Characterization of zinc-binding properties of a novel imidase from Pseudomonas putida YZ-26 |
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Authors: | Ya-Wei Shi Xiao-Qin Liu Peng Shi Xue-Yao Zhang |
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Institution: | Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan 030006, PR China |
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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 × 108 M−1. The activity of the apo-imidase (20 μM) was recovered with the addition of zinc in the lower concentration (0-20 μM), whereas the enzymatic activity is decreased in the presence of high concentration of zinc (above 100 μM). The site-directed mutagenesis of His247, His86 or Cys7, Cys108 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. |
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Keywords: | Imidase Inductively coupled plasma-atomic emission spectrometry Pseudomonas putida YZ-26 Zinc-dependent enzyme Zinc ion 4-(2-Pyridylazo) resorcinol (PAR) |
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