Identification of the Reactive Site of Potato Proteinase Inhibitor I for Various Proteinases

Abstract

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 inhibitor with chymotrypsin causes the cleavage of a single peptide bond, $-Asx57$, 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 that the cleaved peptide bond, Leu-Asx, of the chymotrypsin-modified inhibitor is resynthesized by Nagarse at near neutral pH. It may therefore be concluded that the chymotrypsin-susceptible peptide bond, $-Asx57$, is the main reactive site of the inhibitor for both chymotrypsin and Nagarse. Further, other experimental results indicate that this reactive site is also utilized for the inhibition of the alkaline proteinases of Aspergillus sydowi, Aspergillus sulphureus, and Penicillium chrysogenum.