Reconstructed 19 kDa catalytic domain of gelatinase A is an active proteinase

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Abstract

Matrix metalloproteinases share high protein sequence homology and have defined domain structures. Gelatinases have a unique 19 kDa fibronectin-like insert in the catalytic domain. A synthetic gene was made to express the catalytic domain of human gelatinase A (GCD), in which two polypeptide fragments of the catalytic domain were joined with deletion of the insert. The synthetic gene was highly expressed in Escherichia coli, and the 19 kDa GCD was purified to homogeneity after in vitro refolding. The GCD showed activity at a pH range of 5.5-9 in cleavage of the thiopeptolide Ac-Pro-Leu-Gly-thioester-Leu-Leu-Gly-OEt with optimal activity at neutral pH (Km = 134 μM and kcat = 16 s-1 at pH 7.0). The activity required both zinc and calcium ions, but high concentration of zinc ion showed inhibition. Several stromelysin catalytic domain inhibitors inhibited the GCD with similar specificity. The GCD cleaved the fluorogenic peptides Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 and Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 with catalytic efficiency close to full length human gelatinase A. The reconstructed GCD cleaves not only thiopeptolide and peptide substrates but also protein substrates such as gelatin. These results are consistent with the notion that gelatinases have the same structure for the catalytic domain as other matrix metalloproteinases like stromelysins and collagenases.

Original languageEnglish (US)
Pages (from-to)4702-4708
Number of pages7
JournalBiochemistry
Volume34
Issue number14
StatePublished - 1995
Externally publishedYes

Fingerprint

Matrix Metalloproteinase 2
Matrix Metalloproteinase 3
Gelatinases
Catalytic Domain
Peptide Hydrolases
Matrix Metalloproteinases
Peptides
Zinc
Synthetic Genes
Genes
Ions
prolyl-leucyl-glycine
Collagenases
Substrates
Gelatin
Fibronectins
Escherichia coli
Proteins
Amino Acid Sequence Homology
Calcium

ASJC Scopus subject areas

  • Biochemistry

Cite this

Reconstructed 19 kDa catalytic domain of gelatinase A is an active proteinase. / Ye, Qizhuang.

In: Biochemistry, Vol. 34, No. 14, 1995, p. 4702-4708.

Research output: Contribution to journalArticle

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