Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Mamoru Wakayama, Harutaka Yada, Shun Ichi Kanda, Shin Ichi Hayashi, Yukinori Yatsuda, Kenji Sakai, Mitsuaki Moriguchi

Research output: Contribution to journalArticle

16 Citations (Scopus)

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 languageEnglish
Pages (from-to)1-8
Number of pages8
JournalBioscience, Biotechnology and Biochemistry
Volume64
Issue number1
DOIs
Publication statusPublished - Jan 1 2000
Externally publishedYes

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Alcaligenes
Histidine
Enzymes
Diethyl Pyrocarbonate
Catalysis
aminoacylase I

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 journalArticle

Wakayama, Mamoru ; Yada, Harutaka ; Kanda, Shun Ichi ; Hayashi, Shin Ichi ; Yatsuda, Yukinori ; Sakai, Kenji ; Moriguchi, Mitsuaki. / Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. In: Bioscience, Biotechnology and Biochemistry. 2000 ; Vol. 64, No. 1. pp. 1-8.
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