The canonical methionine 392 of matrix metalloproteinase 2 (gelatinase A) is not required for catalytic efficiency or structural integrity: probing the role of the methionine-turn in the metzincin metalloprotease superfamily

J Biol Chem. 2004 Apr 9;279(15):15615-20. doi: 10.1074/jbc.M312727200. Epub 2004 Jan 19.


Matrix metalloproteinases (MMPs) are an important family of extracellular proteases that process a variety of biologically significant molecules. MMPs are members of the metzincin superfamily of >770 zinc endopeptidases, which includes astacins, serralysins, adamalysins, leishmanolysins, and snapalysins. Metzincins are characterized by an absolutely conserved methionine residue COOH-terminal to the third histidine in the consensus sequence HEXXHXXGXX(H/D), where the histidine residues chelate a catalytic zinc ion. The canonical methionine is part of a tight 1,4-beta-turn that loops the polypeptide chain beneath the catalytic zinc ion, forming a hydrophobic floor to the Zn(2+) ion binding site. The role of this methionine is uncertain, but its absolute conservation indicates an essential catalytic or structural function. To investigate this hypothesis, we replaced Met-392 that forms the Met-turn of human MMP-2 (gelatinase A) by site-directed mutagenesis. The catalytic competence of leucine and serine mutants was assessed. (M392L)MMP-2 and (M392S)MMP-2 cleaved the physiological substrates gelatin, native type I collagen, and the chemokine monocyte chemoattractant protein-3 with similar efficiency to wild-type MMP-2. These mutants also cleaved two quenched fluorescent peptide substrates with a k(cat)/K(m) comparable to wild-type MMP-2 and underwent 4-aminophenylmercuric acetate-induced autoactivation with similar kinetics. (M392L)MMP-2 and (M392S)MMP-2 were inhibited by tissue inhibitor of metalloproteinases (TIMP)-1, -2, and -4 and by the zinc chelators 1,10-phenanthroline and a synthetic hydroxamate inhibitor, Batimastat, similar to the wild-type protein, indicating an unaltered active site topography. A tryptic susceptibility assay also suggested that (M392L)MMP-2 and (M392S)MMP-2 were correctly folded. These results challenge the dogma that this methionine residue and the Met-turn, which are absolutely conserved in all of the subfamilies of the metzincins, play an essential role in catalysis or active site structure.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • CHO Cells
  • Catalysis
  • Collagen / chemistry
  • Cricetinae
  • Dose-Response Relationship, Drug
  • Edetic Acid / pharmacology
  • Electrophoresis, Polyacrylamide Gel
  • Histidine / chemistry
  • Humans
  • Ions
  • Kinetics
  • Leucine / chemistry
  • Matrix Metalloproteinase 2 / chemistry*
  • Matrix Metalloproteinase 2 / metabolism
  • Methionine / chemistry*
  • Mutagenesis, Site-Directed
  • Mutation
  • Oligonucleotides / chemistry
  • Peptides / chemistry
  • Phenylmercuric Acetate / analogs & derivatives*
  • Phenylmercuric Acetate / chemistry
  • Protein Conformation
  • Protein Structure, Tertiary
  • Serine / chemistry
  • Temperature
  • Time Factors
  • Trypsin / pharmacology
  • Zinc / chemistry


  • Ions
  • Oligonucleotides
  • Peptides
  • Serine
  • Histidine
  • 4-aminophenylmercuriacetate
  • Collagen
  • Edetic Acid
  • Methionine
  • Trypsin
  • Matrix Metalloproteinase 2
  • Leucine
  • Zinc
  • Phenylmercuric Acetate