Unexpected crucial role of residue 272 in substrate specificity of fibroblast collagenase.
Degradation of type I collagen by collagenases is an important part of extracellular remodeling. To understand the role of the hinge region of fibroblast collagenase in its collagenolytic activity, we individually substituted the 10 conserved amino acid residues at positions 264, 266, 268, 296, 272, 277, 284, 289, 307, and 313 in this region of the enzyme by their corresponding residues in MMP-3, a noncollagenolytic matrix metalloproteinase. The general proteolytic and triple helicase activities of all of the enzymes were determined, and their abilities to bind to type I collagen were assessed. Among the mutants, only G272D mutant enzyme exhibited a significant change in type I collagenolysis. The alteration of the Gly(272) to Asp reduced the collagenolytic activity of the enzyme to 13% without affecting its general proteolytic activity, substrate specificity, or the collagen binding ability. The catalytic efficiency of the G272D mutant for the triple helical peptide substrate [C(6)-(GP- Hyp)(4)GPL(Mca)GPQGLRGQL(DPN)GVR(GP-HYP)(4)-NH(2)](3) and the peptide substrate Mca-PLGL(Dpa)AR-NH(2) and its dissociation constant for the triple helical collagen were similar to that of the wild type enzyme, indicating that the presence of this residue in fibroblast collagenase is particularly important for the efficient cleavage of type I collagen. Gly(272) is evidently responsible for the hinge-bending motion that is essential for allowing the COOH-terminal domain to present the collagen to the active site.
Tsukada
Hiroki
H
Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163, USA.
Pourmotabbed
Tayebeh
T
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002417:Cattle; D003094:Collagen / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005780:Gelatin / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D020784:Matrix Metalloproteinase 8 / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D001224:Aspartic Acid; D005780:Gelatin; D003094:Collagen; D020784:Matrix Metalloproteinase 8; D005998:Glycine