APMA (4-AMINOPHENYLMERCURIC ACETATE) ACTIVATION OF STROMELYSIN-1 INVOLVES PROTEIN INTERACTIONS IN ADDITION TO THOSE WITH CYSTEINE-75 IN THEPROPEPTIDE

Matrix metalloproteinases (MMPs) can be activated ill vitro by multipl e mechanisms such as treatment with proteases, organomercurials, oxida nts, and detergents, The proposed cysteine switch model for activation suggests that these multiple methods for activation cause the dissoci ation of the single cysteine residue In the propeptide from the active site zinc. In particular, it has been suggested that organomercurials such as 3-aminophenylmercuric acetate (APMA) work by directly reactin g with the sulfhydryl group of this cysteine residue, resulting in its displacement from the active site, However, recent data by Chen et al . [(1993) Biochemistry 32, 10289-10295] demonstrated that modification of this cysteine residue in the propeptide of stromelysin-l by sulfhy dryl reagents did not result in an active enzyme as predicted, To inve stigate the roles that this cysteine residue and the propeptide salt b ridge (R74 to D79) might play in the APMA-induced activation of strome lysin-l, we have changed these residues by site-directed mutagenesis, Wild-type stromelysin-l and the mutants were all expressed at detectab le levels using a recombinant vaccinia virus system and determined to be catalytically competent by zymography, The wild-type stromelysin-l and the cysteine mutants (C75S and C75H) underwent APMA-induced activa tion as determined by the characteristic reduction in molecular weight associated with activation and by their ability to cleave casein only when activated, On the other hand, mutants R74K, D79A, and C75H/D79A did not undergo APMA-induced activation, These results demonstrate tha t APMA-induced activation of stromelysin-l involves protein interactio ns in addition to those with cysteine-75 in the propeptide and also su ggest that the R74 to D79 salt bridge may play a role.