Characterization of zinc-binding properties of a novel imidase from Pseudomonas putida YZ-26.
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.
Shi
Ya-Wei
YW
Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, PR China. yaweishi@sxu.edu.cn
Liu
Xiao-Qin
XQ
Shi
Peng
P
Zhang
Xue-Yao
XY
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*
D017931:DNA Primers; D011994:Recombinant Proteins; D000581:Amidohydrolases; C020047:dihydropyrimidinase; D015032:Zinc