PMID:8226944
| Citation |
Porter, DJ and Austin, EA (1993) Cytosine deaminase. The roles of divalent metal ions in catalysis. J. Biol. Chem. 268:24005-11 |
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| Abstract |
Cytosine deaminase (CDase, EC 3.5.4.1) isolated from Escherichia coli contains a catalytically essential divalent metal ion. Fe2+ was efficiently removed from the enzyme with o-phenanthroline to yield an apoenzyme with less than 5% of the catalytic activity of native enzyme. The time courses for inactivation and for removal of Fe2+ from the enzyme by o-phenanthroline were similar. Apoenzyme reconstituted with Fe2+, Mn2+, Co2+, or Zn2+ (M2+CDase) had kcat values of 185, 88, 50, and 32 s-1, respectively. The Km values of these M2+CDases for cytosine were similar (0.22-0.39 mM). Cytosine potently inhibited reconstitution of the apoenzyme with Fe2+. Fe2+CDase was rapidly inactivated by 1 mM H2O2 (t1/2 < 1 s), whereas Mn2+CDase, Co2+CDase, and Zn2+CDase were not inactivated by H2O2. CDase was also inhibited by excess divalent cations. Cu2+ and Zn2+ reversibly inhibited Fe2+CDase activity with inhibition constants of 1.8 and 5.8 microM, respectively. Cu2+ dissociated slowly from the secondary binding on CDase with a rate constant of 2 x 10(-3) s-1. |
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| Keywords |
Catalysis; Cations, Divalent; Chromatography, Liquid; Culture Media; Cytosine Deaminase; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Hydrogen Peroxide/pharmacology; Metals/pharmacology; Nucleoside Deaminases/antagonists & inhibitors; Nucleoside Deaminases/isolation & purification; Nucleoside Deaminases/metabolism |
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