title: Characterization of thymidine kinase and phosphorylation of deoxyribonucleosides in Chlamydomonas reinhardti.
abstract: Using gel filtration chromatography, we find a single peak of deoxythymidine phosphorylating activity in Chlamydomonas reinhardti. This activity has characteristics of a thymidine kinase, in that (1) it will utilize ATP (or dATP) or CTP (or dCTP) as phosphoryl donor, but not AMP or phenyl phosphate, and (2) it is inhibited by dTTP (and less so by dTDP, dUTP, and dUDP) but is unaffected by 3'-5' cyclic AMP. Partially purified chlamydomonas thymidine kinase has a pH optimum near 8.5, and a molecular weight of 80,000 to 85,000 daltons. Kinetic studies indicate a ping-pong mechanism with a Km for thymidine of 1.5 x 10(-7) moles per liter. 5-Bromo- and 5-fluorodeoxyuridine, and to a lesser degree deoxyuridine, are competitive inhibitors, but significant phosphorylation of these nucleotides could not be demonstrated in vitro by thymidine kinase. While thymidine is phosphorylated to dTMP by crude Chlamydomonas extracts, greater than 80% of the product formed by the partially purified enzyme is dTTP. Further, the gel filtration elution position of the single deoxythymidylate kinase activity present in cell extracts coincides with that of thymidine kinase. These results suggest that a multifunctional enzyme, rather than three separate phosphorylating activities, may be responsible for dTTP formation.
mesh_terms: D000255:Adenosine Triphosphate; D002696:Chlamydomonas / Q000201:enzymology*; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013936:Thymidine; D013937:Thymidine Kinase / Q000378:metabolism*
chemical_list: D000255:Adenosine Triphosphate; D013937:Thymidine Kinase; D013936:Thymidine