Stability of protease inhibitors based on the Bowman-Birk reactive site loop to hydrolysis by proteases

Bowman-Birk proteinase inhibitor proteins contain two inhibitory regions, each of which is encapsulated within nine-residue disulfide-linked loops. It is known that short cyclic peptides that retain the nine-residue disulfide-bridged motif have inhibitory activity, and can be used as models of the natural inhibitor protein. Two factors are important in determining the effectiveness of such inhibitor peptides: the value of the inhibition constant, and rate at which the inhibitor peptide is hydrolyzed by the proteinase. In this paper we report a study of the inhibitory properties and stability towards proteolytic hydrolysis of a family of synthetic peptides derived from the trypsin reactive site loop of the Bowman-Birk inhibitors. The addition of a single amino acid residue to each end of the nine-residue disulfide-linked loop is found to reduce the rate at which the peptide is hydrolyzed. In addition, changing the P2' residue from Asn-->Ile gives inhibitors with considerably enhanced stability to proteolysis, as well as reduced values of Ki. The implications of these factors for the design of inhibitors based on this loop motif is discussed.