THE INHIBITORY SPECIFICITY OF HUMAN PROTEINASE-INHIBITOR-8 IS EXPANDED THROUGH THE USE OF MULTIPLE REACTIVE-SITE RESIDUES

Serine proteinase inhibitors function as regulators of serine proteina se activity in a variety of physiological processes. Proteinase inhibi tor 8 (PI8) is a 45 kDa member of the ovalbumin family of serpins that is an inhibitor of trypsin-like proteinases through the use of Arg(33 9) as the inhibitory P-1 amino acid residue in its reactive site loop. In this study, we have described the inhibitory mechanism of recombin ant human PI8 towards chymotrypsin. PI8 formed an SDS-stable complex w ith and inhibited the amidolytic activity of chymotrypsin via a two-st ep mechanism with an overall equilibrium inhibition constant of 1.7 nM and an overall second-order association rate constant of 1.0 x 10(4) M(-1)s(-1), utilizing Ser(341) as the P-1 residue. The use of separate reactive site loop residues by PI8 to inhibit distinctly different cl asses of proteinases not only supports the hypothesis of the existence of the serpin reactive site as a highly mobile and flexible loop, but also suggests an evolved function in which separate amino acid residu es can be used to broaden the inhibitory specificity of PI8. (C) 1998 Academic Press.