Role of Conserved Histidine Residues inD-Aminoacylase fromAlcaligenes xylosoxydanssubsp.xylosoxydansA-6

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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 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.

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