title: 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 kcat/Km 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 Km, but greatly decreased kcat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The kcat/Km 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 Km and kcat 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.
authors.affiliation: Department of Applied Chemistry, Faculty of Engineering, Oita University, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Wakayama
authors.forename: H
authors.initials: H
authors.lastname: Yada
authors.forename: S
authors.initials: S
authors.lastname: Kanda
authors.forename: S
authors.initials: S
authors.lastname: Hayashi
authors.forename: Y
authors.initials: Y
authors.lastname: Yatsuda
authors.forename: K
authors.initials: K
authors.lastname: Sakai
authors.forename: M
authors.initials: M
authors.lastname: Moriguchi
mesh_terms: D000421:Alcaligenes / Q000187:drug effects / Q000201:enzymology*; D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002942:Circular Dichroism; D003035:Cobalt / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D015032:Zinc / Q000494:pharmacology
chemical_list: D003035:Cobalt; D006639:Histidine; D000581:Amidohydrolases; C019672:aminoacylase I; D015032:Zinc; D004047:Diethyl Pyrocarbonate