Preferential Oxidation of the Methionine Residue near the Active Site of Chymotrypsin.

Hartley (4) has reported the amino acid sequence of a 25-residue peptide from the tryptic digest of X-sulfochymotrypsinogen which contains the “active center” serine as well as both methionine residues of a-chymotrypsin (Fig. 1). By kinetic analysis, Ray et al. (5, 6) deduced that 1 of the 2 methionine residues and 1 of the 2 histidine residues in chymotrypsin are rapidly destroyed on photo-oxidation at pH 7 in the presence of methylene blue (7). In preliminary communications (1,2), we confirmed the kinetic prediction of Ray et al. (5, 6) that 1 of the 2 methionine residues was being rapidly and preferentially photo-oxidized and identified this “fast” methionine as that 3 residues removed from the “active center” serine (Met-3). Recently, Koshland, Strumeyer, and Ray (9) reported that hydrogen peroxide at pH 3 could be used as a specific reagent for oxidation of the methionines in chymotrypsin. In the absence of urea, hydrogen peroxide appeared to oxidize only 1 of the 2 methione residues, but in the presence of 8 M urea at pH 3, both methionines were oxidized. The present paper reports the details of an accurate and quantitative method for identifying the site of oxidation of methionine residues in chymotrypsin. The application of this method to the photo-oxidation of chymotrypsin at pH 7.2 (2) and to hydrogen peroxide oxidation at pH 3 (3) has shown that Met-3 is preferentially oxidized in both cases. Koshland et al. (9) have used the cyanogen bromide method of specific cleavage at methionine residues (10) to identify Met-3 as the site of oxidation by photo-oxidation at pH 7 and Hz02 at pH 3. The method presented in this paper depends upon the isolation of methionine peptides from the oxidized protein. It is shown that spontaneous oxidation of methionine residues occurs during isolation of Hartley’s methionine peptide (Fig. 1) from X-sulfodiisopropylphosphoryl-32P-chymotrypsin. Stabilization of methionine residues was therefore essential before structural studies on oxidized protein could be attempted. This was achieved by exhaustive alkylation of nonoxidized methionine to the sulfonium salt with bromoor iodoacetic acid in the presence of urea at pH 2.8 (11, 12). Since only free methionine and not its oxidized form is alkylated, it is possible to label the nonoxidized methionine as the 14C-carboxymethylsulfonium salt by using I%-labeled alkylating agent and thus determine the position of oxidized methionine by comparison of the peptides prepared from oxidized