Deoxycytidine kinase from human leukemic spleen: preparation and characterization of the homogeneous enzyme

Abstract

Deoxycytidine kinase from human leukemic spleen has been purified 6000-fold to apparent homogeneity with an overall yield of 10%. The purification was achieved by using DEAE chromatography, hydroxylapatite chromatography, and affinity chromatography on dTTP-Sepharose. Only one form of deoxycytidine kinase activity was found during all the chromatographic procedures. The subunit molecular mass, as judged by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, was 30 kilodaltons. The pure enzyme phosphorylates deoxycytidine, deoxyadenosine, and deoxyguanosine, demonstrating for the first time that the same enzyme molecule has the capacity to use these three nucleosides as substrates. The apparent molecular weight of the active enzyme, determined by gel filtration and glycerol gradient centrifugation, was 60,000. Thus, the active form of human deoxycytidine kinase is a dimer. The kinetic behavior of pure human deoxycytidine kinase was studied in detail with regard to four different phosphate acceptors and two different phosphate donors. The apparent Km values were 1, 20, 150, and 120 microM for deoxycytidine, arabinosylcytosine, deoxyguanosine, and deoxyadenosine, respectively. The Vmax values were 5-fold higher for the purine nucleosides as compared to the pyrimidine substrates. We observe competitive inhibition of the phosphorylation of one substrate by the presence of either of the three other substrates, but the apparent Ki values differed greatly from the corresponding Km values, suggesting the existence of allosteric effects. The double-reciprocal plots for ATP-MgCl2 as phosphate donor were convex, indicating negative cooperative effects. In contrast, plots with varying dTTP-MgCl2 concentration as phosphate donor were linear with an apparent Km of 2 microM. The enzyme activity was strongly inhibited by dCTP, in a noncompetitive way with deoxycytidine and in a competitive way with ATP-MgCl2.

Erk5 contributes to maintaining the balance of cellular nucleotide levels and erythropoiesis

“…Metabolic flux through the salvage pathway is regulated by thymidine kinase 1 (TK1), which phosphorylates thymidine, and deoxycytidine kinase, which phosphorylates deoxycytidine, deoxyadenosine and deoxyguanosine. 17,30,31 To test whether thymidine salvaging is increased in Erk5-depleted cells, we incubated cells with [ 3 H]-thymidine and analyzed cellular uptake and TK activity in cell extracts. Although [ 3 H]-thymidine uptake by shErk5 cells was slightly higher, TK activity in shErk5 and control cells was similar (supplementary material Fig.…”

Characterization of the deoxycytidine kinase promoter in human lymphoblast cell lines.

“…deoxycytidine to its 5'-monophosphate and, despite a much higher Km for the purine deoxyribonucleosides, also plays a major role in phosphorylating 2 '-deoxyadenosine and 2 '-deoxyguanosine to their corresponding monophosphates (1)(2)(3)(4). dCK is also central in the activation of anticancer and antiviral agents such as cytosine arabinoside, fludarabine, 2 ',3 '-dideoxycytidine, and 2-chlorodeoxyadenosine (CdA) (5)(6)(7)(8)(9).…”

Deoxynucleoside Kinases Encoded by the yaaG andyaaF Genes of Bacillus subtilis

“…Only 6-oxopurine 2Ј-deoxyribonucleosides (dGuo and dIno) are phosphorylated by the enzyme at significant rates, with a K m for dGuo of 0.6 M and a k cat of 1.4 s Ϫ1 . The homodimeric mammalian mitochondrial dGK, which in addition to dGuo and dIno can use dAdo as a substrate, shows K m and k cat values of 7.6 M and 0.002 s Ϫ1 , respectively, with dGuo as a substrate (9), and the homodimeric human dCK, which uses dCyd, dAdo, and dGuo, phosphorylates dGuo with a K m of 150 -430 M and a k cat of 0.4 -6.0 s Ϫ1 (7,31). Recently, a monomeric multisubstrate dNK from Drosophila melanogaster (Dm-dNK) was characterized (12).…”

“…TK2 can in addition use dCyd as a substrate (4 -6). dCK phosphorylates dCyd, dAdo, and dGuo (7,8), and the mitochondrial dGK is specific for dGuo and dAdo (9,10). A deoxynucleoside kinase with a very different substrate specificity was recently characterized from Drosophila melanogaster.…”