Inter-Faculty Interdisciplinary Doctoral Studies in Natural Sciences and Mathematics, MISMaP College, University of Warsaw, ul. Banacha 2C, 02-097 Warsaw, Poland.
Institute of Experimental Physics, Faculty of Physics, University of Warsaw, ul. Pasteura 5, 02-093 Warsaw, Poland.
Int J Mol Sci. 2021 Mar 6;22(5):2661. doi: 10.3390/ijms22052661.
A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson-Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme-ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the "abortive reaction" inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the rotamer of the imino inhibitor form.
一种同源二聚酶，胸苷酸合酶（TS），长期以来一直是化疗中的分子靶标。为了进一步阐明野生型小鼠胸苷酸合酶（mTS）及其两个单突变体 H190A 和 W103G 的性质和与配体的相互作用，采用了光谱和理论研究。在这些突变体中，位于 190 位的组氨酸和位于 103 位的色氨酸分别被丙氨酸和甘氨酸取代。本文中使用的几种基于发射的光谱方法证明色氨酸 103 在 TS 配体结合中起着特别重要的作用。此外，高级泊松-玻尔兹曼求解器（APBS）的结果表明，与 mTS 家族结构中所有剩余色氨酸残基观察到的分布相比，色氨酸 103 周围的静电势分布存在相当大的差异。光谱和 APBS 结果共同揭示了色氨酸 103 和组氨酸 190 之间可能存在相互作用，这导致 H190A 突变时酶活性降低。与底物 dUMP 和抑制剂 FdUMP 和 N4-OH-dCMP 相比，mTS 复合物及其突变体的静电势比较表明，与 dUMP-mTS 和 FdUMP-mTS 复合物相比，它对 N4OH-dCMP-mTS 中酶-配体相互作用的影响较弱。这种差异对于解释 N4OH-dCMP 对 TS 的“无效反应”抑制机制可能至关重要。此外，基于结构分析和 H190A 突变体在 mTHF 依赖性反应中与 N4-OH-dCMP 形成抗变性复合物的能力，组氨酸 190 显然负责酶活性中心对亚氨基抑制剂形式的构象的强烈偏好。
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