Role of Conserved Histidine Residues in D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

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.