Bioscience, Biotechnology and Biochemistry p. 1 - 8 (2000)
Update date:2022-08-17
Topics:
Wakayama, Mamoru
Yada, Harutaka
Kanda, Shun-Ichi
Hayashi, Shin-Ichi
Yatsuda, Yukinori
Sakai, Kenji
Moriguchi, Mitsuaki
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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Doi:10.1007/BF00959982
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