Abstract

Dual-substrate kinetics have been used to demonstrate that the phosphorylation of 5-azacytidine by a purified preparation of uridine-cytidine kinase is competitive with that of the natural substrates. When 5-azacytidine was combined with either uridine or cytidine in the reaction, the kinetic patterns observed were consistent with the theoretical plots for a single enzyme catalyzing both phosphorylations. In experiments with uridine plus 5-azacytidine, the patterns were those for alternate substrates with significantly disparate V_max values (V_{max, uridine} = 1.82 µmoles phosphorylated/min/mg protein; V_max, _azacytidine = 1.11 µmoles phosphorylated/min/mg protein). In the phosphorylation of cytidine plus 5-azacytidine, the pattern seen was that for alternate substrates with equivalent V_max values (V_{max, cytidine} = 0.97 µmoles phosphorylated/min/mg protein). Furthermore, 5-azacytidine could be shown to be a competitive inhibitor of cytidine phosphorylation, albeit at high ratios of analogue to normal substrate. Apparent K_m’S were found to be: uridine, 0.135 mM; cytidine, 0.067 mM; 5-azacytidine, 8.09 mM. From the kinetic patterns in the dual substrate experiments, it can be concluded that all three nucleosides are phosphorylated by the same enzyme, presumably at a single catalytic site.