Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a "cysteine switch" mechanism for activation.
Latent human fibroblast collagenase (HFC) can be activated by a variety of seemingly disparate means. In addition to the well-characterized activation by trypsin and organomercurial compounds, the enzyme can be activated to various extents by surfactants such as sodium dodecyl sulfate, by chaotropic ions such as SCN-, by disulfide compounds such as oxidized glutathione, by sulfhydryl alkylating agents such as N-ethylmaleimide, and by oxidants such as NaOCl. The underlying basis for these activations is the modification, exposure, or proteolytic release of the Cys73 residue from its habitat in the latent enzyme where it is thought to be complexed to the active-site zinc atom. This residue is not accessible for reaction with small molar excesses of dithionitrobenzoate in native, latent HFC. However, on addition of EDTA, this residue becomes fully exposed and is quantitatively labeled. All modes of activation of latent HFC are believed to involve the dissociation of Cys73 from the active-site zinc atom and its replacement by water, with the concomitant exposure of the active site. This is thought to be the primary event that precedes the well-known autolytic cleavages that are observed following the appearance of collagenase activity. The dissociation of Cys73 from the zinc atom in the latent enzyme "switches" the role of the zinc from a noncatalytic to a catalytic one. This "cysteine switch" mechanism of regulation may be applicable to the entire collagenase gene family.
Springman
E B
EB
Department of Chemistry, Florida State University, Tallahassee 32306.
Angleton
E L
EL
Birkedal-Hansen
H
H
Van Wart
H E
HE
D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D005881:Gingiva / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D003012:Microbial Collagenase / Q000235:genetics / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D015032:Zinc / Q000378:metabolism*
D004228:Dithionitrobenzoic Acid; D003012:Microbial Collagenase; D015032:Zinc; D003545:Cysteine; D005033:Ethylmaleimide