…It was thought that conformational features present in the high-pH modified enzyme might retard the rate of proton transfer to re-form the sulphonium salt. The failure to detect dealkylation after the high-pH enzyme is reversibly heat-denatured under acidic conditions (Glover et al, 1976) appeared to rule out this explanation, as well as others that invoked unusual conformations in the methionine-192 region to rationalize the resistance of the high-pH enzyme to 2-mercaptoethanol. This comprised part of the evidence used to tentatively conclude that the phenacyl moiety in this modified enzyme was not on the methionine residue.…”

“…Radioactively labelled high-pH enzyme (sp. radioactivity 1.03 x 108 d.p.m./mmol) was prepared from (carbonyl-14C]phenacyl bromide by the procedure described previously (Glover et al, 1974(Glover et al, , 1976). Performic acid oxidative cleavage (Hirs, 1956) of the disulphide linkages of this enzyme generated the A, B and C polypeptide chains, which were separated by gel filtration (Hoang et al, 1962).…”

“…We have previously shown that two characteristically different modified enzymes can be produced by the reaction of a-chymotrypsin with phenacyl bromide (Glover et al, 1976). The low-pH enzyme, prepared by reaction at pH4, was shown to be the methionine-192-modified phenacyl sulphonium salt, as proposed originally by Schramm & Lawson (1963) and confirmed later by Naider & Bohak (1972) by using the 2-mercaptoethanol dealkylation technique.…”