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.