Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase
Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase
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DOI：
10.1002/anie.201503968
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发表时间：
2015-07-27
影响因子：
16.6
通讯作者：
Allemann, Rudolf K.
中科院分区：
文献类型：
--
作者：
Luk, Louis Y. P.;Ruiz-Pernia, J. Javier;Allemann, Rudolf K.
化学连接已被用于改变大肠杆菌二氢叶酸还原酶特定区域的运动，并研究局部运动变化对酶催化的影响。制备了两种同位素杂合体;一种具有含有重同位素（H-2、C-13、N-15）的移动的N-末端区段和具有天然同位素丰度的蛋白质的其余部分，另一种仅具有同位素标记的C-末端区段。动力学研究表明，同位素取代的N-末端段只影响一个物理步骤的催化，而酶化学的影响，从C-末端段的蛋白质运动。QM/MM研究支持的想法，催化动力学效应主要来自C-末端片段。同位素杂交的使用提供了深入了解动态耦合的微观机制，这是很难获得与其他研究，并有助于定义的动态网络的分子内相互作用的酶催化。
Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E.coli and to investigate the effects of localized motional changes on enzyme catalysis. Two isotopic hybrids were prepared; one with the mobile N-terminal segment containing heavy isotopes (H-2, C-13, N-15) and the remainder of the protein with natural isotopic abundance, and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only a physical step of catalysis, whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling, which is difficult to obtain with other studies, and helps define the dynamic networks of intramolecular interactions central to enzyme catalysis.