A reinvestigation of the phenacyl bromide modification of alpha-chrymotrypsin

Abstract

The modification of a-chymotrypsin with phenacyl bromide has been reinvestigated over a wide pH range. Evidence is presented which indicates that the nature of the phenacyl modified enzymes prepared by this reaction is dependent upon the pH of the reaction medium. The phenacyl α-chymotrypsin produced at low pH is the Met-192 phenacyl sulfonium salt, 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 enzymatic activity, susceptibility to dealkylation using 2-mercaptoethanol, UV difference absorption spectral properties, and a difference in k(subscript cat) The stoichiometries of both the low and neutral pH modification reactions were determined, using [carbonyl- ¹⁴C]phenacyl bromide, to be one phenacyl group per enzyme molecule. The alkylation of Met-192 was shown to be an obligatory first step in the modification reaction at pH 4.0 and 7.0 by use of Met-192 sulfoxide modified a-chymotrypsin. The site of modification in the low pH and neutral pH phenacyl modified enzymes was determined to be Met-192 by ¹³C-spectroscopy and by thermolytic cleavage of modified enzymes to produce the same N-terminal amino acid. The combined results of these studies have been interpreted in terms of a conformational change in which the (S-phenacyl) methionine residue is inserted into the interior of the protein forming the neutral pH phenacyl modified enzyme.