The soluble catalytic domain of membrane type 1 matrix metalloproteinase cleaves the propeptide of progelatinase A and initiates autoproteolytic activation. Regulation by TIMP-2 and TIMP-3

J Biol Chem. 1996 Jul 19;271(29):17119-23. doi: 10.1074/jbc.271.29.17119.


It has been proposed that the cell-mediated activation of progelatinase A requires binding of the C-terminal domain of the proenzyme to a membrane-associated complex of the membrane type matrix metalloproteinase MT1-MMP and TIMP-2. Subsequent sequential proteolysis of the propeptide by MT1-MMP and gelatinase A is thought to generate the active form of gelatinase A. We have prepared the proform of the catalytic domain of the MT1-MMP and demonstrated that this may be activated in vitro by trypsin proteolysis to yield a functional proteinase capable of cleaving typical metalloproteinase peptide substrates, gelatin and casein. The active catalytic domain of MT1-MMP was also shown to activate progelatinase A to a fully active form. Using the inactive mutant pro-E375A gelatinase A, we dissected the propeptide processing events that occur. MT1-MMP cleaves the propeptide at the sequence Asn37-Leu38 only. Further cleavage of the mutant enzyme propeptide at Asn80-Tyr81, equivalent to that of the active wild type gelatinase A, could only be effected by addition of gelatinase A to the system. TIMP-1 was essentially unable to prevent MT1-MMP processing of wild type or E375A progelatinase A, whereas TIMP-2 and TIMP-3 were good inhibitors of these events. Analysis of the rate of binding of TIMPs to the catalytic domain of MT1-MMP using kinetic methods showed that TIMP-1 is an extremely poor inhibitor of MT1-MMP. In comparison, TIMP-2 and TIMP-3 are excellent inhibitors, binding more rapidly to the catalytic domain of MT1-MMP than to the catalytic domain of gelatinase A. These data demonstrate the basic mechanism of MT1-MMP action on progelatinase A and the reason for the lack of inhibition by TIMP-1 previously demonstrated in cell-based activation studies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Asparagine
  • Binding Sites
  • Catalysis
  • Cloning, Molecular
  • Collagenases / metabolism*
  • Enzyme Activation
  • Enzyme Precursors / metabolism*
  • Gelatinases / metabolism*
  • Humans
  • Kinetics
  • Leucine
  • Matrix Metalloproteinase 1
  • Metalloendopeptidases / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Point Mutation
  • Protein Processing, Post-Translational
  • Proteins / pharmacology*
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Tissue Inhibitor of Metalloproteinase-2
  • Tissue Inhibitor of Metalloproteinase-3
  • Tyrosine


  • Enzyme Precursors
  • Peptide Fragments
  • Proteins
  • Recombinant Proteins
  • Tissue Inhibitor of Metalloproteinase-3
  • Tissue Inhibitor of Metalloproteinase-2
  • Tyrosine
  • Asparagine
  • Collagenases
  • Gelatinases
  • Metalloendopeptidases
  • progelatinase
  • Matrix Metalloproteinase 1
  • Leucine