Deoxycytidine kinase from human leukemic spleen: preparation and characteristics of homogeneous enzyme.
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.
Bohman
C
C
Medical Nobel Institute, Department of Biochemistry I, Karolinska Institute, Stockholm, Sweden.
Eriksson
S
S
D001667:Binding, Competitive; D002852:Chromatography, Ion Exchange; D003842:Deoxycytidine Kinase / Q000032:analysis / Q000302:isolation & purification*; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D007938:Leukemia / Q000201:enzymology*; D010770:Phosphotransferases / Q000302:isolation & purification*; D013154:Spleen / Q000201:enzymology*
D010770:Phosphotransferases; D003842:Deoxycytidine Kinase