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

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.