Characterization of the 46-kDa intermediates of matrix metalloproteinase 3 (stromelysin 1) obtained by site-directed mutation of phenylalanine 83

The precursor of matrix metalloproteinase 3 (MMP-3/stromelysin 1) is activated in vitro by proteinases or mercurial compounds by stepwise processes which include the initial formation of short-lived intermediates and the subsequent intermolecular cleavage of the His(82)-Phe(83) bond to generate the fully activated mature MMP-3 (Nagase, H., Enghild, J. J., Suzuki, K., and Salvesen, G. (1990) Biochemistry 29, 5783-5789), To study the enzymatic properties of the intermediates we have mutated either His(82) or Phe(83) to Arg to obtain a stable MMP-3 intermediate, The mutant proteins were expressed in Chinese hamster ovary K-1 cells using a mammalian expression system. The proMMP-3(H82R) mutant was activated by chymotrypsin, elastase, and 4-aminophenylmercuric acetate to the 45-kDa MMP-3 with similar mechanism and kinetics as the wild-type. In contrast, the activation of the proMMP-3(F83R) mutant by proteinases or 4-aminophenylmercuric acetate resulted in 46-kDa forms, which retained 13, 14, or 15 amino acids of the pro-domain depending on the activators, The proteinase-activated MMP-3(F83R) intermediates exhibited little enzymatic activity, but they were partially active after treatment with SH-reacting reagents. These molecules could bind to the tissue inhibitor of metalloproteinases-1 and alpha(2)-macroglobulin. However, the SH group of Cys(75) of the intermediates was not modified by SH-reagents, indicating that the enzymatic activity generated by SH-reagents resulted from molecular perturbation of the enzyme rather than their interaction with Cys(75). When gelatin and transferrin were digested with the 46-kDa intermediates the products were different from those generated by the wild-type MMP-3, suggesting an alteration in substrate specificity, The treatment of proMMP-3 with trypsin resulted in the formation of a 45-kDa MMP-3 with an NH2-terminal Thr(85), whose activity and substrate specificity were similar to those of the 46-kDa MMP-3(F83R) obtained from the proMMP-3(F83R) mutant. These observations indicate that the correct processing at the His(82)-Phe(83) bond is critical for expression of the full activity and the specificity of MMP-3.