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 Vmax values (Vmax, uridine = 1.82 µmoles phosphorylated/min/mg protein; Vmax, 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 Vmax values (Vmax, 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 Km’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.
ACKNOWLEDGMENTS The authors wish to thank Dr. Edward Steers and Mr. Jon Seeman for their help in quantitating enzyme protein with the amino acid analyzer. We are also indebted to Dr. James Schrode and Mr. Gary Knott for their expert assistance with the MLAB computer program.
- Copyright © 1979 by Academic Press, Inc.