title: Characterization of the 46-kDa intermediates of matrix metalloproteinase 3 (stromelysin 1) obtained by site-directed mutation of phenylalanine 83.
abstract: 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 His82-Phe83 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 His82 or Phe83 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 Cys75 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 Cys75. 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 Thr85, 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 His82-Phe83 bond is critical for expression of the full activity and the specificity of MMP-3.
authors.affiliation: Department of Biochemistry, University of Kansas Medical Center, Kansas City 66160-7421, USA.
authors.forename: U
authors.initials: U
authors.lastname: Benbow
authors.forename: G
authors.initials: G
authors.lastname: Butticè
authors.forename: H
authors.initials: H
authors.lastname: Nagase
authors.forename: M
authors.initials: M
authors.lastname: Kurkinen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002918:Chymotrypsin / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D019272:Leukocyte Elastase; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010196:Pancreatic Elastase / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics*; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds; D019714:Tissue Inhibitor of Metalloproteinases; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism
chemical_list: D004792:Enzyme Precursors; D006023:Glycoproteins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D019714:Tissue Inhibitor of Metalloproteinases; D000511:alpha-Macroglobulins; D010649:Phenylalanine; C010024:4-aminophenylmercuriacetate; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D014357:Trypsin; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D010662:Phenylmercuric Acetate