Ca(2+) and Zn(2+) binding properties of peptide substrates of vertebrate collagenase, MMP-1.
To understand the role of Ca(2+) in vertebrate in the structure and action of collagenase, we have examined peptides that interact with recombinant human fibroblast collagenase for their affinities towards Ca(2+) and Zn(2+) in a non-polar solvent. Two of the peptides, GPQGIAGQ and GNVGLAGA, had sequences in collagen which are, respectively, cleaved and not cleaved by collagenase. A third peptide, PSYFLNAG, had a collagenase-cleaved sequence in ovostatin, a globular protein substrate. Peptides TVGCEECTV and CLPREPGL were derived from TIMP-1; the former competitively inhibits collagenase while the latter does not. The relative rates of hydrolysis of the peptides by collagenase had the order GPQGIAGQ>PSYFLNAG>GNVGLAGA. Circular dichroism spectral data in trifluoroethanol showed that while the TIMP control peptide, CLPREPGL, bound only Zn(2+), the other four peptides bound both Ca(2+) and Zn(2+) with definite stoichiometries. Ca(2+) could displace Zn(2+) in the substrate peptides while Zn(2+) displaced Ca(2+) in the TIMP peptide. GPQGIAGQ, PSYFLNAG and TVGCEECTV formed peptide:Ca(2+):Zn(2+) ternary complexes. Our results suggest that both collagen and globular protein substrates of collagenase may bind Ca(2+) and Zn(2+) in the enzyme's active site. This, in turn, may account for the known importance of the non-catalytic Ca(2+) and Zn(2+) in collagenase activity.
Department of Biochemistry, McMaster University, Hamilton, Ont. L8N 3Z5, Canada.
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Vettakkorumakankav
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Ananthanarayanan
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D017364:Collagenases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D020781:Matrix Metalloproteinase 1; D061965:Matrix Metalloproteinase Inhibitors; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000737:chemistry; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D061965:Matrix Metalloproteinase Inhibitors; D009842:Oligopeptides; D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D017364:Collagenases; D020781:Matrix Metalloproteinase 1; D015032:Zinc; D002118:Calcium