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 effici ently removed from the enzyme with o-phenanthroline to yield an apoenz yme 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+, M n2+, Co2+, or Zn2+ (M2+CDase) had k(cat) values of 185, 88, 50, and 32 s-1, respectively. The K(m) values of these M2+CDases for cytosine we re 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 no t inactivated by H2O2. CDase was also inhibited by excess divalent cat ions. Cu2+ and Zn2+ reversibly inhibited Fe2+CDase activity with inhib ition constants of 1.8 and 5.8 muM, respectively. Cu2+ dissociated slo wly from the secondary binding on CDase with a rate constant of 2 x 10 (-3) s-1.