Mutational analysis for the role of C-terminal region of ecotin, a dimeric inhibitor of pancreatic serine proteases

Abstract

Ecotin is a dimeric molecule that is capable of inhibiting a variety of serine proteases. To clarify the role of the C-terminal region, mutagenesis was performed to delete the C-terminal residues from 130 to 142. The mutant inhibitor behaved as a monomer upon cross-linking analysis followed by gel filtration. The mutation also resulted in a significant increase of the K(i) of ecotin on trypsin, chymotrypsin, and elastase (i.e., by 1 to 2 order of magnitude). The mutant ecotin was slightly more sensitive to heating at 100°C than the wild-type ecotin, but became much more sensitive to the heat treatment upon reduction of the intra-chain disulfide bond in its subunits. In addition, treatment with 4 M urea resulted in complete loss of the activity of the mutant ecotin but not that of the wild-type inhibitor. Thus, the C-terminal region of ecotin seems to be required not only for dimerization of the subunits but also for optimal interaction with target proteases and for maintenance in its structural stability, particularly under reducing or denaturing conditions.