Tight binding of deoxyribonucleotide triphosphates to human thymidine kinase 2 expressed in Escherichia coli.: Purification and partial characterization of its dimeric and tetrameric forms
Tight binding of deoxyribonucleotide triphosphates to human thymidine kinase 2 expressed in Escherichia coli.: Purification and partial characterization of its dimeric and tetrameric forms
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DOI：
10.1021/bi035230f
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发表时间：
2003-12-30
期刊：
影响因子：
2.9
通讯作者：
Flatmark, T
中科院分区：
文献类型：
--
作者：
Barroso, JF;Elholm, M;Flatmark, T
人胸苷激酶2（hTK 2）使用核苷酸三磷酸作为磷酸供体，将嘧啶脱氧核糖核苷磷酸化为相应的核苷单磷酸。在这项研究中，hTK 2的克隆和表达在大肠杆菌中的高水平作为融合蛋白与麦芽糖结合蛋白。乙醇诱导热休克反应以及在28 ℃下共表达质粒编码的GroEL/ES伴侣蛋白最大限度地减少了杂合蛋白的非特异性聚集，并改善了正确折叠酶的三种同源寡聚形式的回收，即，二聚体>四聚体>六聚体。二聚体和四聚体在蛋白水解去除融合伴侣后以稳定和高度纯化的形式分离。两种寡聚体都含有亚化学计量的脱氧核糖核苷酸三磷酸（dTTP > dCTP > dATP），已知是酶的强反馈抑制剂。稳态动力学研究与内源性抑制剂的存在是一致的，两种寡聚体形式揭示了至少类似于5分钟的滞后期，其在与底物（dThd或dCyd）预孵育时被消除。两种低聚物形式的相当相似的动力学性质表明基本功能单元是二聚体。分子对接实验与建模hTK 2的三维结构准确预测的天然底物（dThd，dCyd和ATP）和抑制剂（dTTP和dCTP）的活性位点的结合位置，与所有配体获得高度保守的方向。计算出的相对非键相互作用能与生化数据一致，并表明抑制剂复合物具有较低的稳定化能量（较高的亲和力）比基板。
Human thymidine kinase 2 (hTK2) phosphorylates pyrimidine deoxyribonucleosides to the corresponding nucleoside monophosphates, using a nucleotide triphosphate as a phosphate donor. In this study, hTK2 was cloned and expressed at high levels in Escherichia coli as a fusion protein with maltose-binding protein. Induction of a heat-shock response by ethanol and coexpression of plasmid-encoded GroEL/ES chaperonins at 28 degreesC minimized the nonspecific aggregation of the hybrid protein and improved the recovery of three homooligomeric forms of the properly folded enzyme, i.e., dimer > tetramer > hexamer. The dimer and the tetramer were isolated in stable and highly purified forms after proteolytic removal of the fusion partner. Both oligomers contained a substoichiometric amount of deoxyribonucleotide triphosphates (dTTP > dCTP > dATP), known to be strong feedback inhibitors of the enzyme. Steady-state kinetic studies were consistent with the presence of endogenous inhibitors, and both oligomeric forms revealed a lag phase of at least similar to5 min, which was abolished on preincubation with substrate (dThd or dCyd). The rather similar kinetic properties of the two oligomeric forms indicate that the basic functional unit is a dimer. Molecular docking experiments with a modeled hTK2 three-dimensional structure accurately predicted the binding positions at the active site of the natural substrates (dThd, dCyd, and ATP) and inhibitors (dTTP and dCTP), with highly conserved orientations obtained for all ligands. The calculated relative nonbonded interaction energies are in agreement with the biochemical data and show that the inhibitor complexes have lower stabilization energies (higher affinity) than the substrates.