Swinton D C, Chiang K S
Mol Gen Genet. 1979 Nov;176(3):399-409. doi: 10.1007/BF00333104.
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.
利用凝胶过滤色谱法，我们在莱茵衣藻中发现了一个脱氧胸苷磷酸化活性的单一峰。这种活性具有胸苷激酶的特征，即：（1）它可以利用ATP（或dATP）或CTP（或dCTP）作为磷酸供体，但不能利用AMP或苯基磷酸；（2）它受到dTTP（受dTDP、dUTP和dUDP的抑制作用较小）的抑制，但不受3'-5'环化AMP的影响。部分纯化的衣藻胸苷激酶的最适pH接近8.5，分子量为80,000至85,000道尔顿。动力学研究表明其反应机制为乒乓机制，胸苷的Km为每升1.5×10⁻⁷摩尔。5-溴脱氧尿苷和5-氟脱氧尿苷，以及程度较轻的脱氧尿苷，是竞争性抑制剂，但胸苷激酶在体外无法证明这些核苷酸有显著的磷酸化作用。虽然粗制的衣藻提取物可将胸苷磷酸化为dTMP，但部分纯化的酶形成的产物中超过80%是dTTP。此外，细胞提取物中存在的单一脱氧胸苷酸激酶活性的凝胶过滤洗脱位置与胸苷激酶的洗脱位置一致。这些结果表明，可能是一种多功能酶，而非三种独立的磷酸化活性，负责dTTP的形成。
