Reversible tetramerization of human TK1 to the high catalytic efficient form is induced by pyrophosphate, in addition to tripolyphosphates, or high enzyme concentration.
Thymidine kinase (TK1) is a key enzyme in the salvage pathway of deoxyribonucleotide metabolism, catalyzing the first step in the synthesis of dTTP by transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5'-hydroxyl group of thymidine, forming dTMP. Human TK1 is cytosolic and its activity is absent in resting cells, appears in late G(1), increases in S phase coinciding with the increase in DNA synthesis, and disappears during mitosis. The fluctuation of TK1 through the cell cycle is important in providing a balanced supply of dTTP for DNA replication, and is partly due to regulation of TK1 expression at the transcriptional level. However, TK1 is a regulatory enzyme that can interchange between its dimeric and tetrameric forms, which have low and high catalytic efficiencies, respectively, depending on pre-assay incubation with ATP. Here, the part of ATP that is necessary for tetramerization and how the reaction velocity is influenced by the enzyme concentration are determined. The results show that only two or three of the phosphate groups of ATP are necessary for tetramerization, and that kinetics and tetramerization are closely related. Furthermore, the enzyme concentration was found to have a pivotal effect on catalytic efficiency.