Role of the conserved histidine and aspartic acid residues in activity and stabilization of human gelatinase B: An example of matrix metalloproteinases |
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Authors: | Tayebeh Pourmotabbed Jacob A. Aelion David Tyrrell Karen A. Hasty Chun Hui Bu Carlo L. Mainardi |
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Affiliation: | (1) Department of Biochemistry, University of Tennessee, 38163 Memphis, Tennessee;(2) Department of Medicine, University of Tennessee, 38163 Memphis, Tennessee;(3) Veterans Administration Medical Center, 38104 Memphis, Tennessee;(4) Glycomed Incorporated, 94501 Alameda, California;(5) Department of Anatomy, University of Tessessee, 38163 Memphis, Tennessee |
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Abstract: | Gelatinase B (MMP-9), a member of the matrix metalloproteinase family, is a zinc- and calcium-dependent endopeptidase that is known to play a role in tumor cell invasion and in destruction of cartilage in arthritis. It contains a conserved sequence400His-(X)3-His-(X)28-Asp-Asp-(X)2-436Gly, the function of which is under investigation. The conserved Asp-432 and Asp-433 residues were individually replaced with Gly; these substitutions reduced the gelatinolytic activity of the enzyme to 23% and 0%, respectively. Replacing Asp-433 with Glu, however, decreased the gelatinolytic activity of the enzyme by 93% and proteolytic activity of the enzyme for the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 substrate by 79%. The wild-type and D432G and D433E mutant enzymes had similarKm values for the synthetic substrate and similarKi values for the competitive inhibitor, GM6001. Thekcat/Km values for D432G and D433E mutant enzymes, however, were reduced by a factor of 4 and their KaCa values were increased by four- and sixfold, respectively. The significance of His-400 in the activity of the enzyme was assessed by replacing this residue with Ala and Phe. Both H400A and H400F mutants were inactive toward gelatin substrate. These data demonstrate that Asp-432, Asp-433, and His-400 residues are important for the activity of gelatinase B. His-400 may act as a zinc-binding ligand similar to the His-197 in interstitial collagenase (MMP-7) and Asp-432 and Asp-433 residues are probably involved in stabilization of the active site of the enzyme. The His-400 and Asp-433 residues are conserved in all members of the MMP family. Therefore, our results are relevant to this group as a whole.Abbreviations MMP Matrix metalloproteinase - TIMP tissue inhibitor of metalloproteinase - IPTG isopropyl-D-thiogalactoside - APMA 4-aminophenyl-mercuric acetate - PCR polymerase chain reaction - Dpa 3(2,4-di-nitrophenyl) diaminopropionic acid - Mca 7-methoxycoumarin acetic acid |
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Keywords: | Metalloproteinase gelatinase B active site |
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