Nucleoside Triphosphate Donors for Nucleoside Kinases: Donor Properties of UTP with Human Deoxycytidine Kinase

• Published in: Biochemical and biophysical research communications Vol. 216; no. 1; pp. 42 - 48
• Main Authors: Krawiec, K., Kierdaszuk, B., Eriksson, S., Munchpetersen, B., Shugar, D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.11.1995
• Subjects: Adenosine Triphosphate - metabolism; Deoxyadenosines - metabolism; Deoxycytidine - metabolism; Deoxycytidine Kinase - metabolism; Humans; Kinetics; Leukemia - enzymology; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Spleen - enzymology; Substrate Specificity; Tritium; Uridine Triphosphate - metabolism
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.1995.2589

The reported higher efficiency of UTP, relative to ATP, as phosphate donor for deoxycytidine kinase (dCK), has been extended and found to apply to both dCyd and dAdo as accepters. UTP as phosphate donor was shown to follow strictly Michaelis kinetics, with Km = 1 μM, in striking contrast tb ATP, which exhibits marked negative cooperativity (Hill coef. = 0.7) with a several-fold higher Kappm = 15 μM. Phosphate transfer was followed directly with use of mixtures of [γ-32P]ATP and cold UTP as donors, or with 3H-labeled accepters and cold donors. With equimolar concentrations of ATP and UTP (50 μM or 1 mM each), and dCyd or dAdo as acceptor, only minimal phosphate transfer occurred from ATP (3-10%). With a 6:1 ratio of ATP:UTP, hence exceeding the intracellular ratio, phosphate transfer from ATP increased, but still did not exceed 25-40% with either dCyd or dAdo as acceptor. Moreover, relative ATP transfer is dependent on the dCyd concentration. We conclude that the major intracellular phosphate donor for dCK is not ATP, but UTP. Preliminary data for human thymidine kinases (TK1 and TK2) exhibit quite different behaviour. The foregoing, together with literature data, are highly relevant to in vitro studies on the properties of the nucleoside kinases, and to the design of chemotherapeutically active nucleoside analogues.