J. Biol. Chem., Vol. 258, Issue 12, 7490-7498, Jun, 1983
Ovostatin: a novel proteinase inhibitor from chicken egg white. II. Mechanism of inhibition studied with collagenase and thermolysin
H Nagase and ED Harris Jr
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.
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