Department of Chemistry, Stanford University, Stanford, California 94305, USA.
J Am Chem Soc. 2009 Apr 22;131(15):5488-94. doi: 10.1021/ja808244t.
We describe the use of nonpolar nucleoside analogues of systematically varied size and shape to probe the mechanisms by which the two human thymidine kinases (TK1 and TK2) recognize and phosphorylate their substrate, thymidine. Comparison of polar thymidine with a nonpolar isostere, 2,4-difluorotoluene deoxyriboside, as substrates for the two enzymes establishes that TK1 requires electrostatic complementarity to recognize the thymine base with high efficiency. Conversely, TK2 does not and phosphorylates the hydrophobic shape mimic with efficiency nearly the same as the natural substrate. To test the response to nucleobase size, thymidine-like analogues were systematically varied by replacing the 2,4 substituents on toluene with hydrogen and the halogen series (H, F, Cl, Br, I). Both enzymes showed a distinct preference for substrates having the natural size. To examine the shape preference, we prepared four mono- and difluorotoluene deoxyribosides with varying positions of substitutions. While TK1 did not accept these nonpolar analogues as substrates, TK2 did show varying levels of phosphorylation of the shape-varied set. This latter enzyme preferred toluene nucleoside analogues having steric projections at the 2 and 4 positions, as is found in thymine, and strongly disfavored substitution at the 3-position. Steady-state kinetics measurements showed that the 4-fluoro compound (7) had an apparent V(max)/K(m) value within 14-fold of the natural substrate, and the 2,4-difluoro compound (1), which is the closest isostere of thymidine, had a value within 2.5-fold. The results establish that nucleoside recognition mechanisms for the two classes of enzymes are very different. On the basis of these data, nonpolar nucleosides are likely to be active in the nucleotide salvage pathway in human cells, suggesting new designs for future bioactive molecules.
我们描述了使用大小和形状系统变化的非极性核苷类似物来探测两种人类胸苷激酶（TK1 和 TK2）识别和磷酸化其底物胸苷的机制。将极性胸苷与非极性等排体 2,4-二氟甲苯脱氧核苷进行比较，作为两种酶的底物，确定 TK1 需要静电互补才能高效识别胸腺嘧啶碱基。相反，TK2 不需要，并且以与天然底物几乎相同的效率磷酸化疏水性形状模拟物。为了测试对核苷碱基大小的反应，我们系统地改变了类似于胸腺嘧啶的类似物，通过将甲苯上的 2,4 取代基用氢和卤素系列（H、F、Cl、Br、I）取代。两种酶都明显偏爱具有天然大小的底物。为了检查形状偏好，我们制备了四个具有不同取代位置的单氟和二氟甲苯脱氧核苷。虽然 TK1 不能接受这些非极性类似物作为底物，但 TK2 确实表现出对形状变化集的不同程度的磷酸化。后一种酶更喜欢在 2 和 4 位具有空间突出的甲苯核苷类似物，如胸腺嘧啶中存在的那样，并强烈不喜欢在 3 位取代。稳态动力学测量表明，4-氟化合物（7）的表观 V(max)/K(m)值与天然底物相差约 14 倍，而 2,4-二氟化合物（1）是胸腺嘧啶的最接近的等排体，其值相差约 2.5 倍。结果表明，两种酶类的核苷识别机制非常不同。基于这些数据，非极性核苷很可能在人类细胞的核苷酸补救途径中具有活性，这为未来的生物活性分子设计提供了新的思路。
