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Enzymatic Phosphorylation of 1-ß-D-Arabinofuranosylcytosine¹

Department of Biochemistry, Institute for Virus Research, Kyoto University, Kyoto, Japan

1-ß-D-Arabinofuranosylcytosine (ara-C) was phosphorylated by three successive enzymatic reactions to 1-ß-D-arabinofuranosylcytosine 5'-triphosphate via the intermediate formation of 1-ß-D-arabinofuranosyl-CMP and 1-ß-D-arabinofuranosyl-CDP. The enzymes concerned were deoxycytidine (CdR) kinase, deoxycytidine monophosphokinase, and nucleoside diphosphokinase of calf thymus.

In the initial two successive reactions, K_m values for ara-C and 1-ß-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-ß-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, K_m values of nucleoside diphosphokinases for 1-ß-D-arabinofuranosyl-CDP, 5'-dCDP, and 5'-CDP were of the same order of magnitude (10^-4 M).

¹ This investigation was supported in part by a research grant from the Ministry of Education of Japan. A preliminary account of this paper was presented at the 14th General Meeting of The Japanese Biochemical Society, Osaka, November 1966.

² This work was done in partial fulfillment of the requirements for the degree of Doctor of Science, Kyoto University. Present address: Biological Research Laboratories, Research and Development Division, Takeda Chemical Industries, Ltd., Osaka, Japan.

Received 12/17/70. Accepted 5/20/71.