The role of exon 5 in fibroblast collagenase (MMP-1) substrate specificity and inhibitor selectivity.
Interstitial collagen is degraded by members of the matrix metalloproteinase (MMP) family, including MMP-1. Previous work has shown that the region of MMP-1 coded for by exon 5 is implicated both in substrate specificity and inhibitor selectivity. We have constructed a chimeric enzyme, the exon 5 chimera, consisting primarily of MMP-1, with the region coded for by exon 5 replaced with the equivalent region of MMP-3, a noncollagenolytic MMP. Unlike MMP-3, the exon 5 chimera is capable of cleaving type I collagen, but the activity is only 2.2% of trypsin-activated MMP-1. 'Superactivation' of the chimera has no discernible effect, suggesting that the salt bridge formed in 'superactive' MMP-1 is not present. The kinetics for exon 5 chimera cleavage of two synthetic substrates display an MMP-3 phenotype, however, cleavage of gelatin is slightly impaired as compared to the parent enzymes. The K(iapp) values for the exon 5 chimera complexed with synthetic inhibitors and N-terminal TIMP-2 also show a more MMP-3-like behaviour. However, the k(on) values for N-terminal TIMP-1 and N-terminal TIMP-2 are more comparable to those for MMP-1. These data show that the region of MMP-1 coded for by exon 5 is involved in both substrate specificity and inhibitor selectivity and the structural basis for our findings is discussed.
University of East Anglia, School of Biological Sciences, Norwich, UK. vk8@york.ac.uk
V
V
Knäuper
M L
ML
Patterson
F X
FX
Gomis-Rüth
B
B
Smith
A
A
Lyons
A J
AJ
Docherty
G
G
Murphy
D000595:Amino Acid Sequence; D005091:Exons; D005347:Fibroblasts / Q000201:enzymology; D007700:Kinetics; D020781:Matrix Metalloproteinase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011480:Protease Inhibitors / Q000494:pharmacology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D020781:Matrix Metalloproteinase 1