Nucleotide specificity of human deoxycytidine kinase.
The ability of deoxycytidine kinase (dCK) to phosphorylate 2'-deoxycytidine (dCyd) and its analogs in the presence of eight nucleoside triphosphates (NTPs), simulating the cellular milieu, was investigated. Using highly purified dCK from MOLT-4 T lymphoblasts, Km and Vmax values were determined for the phosphorylation of dCyd in the presence of cellular concentrations of the eight endogenous NTPs. The results demonstrated that the efficiency of dCyd phosphorylation was greatest in the presence of all eight nucleotides, relative to ATP alone, according to relative Vmax/Km values. UTP was a better phosphate donor than ATP but was less efficient than the NTP mixture. The greater efficacy of the NTP mixture, compared with ATP alone, was due in large part to the presence of UTP, although the results suggested that the presence of other nucleotide(s) also enhanced dCyd phosphorylation. Previous results demonstrated that dCTP was a potent competitive or noncompetitive (with respect to dCyd) inhibitor of dCK, with a Ki value of approximately 1 microM. In contrast, the results presented here demonstrated that, in the presence of either the NTP mixture or UTP, inhibition of dCK was uncompetitive with respect to dCyd, with a Ki value of approximately 60 microM. Furthermore, the results demonstrated that the clinically relevant nucleoside analogs 1-beta-D-arabinofuranosylcytosine, 2',2'-difluoro-2'-deoxycytidine (dFdC), and 9-beta-D-arabinofuranosyl-2-fluoroadenine also preferred UTP or the NTP mixture, compared with ATP alone, as a phosphate donor. Of the three nucleoside analogs tested, dFdC was the most efficient dCK substrate. These data indicate that the preferred phosphate donor for dCK is UTP or a combination of UTP and another nucleotide. Furthermore, the dCTP concentration in intact cells, which is typically 10-20 microM, is not sufficient to cause substantial inhibition of dCK, due to the presence of UTP. Strategies to increase cellular dCK activity should focus on optimizing UTP concentrations.
Shewach
D S
DS
Department of Pharmacology, University of Michigan Medical Center, Ann Arbor 48109.
Reynolds
K K
KK
Hertel
L
L
D000255:Adenosine Triphosphate / Q000378:metabolism; D003841:Deoxycytidine / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000494:pharmacology; D003848:Deoxyguanine Nucleotides / Q000378:metabolism / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000201:enzymology; D014544:Uridine Triphosphate / Q000378:metabolism
D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D009711:Nucleotides; D010710:Phosphates; D003841:Deoxycytidine; C024107:2'-deoxycytidine 5'-triphosphate; D006160:Guanosine Triphosphate; C029603:deoxyguanosine triphosphate; D000255:Adenosine Triphosphate; D003842:Deoxycytidine Kinase; D014544:Uridine Triphosphate