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 fibrobl ast-type collagenase have been expressed in Escherichia coli. Replacem ent of each of the three zinc ligands, His-199, His-203, and His-209, in the active site sequence: VAAHEXGHXXGXXH, not only destroyed cataly tic activity but also led to improper folding of the polypeptide, sugg esting that this sequence also serves as a structural zinc-binding sit e. By comparison, mutation of His-194 immediately preceding this seque nce had no measurable effect on catalytic activity or on folding. Repl acement of Glu-200 in the active site yielded enzymes that either were completely inactive (E200Q) or had greatly diminished (E200D) catalyt ic activity. Both Glu-200 mutants, however, were fully capable of form ing complexes with tissue inhibitor of metalloproteinases-1 (TIMP-1) a fter reaction with organomercurials. Formation of complexes with TIMP- 1 appear to require a properly folded, but not necessarily catalytical ly competent, active site. By contrast, complexes with alpha(2)-macrog lobulin form only with mutants with a catalytically competent active s ite. Two mutants identified in this study (E200Q and D212E) appeared t o be properly folded but unable to generate any catalytic activity whe n exposed to either p-aminophenylmercuric acetate, trypsin, or SDS.