Oxidation of methionine residues of porcine and bovine pepsins.
By treating porcine and bovine pepsins with H2O2 at pH 3.2, 3.5 of the 4 methionine residues of porcine pepsin and 1.6 of the 3 residues of bovine pepsin were oxidized to methionine sulfoxide. The effect of modification on activity varied with the substrate. There were no significant changes in catalytic constants in the hydrolysis of acetyl-L-phenylalanyl-L-tyrosine by both pepsins and in the hydrolysis of benzyloxycarbonyl-L-glutamyl-L-tyrosine by porcine pepsin. Hydrolysis of benzyloxycarbonyl-L-glutamyl-L-tyrosine by bovine pepsin was too slow to measure. With benzyloxycarbonyl-L-histidyl-L-phenylalanyl-L-tryptophan ethyl ester as substrate, the modification decreased the catalytic efficiency (kcat/Km) by two-thirds for porcine pepsin and by half for bovine pepsin. With hemoglobin substrate, digestion was significantly less than with native pepsin for modified porcine pepsin, and slightly less for modified bovine pepsin. The results are interpreted as indicating the presence of a methionine residue that participates in the binding of long substrates, but is not close enough to the active site to reach short substrates. Cleavage of the modified pepsins with cyanogen bromide identified the methionine nearest the carboxyl terminus of both pepsins as a resiude that remained partially unmodified.