Identification of the trypsin-reactive site of the Bowman-Birk soybean inhibitor

Abstract

Succinylation of the 5 lysine residues of the Bowman-Birk inhibitor abolished its antitryptic activity without affecting its ability to inhibit chymotrypsin. Guanidination of the 5 lysine residues and modification of 1 of the 2 arginine residues, however, had no effect on either activity. Guanidination of the Bowman-Birk inhibitor-trypsin complex followed by dissociation of the complex revealed that one of the lysine residues of Bowman-Birk inhibitor had been protected from modification while in the complex. This particular lysine residue was identified as lysine-17 by isolation and characterization of two fragments from trypsin-modified Bowman-Birk inhibitor which had been reduced and alkylated. From end-group analysis of the trypsin-modified Bowman-Birk inhibitor and of the two fragments isolated therefrom, a limited sequence of amino acids in the immediate vicinity of the trypsin-sensitive bond of Bowman-Birk inhibitor was derived. This sequence was found to bear a striking degree of homology to the proposed antitrypsin site of the lima bean inhibitor.