Role of the conserved histidine and aspartic acid residues in activity and stabilization of human gelatinase B: an example of matrix metalloproteinases.
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 sequence. 400His-(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 similar Km values for the synthetic substrate and similar Ki values for the competitive inhibitor, GM6001. The kcat/Km values for D432G and D433E mutant enzymes, however, were reduced by a factor of approximately 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.
Pourmotabbed
T
T
Department of Biochemistry, University of Tennessee, Memphis 38163, USA.
Aelion
J A
JA
Tyrrell
D
D
Hasty
K A
KA
Bu
C H
CH
Mainardi
C L
CL
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D017364:Collagenases / Q000737:chemistry* / Q000378:metabolism; D017124:Conserved Sequence; D004795:Enzyme Stability; D006639:Histidine / Q000378:metabolism; D006801:Humans; D020780:Matrix Metalloproteinase 9; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D001224:Aspartic Acid; D006639:Histidine; D017364:Collagenases; D020780:Matrix Metalloproteinase 9; D015032:Zinc; D002118:Calcium