Identification of metal ligands in the Clostridium histolyticum ColH collagenase

A Clostridium histolyticum 116-kDa collagenase has an H⁴¹⁵EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases. To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues. The mutation affecting His⁴¹⁵ or His⁴¹⁹ abolished catalytic activity and zinc binding, while that affecting Glu⁴¹⁶ did the former but not the latter. These results suggest that the motif forms the catalytic site. We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands. The mutation affecting Glu⁴⁴⁷ decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu⁴⁴⁶ or Glu⁴⁵¹ had smaller effects on activity and zinc binding. These mutations caused a decrease in k(cat) but no significant change in K(m). These results are consistent with the hypothesis that Glu⁴⁴⁷ is the third zinc ligand. The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily. The effects of mutations affecting Glu⁴⁴⁶ and Glu⁴⁵¹ suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.