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 or by the addition of aminophenylmercuric acetate (APMA). By using an inhibitor of mature stromelysin to trap intermediates, it was found that the two methods of activation occurred by different mechanisms. Heat activation was achieved by a single-step bimolecular cleavage which was dependent on the presence of a small amount of mature enzyme. In contrast, APMA activation occurred by a complex multistep mechanism which consisted of intramolecular cleavages within the NH2-terminal pro portion of the molecule followed by a bimolecular cleavage at the NH2-terminus of the mature stromelysin. In spite of the different mechanisms of activation, both methods generate indistinguishable active enzymes.