USE OF AN ACTIVE-SITE INHIBITOR OF STROMELYSIN TO ELUCIDATE THE MECHANISM OF PROSTROMELYSIN ACTIVATION

A COOH-terminally truncated recombinant form of prostromelysin-1 (MMP- 3; EC 3.4.27.17) was activated by incubation at elevated temperature o r by the addition of aminophenylmercuric acetate (APMA). By using an i nhibitor of mature stromelysin to trap intermediates, it was found tha t the two methods of activation occurred by different mechanisms. Heat activation was achieved by a single-step bimolecular cleavage which w as dependent on the presence of a small amount of mature enzyme. In co ntrast, APMA activation occurred by a complex multistep mechanism whic h consisted of intramolecular cleavages within the NH2-terminal pro po rtion of the molecule followed by a bimolecular cleavage at the NH2-te rminus of the mature stromelysin. In spite of the different mechanisms of activation, both methods generate indistinguishable active enzymes . (C) 1995 Academic Press, Inc.