Different affinity of the two forms of human cytosolic thymidine kinase towards pyrimidine analogs

Recent results showed that ATP enables a kinetically slow shift from a low affinity form to a high affinity form of human cytosolic thymidine kinase (TK1), as reflected by the respective apparent Km values for thymidine of 15 microM and 0.7 microM. The shift is dependent on the concentration of enzyme protein, and calculations indicate that the low affinity form is predominant in G1 cells, and the high affinity form is predominant in S-phase cells. Here, we report that the two forms of TK1 differ manyfold in affinity to the substrate ATP, to the inhibitor dTTP and to various analogs of thymidine substituted in the pyrimidine or sugar. Furthermore, the kinetic reaction mechanisms suggest that the nucleoside analog. 3'-azidothymidine, used for treatment of infections with human immune deficiency virus (HIV), is not a substrate for the low affinity form of TK1.