Abstract
D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67N mutant was barely active, with a kcat/Km 6.3 × 104 times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity. The H67N mutant had almost constant Km, but greatly decreased kcat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The kcat/Km of H250N mutant was reduced by a factor of 2.5 × 104-old as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the Km and kcat was found. The Zn content of H250N mutant was nearly half of that of wild-type enzyme. These results suggest that the His250 residue might be essential to catalysis via Zn binding.
| Original language | English |
|---|---|
| Pages (from-to) | 1-8 |
| Number of pages | 8 |
| Journal | Bioscience, Biotechnology and Biochemistry |
| Volume | 64 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - Jan 1 2000 |
| Externally published | Yes |
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All Science Journal Classification (ASJC) codes
- Biotechnology
- Analytical Chemistry
- Biochemistry
- Applied Microbiology and Biotechnology
- Molecular Biology
- Organic Chemistry
Cite this
Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. / Wakayama, Mamoru; Yada, Harutaka; Kanda, Shun Ichi; Hayashi, Shin Ichi; Yatsuda, Yukinori; Sakai, Kenji; Moriguchi, Mitsuaki.
In: Bioscience, Biotechnology and Biochemistry, Vol. 64, No. 1, 01.01.2000, p. 1-8.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6
AU - Wakayama, Mamoru
AU - Yada, Harutaka
AU - Kanda, Shun Ichi
AU - Hayashi, Shin Ichi
AU - Yatsuda, Yukinori
AU - Sakai, Kenji
AU - Moriguchi, Mitsuaki
PY - 2000/1/1
Y1 - 2000/1/1
N2 - D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67N mutant was barely active, with a kcat/Km 6.3 × 104 times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity. The H67N mutant had almost constant Km, but greatly decreased kcat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The kcat/Km of H250N mutant was reduced by a factor of 2.5 × 104-old as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the Km and kcat was found. The Zn content of H250N mutant was nearly half of that of wild-type enzyme. These results suggest that the His250 residue might be essential to catalysis via Zn binding.
AB - D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67N mutant was barely active, with a kcat/Km 6.3 × 104 times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity. The H67N mutant had almost constant Km, but greatly decreased kcat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The kcat/Km of H250N mutant was reduced by a factor of 2.5 × 104-old as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the Km and kcat was found. The Zn content of H250N mutant was nearly half of that of wild-type enzyme. These results suggest that the His250 residue might be essential to catalysis via Zn binding.
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UR - http://www.scopus.com/inward/citedby.url?scp=0033632280&partnerID=8YFLogxK
U2 - 10.1271/bbb.64.1
DO - 10.1271/bbb.64.1
M3 - Article
VL - 64
SP - 1
EP - 8
JO - Bioscience, Biotechnology and Biochemistry
JF - Bioscience, Biotechnology and Biochemistry
SN - 0916-8451
IS - 1
ER -