Cytosine deaminase. The roles of divalent metal ions in catalysis.
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.
Porter
D J
DJ
Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Austin
E A
EA
D002384:Catalysis; D002413:Cations, Divalent; D002853:Chromatography, Liquid; D003470:Culture Media; D043525:Cytosine Deaminase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D009700:Nucleoside Deaminases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D002413:Cations, Divalent; D003470:Culture Media; D008670:Metals; D006861:Hydrogen Peroxide; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase