Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6
2000
Wakayama, M. (Oita Univ. (Japan)) | Yada, H. | Kanda, S. | Hayashi, S. | Yatsuda, Y. | Sakai, K. | Moriguchi, M.
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 x 10(4) 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 x 10(4)-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
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