title: Escherichia coli cytosine deaminase: the kinetics and thermodynamics for binding of cytosine to the apoenzyme and the Zn(2+) holoenzyme are similar.
abstract: Recombinant Escherichia coli cytosine deaminase is purified as a mixture of Zn(2+) and Fe(2+) forms of the enzyme. Fe(2+) is removed readily by o-phenanthroline to yield apoenzyme (apoCDase) that contains <0.2 mol of Zn(2+)per mol of subunit. ApoCDase was efficiently reconstituted to Zn(2+)CDase by treatment with ZnCl(2). The interaction of cytosine with apoCDase and Zn(2+)CDase was investigated at pH 7.5 and 25 degrees C by monitoring changes in intrinsic protein fluorescence. The values for the kinetic data K(1), k(2), and k(3) for Zn(2+)CDase were 0.25 mM, 80 s(-1), and 38 s(-1), respectively. The value for k(-2) was statistically indistinguishable from zero. The analogous values for K(1), k(2), and k(-2), (k(3)=0) for apoCDase were 0.157 mM, 186 s(-1) and approximately 0.8 s(-1), respectively. The overall dissociation constant of apoCDase for cytosine was 0.00069 mM, whereas the K(m) of Zn(2+)CDase for cytosine was 0.20 mM. The pre-steady state phase of the reaction was associated with an absorbance increase at 280 nm that was attributed to solvent perturbation of the spectrum of cytosine or enzyme. Formation of the Fe(2+)CDase-cytosine complex was too rapid to monitor by these techniques.
authors.affiliation: Glaxo Wellcome, 5 Moore Drive, Research Triangle Park, NC 27709, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Porter
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D003596:Cytosine / Q000378:metabolism; D043525:Cytosine Deaminase; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009700:Nucleoside Deaminases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D015032:Zinc / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D003596:Cytosine; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D015032:Zinc