Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp xylosoxydans A-6

D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcal igenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67 N 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 detect able activity, The H67N mutant had almost constant K-m, but greatly decreas ed k(cat). These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fract ion. 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 betw een 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. Th ese results suggest that the His250 residue might be essential to catalysis via Zn binding.