Identification of the active site of gelatinase B as the structural element sufficient for converting a protein to a metalloprotease

Kuljeet Kaur, Kijeen Zhu, Marilyn S. Whittemore, Richard L. Petersen, Andrea Lichte, Harald Tschesche, Tayebeh Pourmotabbed

Research output: Contribution to journalArticle

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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-NH2 and DNP-PLGLWA-(D)-R-NH2. The fusion protein hydrolyzed the Mca-PLGL(Dpa)AR-NH2 peptide with the same efficiency as that of the stromelysin, kcat/Km ≃ 1.07 × 106 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 Zn2+. 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.

Original languageEnglish (US)
Pages (from-to)4789-4797
Number of pages9
JournalBiochemistry
Volume41
Issue number15
DOIs
StatePublished - Apr 16 2002

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Matrix Metalloproteinase 9
Metalloproteases
Catalytic Domain
Fusion reactions
Proteins
Maltose-Binding Proteins
Gelatin
Collagen Type X
Collagen Type V
Matrix Metalloproteinase 3
Peptides
Molecules
Tissue Inhibitor of Metalloproteinase-1
Elastin
Substrates
Matrix Metalloproteinases
Edetic Acid
Paramagnetic resonance
Metals
Amino Acids

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Identification of the active site of gelatinase B as the structural element sufficient for converting a protein to a metalloprotease. / Kaur, Kuljeet; Zhu, Kijeen; Whittemore, Marilyn S.; Petersen, Richard L.; Lichte, Andrea; Tschesche, Harald; Pourmotabbed, Tayebeh.

In: Biochemistry, Vol. 41, No. 15, 16.04.2002, p. 4789-4797.

Research output: Contribution to journalArticle

Kaur, Kuljeet ; Zhu, Kijeen ; Whittemore, Marilyn S. ; Petersen, Richard L. ; Lichte, Andrea ; Tschesche, Harald ; Pourmotabbed, Tayebeh. / Identification of the active site of gelatinase B as the structural element sufficient for converting a protein to a metalloprotease. In: Biochemistry. 2002 ; Vol. 41, No. 15. pp. 4789-4797.
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