Enzymatic phosphorylation of 1-beta-D-arabinofuranosylcytosine.

1-β-d-Arabinofuranosylcytosine (ara-C) was phosphorylated by three successive enzymatic reactions to 1-β-d-arabinofuranosylcytosine 5′-triphosphate via the intermediate formation of 1-β-d-arabinofuranosyl-CMP and 1-β-d-arabinofuranosyl-CDP. The enzymes concerned were deoxycytidine (CdR) kinase, deoxycytidine monophosphokinase, and nucleoside diphosphokinase of calf thymus. In the initial two successive reactions, Km values for ara-C and 1-β-d-arabinofuranosyl-CMP were intermediate between those of the corresponding deoxyribosidic and ribosidic derivatives. Calf thymus extract contained a cytidine kinase which was separable from the CdR kinase in question. The former enzyme catalyzed neither ara-C nor CdR, while the latter phosphorylated ara-C as well as CdR. In the second reaction, the addition of reduced or oxidized glutathione to the enzyme preparation effects the reaction rate in varying degrees depending on the phosphate acceptor used. By adjusting the ratio between concentrations of reduced and oxidized glutathione, it was possible to abolish the dCMP and 1-β-d-arabinofuranosyl-CMP kinase activities concomitantly without causing appreciable change in CMP kinase activity. These results taken together clearly indicate that ara-C and its phosphorylated derivatives are CdR analogs rather than cytidine analogs. In the last reaction, Km values of nucleoside diphosphokinases for 1-β-d-arabinofuranosyl-CDP, 5′-dCDP, and 5′-CDP were of the same order of magnitude (10-4 m).