Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 2816-2822 |
| Number of pages | 7 |
| Journal | Journal of Bacteriology |
| Volume | 181 |
| Issue number | 9 |
| Publication status | Published - 1999 |
| Externally published | Yes |
Fingerprint
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology
- Immunology
Cite this
Identification of metal ligands in the Clostridium histolyticum ColH collagenase. / Jung, Chang Min; Matsushita, Osamu; Katayama, Seiichi; Minami, Junzaburo; Sakurai, Jun; Okabe, Akinobu.
In: Journal of Bacteriology, Vol. 181, No. 9, 1999, p. 2816-2822.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Identification of metal ligands in the Clostridium histolyticum ColH collagenase
AU - Jung, Chang Min
AU - Matsushita, Osamu
AU - Katayama, Seiichi
AU - Minami, Junzaburo
AU - Sakurai, Jun
AU - Okabe, Akinobu
PY - 1999
Y1 - 1999
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0032947997&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032947997&partnerID=8YFLogxK
M3 - Article
VL - 181
SP - 2816
EP - 2822
JO - Journal of Bacteriology
JF - Journal of Bacteriology
SN - 0021-9193
IS - 9
ER -