Munch-Petersen B, Cloos L, Jensen H K, Tyrsted G
Institute of Life Sciences and Chemistry Roskilde University, Denmark.
Adv Enzyme Regul. 1995;35:69-89. doi: 10.1016/0065-2571(94)00014-t.
In mammalian cells, salvage pathway phosphorylation of thymidine is catalyzed by two thymidine kinases: the cell-cycle regulated cytoplasmic TK1 and the constitutively expressed mitochondrial TK2. Since TK1 is virtually absent in non-dividing cells, TK2 is probably the only thymidine kinase present in these cells. In cellular metabolism, TK1 and TK2 presumably serve to maintain sufficient dTTP for DNA replication and repair. TK1 purified from phytohemagglutinin-stimulated human lymphocytes is a dimer in the absence and a tetramer in the presence of ATP. In addition to the molecular weight transition, incubation with ATP at 4 degrees C or storage with ATP induces a reversible, enzyme concentration-dependent, kinetically slow transition from a low to a high affinity form of TK1, with Km values of 14 microM and 0.5 microM, respectively. This affinity difference implies that at cellular thymidine concentrations, the difference in catalytic activity between the two TK1 forms will be 3-5-fold. Calculations of cellular TK1 concentration suggested that the low affinity dimer form was dominant in G0/G1 cells and the high affinity tetramer form in S-phase cells. Hence, the transition may serve to fine-tune the cell-cycle regulation of thymidine kinase activity on the post-translational level. To study the ATP effect on the molecular level, an IPTG inducible T7 RNA polymerase-dependent expression system for the entire human TK1 polypeptide in E. coli was established. The recombinant TK1 has the same subunit mass and specific activity as the native enzyme. However, the recombinant TK1 solely displayed the kinetics of the high affinity form, with Km values of 0.3-0.4 microM regardless of pre-exposure to ATP, indicating that the ATP effect may be dependent on post-translational modifications absent in E. coli. Surprisingly, we did not observe any effect of ATP on TK1 purified from bone-marrow cells from a patient with acute monocytic leukemia (AMOL). Furthermore, the Km values of TK1 from these cells were 45 microM for the ATP-free enzyme and 65 microM for the ATP-incubated enzyme. With TK1 purified from HL-60 cells, we obtained the same pattern and kinetic values as for TK1 from lymphocytes. In the light of the results with the recombinant TK1, we presume that the lack of ATP effect and very high Km values observed for the AMOL TK1 may be due to changes in post-translational regulatory mechanisms in acute monocytic cells.
在哺乳动物细胞中，胸苷的补救途径磷酸化由两种胸苷激酶催化：细胞周期调控的细胞质TK1和组成性表达的线粒体TK2。由于TK1在非分裂细胞中几乎不存在，TK2可能是这些细胞中唯一存在的胸苷激酶。在细胞代谢中，TK1和TK2可能用于维持足够的dTTP用于DNA复制和修复。从植物血凝素刺激的人淋巴细胞中纯化的TK1在不存在ATP时是二聚体，在存在ATP时是四聚体。除了分子量转变外，在4℃下与ATP孵育或与ATP一起储存会诱导TK1从低亲和力形式到高亲和力形式的可逆的、酶浓度依赖性的、动力学缓慢的转变，其Km值分别为14μM和0.5μM。这种亲和力差异意味着在细胞胸苷浓度下，两种TK1形式之间的催化活性差异将为3至5倍。细胞TK1浓度的计算表明，低亲和力二聚体形式在G0/G1期细胞中占主导，高亲和力四聚体形式在S期细胞中占主导。因此，这种转变可能有助于在翻译后水平上微调胸苷激酶活性的细胞周期调控。为了在分子水平上研究ATP的作用，在大肠杆菌中建立了用于整个人TK1多肽的IPTG诱导的T7 RNA聚合酶依赖性表达系统。重组TK1具有与天然酶相同的亚基质量和比活性。然而，重组TK1仅表现出高亲和力形式的动力学，无论是否预先暴露于ATP，其Km值均为0.3 - 0.4μM，这表明ATP的作用可能取决于大肠杆菌中不存在的翻译后修饰。令人惊讶的是，我们没有观察到ATP对从急性单核细胞白血病（AMOL）患者的骨髓细胞中纯化的TK1有任何影响。此外，这些细胞中TK1的Km值对于无ATP的酶为45μM，对于与ATP孵育的酶为65μM。对于从HL - 60细胞中纯化的TK1，我们获得了与淋巴细胞中TK1相同的模式和动力学值。根据重组TK1的结果，我们推测在急性单核细胞中观察到的AMOL TK1缺乏ATP效应和非常高的Km值可能是由于翻译后调控机制的变化。
