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

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

Research output: Contribution to journalArticle

50 Scopus citations

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Mutational analysis of residues in and around the active site of human fibroblast-type collagenase'. Together they form a unique fingerprint.

  • Cite this

    Windsor, L. J., 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.