title: Characterization of zinc-binding properties of a novel imidase from Pseudomonas putida YZ-26.
abstract: The imidase from Pseudomonas putida YZ-26 consisting of 293-amino acid residues is a novel imidase with four subunits as the holo-enzyme and low molecular weight which is significantly different from known mammalian imidase. This study measured the zinc-binding properties of the imidase using inductively coupled plasma-atomic emission spectrometry and competition assay combined with activity determinations. Results show that each subunit of the imidase binds the zinc ion by 1:1 stoichiometry with apparent binding constant of 9.5 x 10(8)M(-1). The activity of the apo-imidase (20 microM) was recovered with the addition of zinc in the lower concentration (0-20 microM), whereas the enzymatic activity is decreased in the presence of high concentration of zinc (above 100 microM). The site-directed mutagenesis of His(247), His(86) or Cys(7), Cys(108) in imidase resulted in loss of activity and zinc-binding abilities at different degrees, showing that these residues may critically affect both enzymatic activity and conformation.
authors.affiliation: Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, PR China. yaweishi@sxu.edu.cn
authors.forename: Ya-Wei
authors.initials: YW
authors.lastname: Shi
authors.forename: Xiao-Qin
authors.initials: XQ
authors.lastname: Liu
authors.forename: Peng
authors.initials: P
authors.lastname: Shi
authors.forename: Xue-Yao
authors.initials: XY
authors.lastname: Zhang
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D011485:Protein Binding; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D000581:Amidohydrolases; C020047:dihydropyrimidinase; D015032:Zinc