Mani Rajam S, Usova Elena V, Eriksson Staffan, Cass Carol E
Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada.
Nucleosides Nucleotides Nucleic Acids. 2003 Feb;22(2):175-92. doi: 10.1081/NCN-120019513.
Deoxycytidine kinase (dCK), a cytosolic enzyme with broad substrate specificity, plays a key role in the activation of therapeutic nucleoside analogues by their 5'-phosphorylation. The structure of human dCK is still not known and the current work was undertaken to determine its oligomeric and secondary structure. Biophysical studies were conducted with purified recombinant human dCK. The Mr determined by low-speed sedimentation equilibrium under nondenaturing conditions was 60,250 +/- 1,000, indicating that dCK, which has a predicted Mr of 30,500, exists in solution as a dimer. Analysis of circular dichroism spectra revealed the presence of two negative dichroic bands located at 222 and 209 nm with ellipticity values of -11,900 +/- 300 and -12,500 +/- 300 deg x cm2 x dmol(-1), respectively, indicating the presence of approximately 40% alpha-helix and 50% beta-structure. Circular Dichroism studies in the aromatic and far-ultraviolet range and UV difference spectroscopy indicated that binding of substrates to dCK reduced its alpha-helical content and perturbed tryptophan and tyrosine. Steady-state fluorescence demonstrated that deoxycytidine (the phosphate acceptor) and ATP (the phosphate donor) bound to different sites on dCK and fluorescence quenching revealed bimodal binding of deoxycytidine and unimodal binding of ATP. Spectroscopic studies indicated that substrate binding induced conformational changes, with the result that dCK exhibited different affinities for various substrates. These results are consistent with a random bi-bi kinetic mechanism of phosphorylation of dCyd with either ATP or UTP.
脱氧胞苷激酶（dCK）是一种具有广泛底物特异性的胞质酶，通过对治疗性核苷类似物进行5'-磷酸化在其激活过程中起关键作用。人dCK的结构尚不清楚，目前开展的这项工作旨在确定其寡聚体结构和二级结构。使用纯化的重组人dCK进行了生物物理研究。在非变性条件下通过低速沉降平衡测定的相对分子质量为60,250±1,000，这表明预测相对分子质量为30,500的dCK在溶液中以二聚体形式存在。圆二色光谱分析显示在222和209 nm处存在两个负二色性带，椭圆率值分别为-11,900±300和-12,500±300 deg·cm²·dmol⁻¹，表明存在约40%的α-螺旋和50%的β-结构。在芳香族和远紫外区域的圆二色性研究以及紫外差光谱表明，底物与dCK的结合降低了其α-螺旋含量，并干扰了色氨酸和酪氨酸。稳态荧光表明脱氧胞苷（磷酸受体）和ATP（磷酸供体）结合到dCK的不同位点，荧光猝灭显示脱氧胞苷为双峰结合而ATP为单峰结合。光谱研究表明底物结合诱导了构象变化，结果dCK对各种底物表现出不同的亲和力。这些结果与dCyd与ATP或UTP磷酸化的随机双底物双产物动力学机制一致。
