Role of Conserved Histidine Residues in D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6
2000
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 k cₐₜ/K ₘ 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 K ₘ, but greatly decreased k cₐₜ. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The k cₐₜ/K ₘ of H250N mutant was reduced by a factor of 2.5×10⁴-fold as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the K ₘ and k cₐₜ 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.
اظهر المزيد [+] اقل [-]الكلمات المفتاحية الخاصة بالمكنز الزراعي (أجروفوك)
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