Unexpected crucial role of residue 272 in substrate specificity of fibroblast collagenase

Hiroki Tsukada, Tayebeh Pourmotabbed

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Degradation of type I collagen by collagenases is an important part of extracellular remodeling. To understand the role of the hinge region of fibroblast collagenase in its collagenolytic activity, we individually substituted the 10 conserved amino acid residues at positions 264, 266, 268, 296, 272, 277, 284, 289, 307, and 313 in this region of the enzyme by their corresponding residues in MMP-3, a noncollagenolytic matrix metalloproteinase. The general proteolytic and triple helicase activities of all of the enzymes were determined, and their abilities to bind to type I collagen were assessed. Among the mutants, only G272D mutant enzyme exhibited a significant change in type I collagenolysis. The alteration of the Gly272 to Asp reduced the collagenolytic activity of the enzyme to 13% without affecting its general proteolytic activity, substrate specificity, or the collagen binding ability. The catalytic efficiency of the G272D mutant for the triple helical peptide substrate [C6-(GP-Hyp)4GPL(Mca) GPQGLRGQL(DPN)GVR(GP-HYP)4-NH2]3 and the peptide substrate Mca-PLGL(Dpa)AR-NH2 and its dissociation constant for the triple helical collagen were similar to that of the wild type enzyme, indicating that the presence of this residue in fibroblast collagenase is particularly important for the efficient cleavage of type I collagen. Gly272 is evidently responsible for the hinge-bending motion that is essential for allowing the COOH-terminal domain to present the collagen to the active site.

Original languageEnglish (US)
Pages (from-to)27378-27384
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number30
DOIs
StatePublished - Jul 26 2002

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Matrix Metalloproteinase 8
Substrate Specificity
Collagen Type I
Substrates
Enzymes
Collagen
Hinges
Matrix Metalloproteinases
Peptides
Collagenases
Viperidae
NAD
Catalytic Domain
Amino Acids
Degradation

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Unexpected crucial role of residue 272 in substrate specificity of fibroblast collagenase. / Tsukada, Hiroki; Pourmotabbed, Tayebeh.

In: Journal of Biological Chemistry, Vol. 277, No. 30, 26.07.2002, p. 27378-27384.

Research output: Contribution to journalArticle

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N2 - Degradation of type I collagen by collagenases is an important part of extracellular remodeling. To understand the role of the hinge region of fibroblast collagenase in its collagenolytic activity, we individually substituted the 10 conserved amino acid residues at positions 264, 266, 268, 296, 272, 277, 284, 289, 307, and 313 in this region of the enzyme by their corresponding residues in MMP-3, a noncollagenolytic matrix metalloproteinase. The general proteolytic and triple helicase activities of all of the enzymes were determined, and their abilities to bind to type I collagen were assessed. Among the mutants, only G272D mutant enzyme exhibited a significant change in type I collagenolysis. The alteration of the Gly272 to Asp reduced the collagenolytic activity of the enzyme to 13% without affecting its general proteolytic activity, substrate specificity, or the collagen binding ability. The catalytic efficiency of the G272D mutant for the triple helical peptide substrate [C6-(GP-Hyp)4GPL(Mca) GPQGLRGQL(DPN)GVR(GP-HYP)4-NH2]3 and the peptide substrate Mca-PLGL(Dpa)AR-NH2 and its dissociation constant for the triple helical collagen were similar to that of the wild type enzyme, indicating that the presence of this residue in fibroblast collagenase is particularly important for the efficient cleavage of type I collagen. Gly272 is evidently responsible for the hinge-bending motion that is essential for allowing the COOH-terminal domain to present the collagen to the active site.

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