Identification of the Active Site of Gelatinase B as the Structural Element Sufficient for Converting a Protein to a Metalloprotease
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- 作者：Kuljeet Kaur ; Kijeen Zhu ; Marilyn S. Whittemore ; Richard L. Petersen ; Andrea Lichte ; Harald Tschesche ; and Tayebeh Pourmotabbed
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：April 16, 2002
- 年：2002
- 卷：41
- 期：15
- 页码：4789 - 4797
- 全文大小：252K
- 年卷期：v.41,no.15(April 16, 2002)
- ISSN：1520-4995
文摘
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 proteinconsisting of maltose binding protein and the active site region of gelatinase B. We found that additionof the active site of gelatinase B, which corresponds to 12% of the total protein molecule, to maltosebinding 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 activityof the protein by EDTA suggested that its activity was metal dependent. ESR analyses indicated that thefusion protein bound one molecule of Zn2+. In addition, Z-Pro-Leu-Gly-hydroxamate and TIMP-1 inhibitedthe activity of the protein, suggesting that the structure of the active site of the fusion protein is similarto that of the other metalloproteinases. These data provide fundamental information about the structuralelements required for transforming a protein to a metalloprotease.
1.07 × 106 M-1 h-1.The fusion protein, however, was not able to degrade the large substrate, gelatin. Inhibition of the activityof the protein by EDTA suggested that its activity was metal dependent. ESR analyses indicated that thefusion protein bound one molecule of Zn2+. In addition, Z-Pro-Leu-Gly-hydroxamate and TIMP-1 inhibitedthe activity of the protein, suggesting that the structure of the active site of the fusion protein is similarto that of the other metalloproteinases. These data provide fundamental information about the structuralelements required for transforming a protein to a metalloprotease.