Kinetic studies with 5-azacytidine-5'-triphosphate and DNA-dependent RNA polymerase.

In order to further understand the biochemical mode of action of 5-azacytidine, a potent antileukemic agent, kinetic studies were performed with 5-azacytidine-5'-triphosphate (5-aza-CTP) and purified DNA-dependent RNA polymerase from Escherichia coli and calf thymus. RNA polymerase could catalyze the incorporation of the fradulent nucleotide, 5-aza-CTP, into RNA. The apparent K_m value for 5-aza-CTP was estimated to be 350 and 390 for the E. coli and calf thymus enzymes respectively. The K_m value for 5-aza-CTP was about 18-fold greater than the K_m value for CTP (20 μM). The apparent V_max value for CTP was about 2-fold greater than the V_max value for 5-aza-CTP. 5-Aza-CTP was a weak competitive inhibitor with respect to CTP; the apparent K_i value for 5-aza-CTP was estimated to be 680 and 810 μM for the E. coli and calf thymus enzymes respectively. On the other hand, CTP was a potent competitive inhibitor with respect to 5-aza-CTP; the apparent K_i value of CTP was estimated to be 16 μM. 5-Aza-CTP did not appear to inhibit the incorporation of UTP into RNA in the reaction catalyzed by RNA polymerase. These data suggest that the inhibition of RNA synthesis in cells by 5-aza-cytidine is not produced by the inhibition of RNA polymerase by 5-aza-CTP.