Ovostatin: a novel proteinase inhibitor from chicken egg white. II. Mechanism of inhibition studied with collagenase and thermolysin

Abstract

The inhibition mechanism of ovostatin was studied using rabbit synovial collagenase and thermolysin. When enzymes were complexed with ovostatin, only the proteolytic activity towards high molecular weight substrates was inhibited. Activity towards low molecular weight substrates was partially modified: the catalytic activity of collagenase bound to ovostatin was inhibited by only 40% towards 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg and that of thermolysin bound to ovostatin was activated about 2.6-fold towards benzyloxycarbonyl-Gly-Leu-NH2 and benzyloxycarbonyl-Gly-Phe-NH2. Collagenase-ovostatin complexes failed to react with anti-(collagenase) antibody. Saturation of ovostatin with thermolysin prevented the subsequent binding of collagenase. Ovostatin-proteinase complexes ran faster than free ovostatin on 5% polyacrylamide gel electrophoresis. Complexing ovostatin with either collagenase or thermolysin resulted in the cleavage of the quarter-subunit of ovostatin (Mr = 165,000) into two fragments with Mr = 88,000 and 78,000. On the other hand, when the inhibitory capacity of ovostatin was tested with trypsin, chymotrypsin, and papain, only partial inhibition of their proteolytic activities was observed towards azocasein. Stronger inhibition was noted when Azocoll was a substrate, however. Analyses of ovostatin-enzyme complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the quarter-subunit of ovostatin was cleaved into several fragments by those enzymes. These results led us to propose that ovostatin inhibits metalloproteinases in preference to proteinases of other classes in a manner similar to alpha 2-macroglobulin; hydrolysis of a peptide bond by a proteinase in the susceptible region of the ovostatin polypeptide chain triggers a conformational change in the ovostatin molecule and the enzyme becomes bound to ovostatin in such a way that the proteinase is sterically hindered from access to large protein substrates and yet is accessible to small synthetic substrates. A kinetic study of collagenase binding to ovostatin gave the value of k2/Ki = 6.3 X 10(5) M-1 min-1. The results indicate that ovostatin is equally as good a substrate for collagenase as type I collagens.