Mutational analysis of residues in and around the active site of human fibroblast-type collagenase

L. Windsor, M. Kirby Bodden, Bente Birkedal-Hansen, Jeffrey A. Engler, Henning Birkedal-Hansen

Research output: Contribution to journalArticle

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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 α2-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.

Original languageEnglish (US)
Pages (from-to)26201-26207
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number42
StatePublished - Oct 21 1994
Externally publishedYes

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Matrix Metalloproteinase 8
Catalyst activity
Catalytic Domain
Zinc
Tissue Inhibitor of Metalloproteinase-1
Binding Sites
Macroglobulins
Trypsin
Escherichia coli
Acetates
Ligands
Peptides
Mutation
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Windsor, L., Bodden, M. K., Birkedal-Hansen, B., Engler, J. A., & Birkedal-Hansen, H. (1994). Mutational analysis of residues in and around the active site of human fibroblast-type collagenase. Journal of Biological Chemistry, 269(42), 26201-26207.

Mutational analysis of residues in and around the active site of human fibroblast-type collagenase. / Windsor, L.; Bodden, M. Kirby; Birkedal-Hansen, Bente; Engler, Jeffrey A.; Birkedal-Hansen, Henning.

In: Journal of Biological Chemistry, Vol. 269, No. 42, 21.10.1994, p. 26201-26207.

Research output: Contribution to journalArticle

Windsor, L, Bodden, MK, Birkedal-Hansen, B, Engler, JA & Birkedal-Hansen, H 1994, 'Mutational analysis of residues in and around the active site of human fibroblast-type collagenase', Journal of Biological Chemistry, vol. 269, no. 42, pp. 26201-26207.
Windsor L, Bodden MK, Birkedal-Hansen B, Engler JA, Birkedal-Hansen H. Mutational analysis of residues in and around the active site of human fibroblast-type collagenase. Journal of Biological Chemistry. 1994 Oct 21;269(42):26201-26207.
Windsor, L. ; Bodden, M. Kirby ; Birkedal-Hansen, Bente ; Engler, Jeffrey A. ; Birkedal-Hansen, Henning. / Mutational analysis of residues in and around the active site of human fibroblast-type collagenase. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 42. pp. 26201-26207.
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