title: Reconstructed 19 kDa catalytic domain of gelatinase A is an active proteinase.
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 microM 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.
authors.affiliation: Department of Biochemistry, Parke-Davis Pharmaceutical Research, Division of Warner-Lamber Company, Ann Arbor, Michigan 48105, USA.
authors.forename: Q Z
authors.initials: QZ
authors.lastname: Ye
authors.forename: L L
authors.initials: LL
authors.lastname: Johnson
authors.forename: A E
authors.initials: AE
authors.lastname: Yu
authors.forename: D
authors.initials: D
authors.lastname: Hupe
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli / Q000235:genetics; D018093:Gelatinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005813:Genes, Synthetic; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020778:Matrix Metalloproteinase 2; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000494:pharmacology
chemical_list: D004247:DNA; D018093:Gelatinases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D015032:Zinc; D002118:Calcium