Catalytic Activities and Substrate Specificity of the Human Membrane Type 4 Matrix Metalloproteinase Catalytic Domain

• Published in: The Journal of biological chemistry 1999-11, Vol.274 (46), p.33043-33049
• Main Authors: Wang, Yahong, Johnson, Adam R., Ye, Qi-Zhuang, Dyer, Richard D.
• Format: Article
• Language: English
• Subjects: 1,10-phenanthroline; Amino Acid Sequence; Binding Sites; Chelating Agents - pharmacology; Cloning, Molecular; Enzyme Activation; Enzyme Inhibitors - pharmacology; Enzyme Precursors - metabolism; Escherichia coli; gelatin; Gelatin - metabolism; Gelatinases - metabolism; Humans; Hydrogen-Ion Concentration; Kinetics; Matrix Metalloproteinases - chemistry; Matrix Metalloproteinases - genetics; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases - metabolism; Molecular Sequence Data; Protein Folding; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Substrate Specificity; tissue inhibitor of metalloproteinase 1; tissue inhibitor of metalloproteinase 2; Zinc - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.46.33043

Membrane type (MT) matrix metalloproteinases (MMPs) are recently recognized members of the family of Zn2+- and Ca2+-dependent MMPs. To investigate the proteolytic capabilities of human MT4-MMP (i.e. MMP-17), we have cloned DNA encoding its catalytic domain (CD) from a breast carcinoma cDNA library. Human membrane type 4 MMP CD (MT4-MMPCD) protein, expressed as inclusion bodies inEscherichia coli, was purified to homogeneity and refolded in the presence of Zn2+ and Ca2+. While MT4-MMPCD cleaved synthetic MMP substrates Ac-PLG-[2-mercapto-4-methylpentanoyl]-LG-OEt and Mca-PLGL-Dpa-AR-NH2 with modest efficiency, it catalyzed with much higher efficiency the hydrolysis of a pro-tumor necrosis factor-α converting enzyme synthetic substrate, Mca-PLAQAV-Dpa-RSSSR-NH2. Catalytic efficiency with the pro-tumor necrosis factor-α converting enzyme substrate was maximal at pH 7.4 and was modulated by three ionizable enzyme groups (pKa3 = 6.2, pKa2 = 8.3, and pKa1 = 10.6). MT4-MMPCD cleaved gelatin but was inactive toward type I collagen, type IV collagen, fibronectin, and laminin. Like all known MT-MMPs, MT4-MMPCD was also able to activate 72-kDa progelatinase A to its 68-kDa form. EDTA, 1,10-phenanthroline, reference hydroxamic acid MMP inhibitors, tissue inhibitor of metalloproteinases-1, and tissue inhibitor of metalloproteinases-2 all potently blocked MT4-MMPCD enzymatic activity. MT4-MMP is, therefore, a competent Zn2+-dependent MMP with unique specificity among synthetic substrates and the capability to both degrade gelatin and activate progelatinase A.