title: Mutational analysis of residues in and around the active site of human fibroblast-type collagenase.
abstract: Mutants in and around the catalytic zinc-binding site of human fibroblast-type collagenase have been expressed in Escherichia coli. Replacement of each of the three zinc ligands, His-199, His-203, and His-209, in the active site sequence: VAAHEXGHXXGXXH, not only destroyed catalytic activity but also led to improper folding of the polypeptide, suggesting that this sequence also serves as a structural zinc-binding site. By comparison, mutation of His-194 immediately preceding this sequence had no measurable effect on catalytic activity or on folding. Replacement of Glu-200 in the active site yielded enzymes that either were completely inactive (E200Q) or had greatly diminished (E200D) catalytic activity. Both Glu-200 mutants, however, were fully capable of forming complexes with tissue inhibitor of metalloproteinases-1 (TIMP-1) after reaction with organomercurials. Formation of complexes with TIMP-1 appear to require a properly folded, but not necessarily catalytically competent, active site. By contrast, complexes with alpha 2-macroglobulin form only with mutants with a catalytically competent active site. Two mutants identified in this study (E200Q and D212E) appeared to be properly folded but unable to generate any catalytic activity when exposed to either p-aminophenylmercuric acetate, trypsin, or SDS.
authors.affiliation: Department of Oral Biology, University of Alabama School of Dentistry, Birmingham.
authors.forename: L J
authors.initials: LJ
authors.lastname: Windsor
authors.forename: M K
authors.initials: MK
authors.lastname: Bodden
authors.forename: B
authors.initials: B
authors.lastname: Birkedal-Hansen
authors.forename: J A
authors.initials: JA
authors.lastname: Engler
authors.forename: H
authors.initials: H
authors.lastname: Birkedal-Hansen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002364:Caseins / Q000378:metabolism; D017364:Collagenases / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D017510:Protein Folding; D019714:Tissue Inhibitor of Metalloproteinases; D014357:Trypsin / Q000494:pharmacology; D015032:Zinc / Q000378:metabolism*
chemical_list: D002364:Caseins; D006023:Glycoproteins; D019714:Tissue Inhibitor of Metalloproteinases; C010024:4-aminophenylmercuriacetate; D014357:Trypsin; D017364:Collagenases; D015032:Zinc; D010662:Phenylmercuric Acetate