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

Abstract

A reinvestigation of the modification reactions of alpha-chymotrypsin with phenacyl bromide was carried out. Results conclusively demonstrate that the chemically and physically different modified enzymes prepared at pH 4 and at pH 7 both contain the phenacyl group at methionine-192 in the sulphonium salt form. Evidence to suppoort this conclusion derives from 13C nuclear-magnetic-resonance spectroscopic observations on [methylene-13C]phenacyl-enriched enzymes. More conclusively, the methionine-192-containing C-chain, derived by performic acid oxidative cleavage of radioactively-labelled enzyme prepared at pH 7, was shown to contain the phenacyl moiety and to undergo dealkylation by 2-mercaptoethanol with loss of this moiety. In addition, thermolytic cleavage of the high-pH enzyme results in fragmentation of the polypeptide chain in a fashion analogous to model reactions of phenacylmethionyl dipeptides and other methionine-192 sulphonium salts. A rationalization of the unusual nature of the high-pH phenacyl-modified enzyme based on the irreversible formation of stable conformation in which the phenacyl moiety is rigidly located in interior regions of the enzyme is presented and discussed.

Identification of methionyl and cysteinyl residues in the substrate binding site of carboxypeptidase Y

“…It is apparent that the predominant part of the radioactivity eluted in Fraction II indicating that the alkylated methionyl residue is located in the peptide with Lys-Pro-TyrHis-Thr-as the N-terminal sequence and the amino acid composition indicated that no cleavage had taken place at Met-398. To demonstrate conclusively the location of the alkylated methionyl residue it was utilized that peptides may be cleaved under mild conditions at alkylated methionyl residues by a mechanism similar to that responsible for the cleavage with cyanogen bromide ( 13,14). When fraction II was boiled in water cleavage took place at Met-398 producing a new N-terminal sequence, Val-Pro-Phe-AspVal-.…”

Reactions of .alpha.-chymotrypsin with 4-(trifluoromethyl)-.alpha.-bromoacetanilide

“…It has been noted that chymotrypsin modified with a-bromoacetophenone can be isolated at two conformational isomers, depending upon the solution pH used in preparation of the derivative (Mariano et al, 1978). In this work attempts to detect equilibration between 4-(trifluoromethyl)-a-bromoacetanilide-modified proteins prepared at pH 4.4 and 7 by fluorine spectroscopy produced no evidence for conformational isomerization.…”