Krawiec K, Kierdaszuk B, Eriksson S, Munch-Petersen B, Shugar D
University of Warsaw, Department of Biophysics, Poland.
Biochem Biophys Res Commun. 1995 Nov 2;216(1):42-8. 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 acceptors. UTP as phosphate donor was shown to follow strictly Michaelis kinetics, with Km = 1 microM, in striking contrast to ATP, which exhibits marked negative cooperativity (Hill coef. = 0.7) with a several-fold higher Kmapp = 15 microM. Phosphate transfer was followed directly with use of mixtures of [gamma-32P]ATP and cold UTP as donors, or with 3H-labeled acceptors and cold donors. With equimolar concentrations of ATP and UTP (50 microM 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.
据报道，相对于ATP，尿苷三磷酸（UTP）作为脱氧胞苷激酶（dCK）的磷酸供体具有更高的效率，这一发现已得到扩展，并发现其适用于脱氧胞苷（dCyd）和脱氧腺苷（dAdo）作为受体的情况。结果表明，UTP作为磷酸供体严格遵循米氏动力学，米氏常数（Km） = 1微摩尔，这与ATP形成鲜明对比，ATP表现出明显的负协同性（希尔系数 = 0.7），表观米氏常数（Kmapp）高出数倍，为15微摩尔。使用[γ-32P]ATP和冷UTP的混合物作为供体，或使用3H标记的受体和冷供体，可直接跟踪磷酸转移。当ATP和UTP浓度相等（各为50微摩尔或1毫摩尔），且dCyd或dAdo作为受体时，ATP的磷酸转移极少（3 - 10%）。当ATP与UTP的比例为6:1，即超过细胞内比例时，以dCyd或dAdo作为受体，ATP的磷酸转移增加，但仍不超过25 - 40%。此外，相对ATP转移取决于dCyd浓度。我们得出结论，dCK在细胞内的主要磷酸供体不是ATP，而是UTP。人胸苷激酶（TK1和TK2）的初步数据显示出截然不同的行为。上述内容以及文献数据，对于核苷激酶性质的体外研究以及化疗活性核苷类似物的设计具有高度相关性。
