Matrix metalloproteinases (MMPs) can be activated in vitro by multiple mechanisms such as treatment with proteases, organomercurials, oxidants, and detergents. The proposed cysteine switch model for activation suggests that these multiple methods for activation cause the dissociation of the single cysteine residue in the propeptide from the active site zinc. In particular, it has been suggested that organomercurials such as 4-aminophenylmercuric acetate (APMA) work by directly reacting 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-I by sulfhydryl reagents did not result in an active enzyme as predicted. To investigate the roles that this cysteine residue and the propeptide salt bridge (R74 to D79) might play in file APMA-induced activation of stromelysin-I, we have changed these residues by site-directed mutagenesis. Wild type stromelysin-I and the mutants were all expressed at detectable levels using a recombinant vaccinia virus system and determined to be catalytically competent by zymography. The wild-type stromelysin-I and the cysteine mutants (C75S and C75H) underwent APMA-induced activation 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 that APMA-induced activation of stromelysin-I involves protein interactions in addition to those with cysteine-75 in the propeptide and also suggest that the R74 to D79 salt bridge may play a role.
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