Identification of the reactive site of potato proteinase inhibitor I for various proteases

A specific peptide bond of potato proteinase inhibitor I is hydrolyzed by incubation with a catalytic amount of chymotrypsin [EC 3.4.4.5] at pH 3: The inhibitor thus modified is separated into two fragments by reduction, S-carboxymethylation and subsequent gel filtration on a Sephadex G-50 column. The terminal amino acids and amino acid compositions of these two fragments show that incubation of the 56 inhibitor with chymotrypsin causes the cleavage of a single peptide bond, Leu— 57 Asx, within a disulfide loop of the inhibitor molecule. An inhibitor-enzyme complex is formed by incubation of the chymotrypsin-modified inhibitor with equi-molar Nagarse at pH 7.6. Amino- and carboxyl-terminal amino acids and the amino acid composition of inhibitor which was dissociated from this inhibitor-enzyme complex at acid pH, are consistent with those of native inhibitor. This fact shows 56 57 that the cleaved peptide bond, Leu—Asx, of the chymotrypsin-modified inhibito