tailieunhanh - Báo cáo khoa học: 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 deoxy-ribonucleotide metabolism, catalyzing the first step in the synthesis of dTTP by transfer of ac-phosphate group from a nucleoside triphosphate to the 5¢-hydroxyl group of thymidine, forming dTMP. | ỊFEBS Journal Reversible tetramerization of human TK1 to the high catalytic efficient form is induced by pyrophosphate in addition to tripolyphosphates or high enzyme concentration Birgitte Munch-Petersen Department of Science Systems and Models Roskilde University Denmark Keywords ATP gel filtration kinetics tetramerization thymidine kinase Correspondence B. Munch-Petersen Department of Science Systems and Models Universitetsvej1 Building Roskilde University DK-4000 Roskilde Denmark Fax 45 4674 3011 Tel 45 4674 2419 E-mail bmp@ Website http nsm Received 5 August 2008 revised 5 November 2008 accepted 17 November 2008 doi 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 y-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 G1 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. For decades it has been the general belief that the building