Université Paris Sud-CNRS, UMR 8000, bâtiments 201 P2 and 350, 91405 Orsay, France.
Univ. Grenoble Alpes, CEA, CNRS, IBS, 38000 Grenoble, France.
J Struct Biol. 2020 May 1;210(2):107478. doi: 10.1016/j.jsb.2020.107478. Epub 2020 Feb 19.
L-Lactate dehydrogenase (LDH) is a model protein allowing to shed light on the fundamental molecular mechanisms that drive the acquisition, evolution and regulation of enzyme properties. In this study, we test the hypothesis of a link between thermal stability of LDHs and their capacity against unfolding induced by reactive oxygen species (ROS) generated by γ-rays irradiation. By using circular dichroism spectroscopy, we analysed that high thermal stability of a thermophilic LDH favours strong resistance against ROS-induced unfolding, in contrast to its psychrophilic and mesophilic counterparts that are less resistant. We suggest that a protein's phenotype linking strong thermal stability and resistance against ROS damages would have been a selective evolutionary advantage. We also find that the enzymatic activity of the thermophilic LDH that is strongly resistant against ROS-unfolding is very sensitive to inactivation by irradiation. To address this counter-intuitive observation, we combined mass spectrometry analyses and enzymatic activity measurements. We demonstrate that the dramatic change on LDH activity was linked to remote chemical modifications away from the active site, that change the equilibrium between low-affinity tense (T-inactive) and high-affinity relaxed (R-active) forms. We found the T-inactive thermophilic enzyme obtained after irradiation can recover its LDH activity by addition of the allosteric effector 1, 6 fructose bis phosphate. We analyse our data within the general framework of allosteric regulation, which requires that an enzyme in solution populates a large diversity of dynamically-interchanging conformations. Our work demonstrates that the radiation-induced inactivation of an enzyme is controlled by its dynamical properties.
L-乳酸脱氢酶（LDH）是一种模型蛋白，可以帮助我们深入了解驱动酶特性获得、进化和调节的基本分子机制。在这项研究中，我们检验了 LDH 的热稳定性与其对由γ射线辐照产生的活性氧物种（ROS）诱导的解折叠能力之间存在关联的假设。我们使用圆二色性光谱分析，发现嗜热 LDH 的高热稳定性有利于其强烈抵抗 ROS 诱导的解折叠，而其嗜冷和中温对应物的抵抗能力较弱。我们认为，将蛋白质的热稳定性与对 ROS 损伤的抵抗力联系起来的表型可能是一种选择性进化优势。我们还发现，对 ROS 解折叠具有很强抵抗力的嗜热 LDH 的酶活性对辐照失活非常敏感。为了解决这个违反直觉的观察结果，我们结合了质谱分析和酶活性测量。我们证明，LDH 活性的巨大变化与远离活性部位的远程化学修饰有关，这些修饰改变了低亲和力紧张（T-无活性）和高亲和力松弛（R-活性）形式之间的平衡。我们发现，辐照后获得的 T-无活性嗜热酶可以通过添加别构效应物 1,6 果糖二磷酸恢复其 LDH 活性。我们在变构调节的一般框架内分析我们的数据，该框架要求溶液中的酶具有很大的多样性，能够动态地相互转换构象。我们的工作表明，酶的辐射失活受其动力学特性的控制。
