title: Identification of the active site of gelatinase B as the structural element sufficient for converting a protein to a metalloprotease.
abstract: Gelatinase B is a member of the matrix metalloproteinase family that efficiently cleaves gelatin, elastin, and types V and X collagen. To understand the contribution of the active site of the enzyme (amino acid residues 373-456) in these activities, we studied catalytic properties of a fusion protein consisting of maltose binding protein and the active site region of gelatinase B. We found that addition of the active site of gelatinase B, which corresponds to 12% of the total protein molecule, to maltose binding protein is sufficient to endow the protein with the ability to cleave the peptide substrates Mca-PLGL(Dpa)AR-NH(2) and DNP-PLGLWA-(D)-R-NH(2). The fusion protein hydrolyzed the Mca-PLGL(Dpa)AR-NH(2) peptide with the same efficiency as that of the stromelysin, k(cat)/K(m) approximately 1.07 x 10(6) M(-)(1) h(-)(1). The fusion protein, however, was not able to degrade the large substrate, gelatin. Inhibition of the activity of the protein by EDTA suggested that its activity was metal dependent. ESR analyses indicated that the fusion protein bound one molecule of Zn(2+). In addition, Z-Pro-Leu-Gly-hydroxamate and TIMP-1 inhibited the activity of the protein, suggesting that the structure of the active site of the fusion protein is similar to that of the other metalloproteinases. These data provide fundamental information about the structural elements required for transforming a protein to a metalloprotease.
authors.affiliation: Department of Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
authors.forename: Kuljeet
authors.initials: K
authors.lastname: Kaur
authors.forename: Kijeen
authors.initials: K
authors.lastname: Zhu
authors.forename: Marilyn S
authors.initials: MS
authors.lastname: Whittemore
authors.forename: Richard L
authors.initials: RL
authors.lastname: Petersen
authors.forename: Andrea
authors.initials: A
authors.lastname: Lichte
authors.forename: Harald
authors.initials: H
authors.lastname: Tschesche
authors.forename: Tayebeh
authors.initials: T
authors.lastname: Pourmotabbed
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D058619:Maltose-Binding Proteins; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry* / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D003094:Collagen; D008666:Metalloendopeptidases; D020780:Matrix Metalloproteinase 9; D015032:Zinc