Internal dynamics in the enzyme barnase

芽孢杆菌RNA酶的内部动力学

• 批准号: BB/J014966/1
• 负责人: Michael Williamson
• 金额: $ 51.57万
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FJ014966%2F1
• 关键词: Internal; dynamics; enzyme; barnase

Enzymes are the catalysts that carry out all of the reactions in nature. We have known for over 100 years that the structure of an enzyme has to be matched closely to the structures of the molecules that are reacting ('substrates'), and X-ray and NMR structures have shown how this is achieved in detail for many enzymes (the lock and key hypothesis). However, our attempts to design new enzymes have so far been rather pathetic in comparison with the impressive catalytic ability of real enzymes. The best rationally designed enzymes are at least a million times slower than the real thing. Partly this is because the structure has to be very accurately correct. However, another reason, which we are only just beginning to come to grips with, is that an enzyme is not just a static framework, but it moves constantly, mainly as a result of continual bombardment by solvent molecules. This provides it with a lot of kinetic energy, and it appears that somehow this random thermal kinetic energy is channeled into a few very specific motions in order to help the enzyme perform its catalysis. One of the main ways in which this is achieved is that the 'normal' or resting state of an enzyme is an 'open' state, in which the active site (where the reaction occurs) is not in its optimum configuration. Motion within the enzyme very specifically closes the active site, and is precisely tuned so that only a few percent of enzyme molecules are in this active or 'closed' state at any one time. The substrates bind more tightly to the closed state than the open one, and therefore the presence of substrate pulls almost all of the enzyme molecules over into the more active closed state. This model is a refinement of the induced fit hypothesis, and is called conformational selection. It is not clear why enzymes need to do this. In some cases it is because the substrate cannot get into the closed state, but the more general reason may be that evolution does not want the enzyme to be active unless there are substrates bound, to avoid unwanted reactions.This proposal aims to understand these motions for a model enzyme called barnase, which digests RNA. We have shown that in barnase there are two different motions required to make the closed state. One of these is a simple bending of the enzyme, like a hinge closing, and is a low-energy and common motion. The other requires several loops around the active site to close up together, rather like the fingers of a hand closing, and cannot occur efficiently unless the hinge is closed already. We have good evidence that this happens, but we need more details in order to understand it properly: we need to know rates, energies and structures, and how these motions are determined by the structure of barnase. Exactly what does it do and how does it do it? The first motion is easy to understand, but the second is not. Once we have understood it, we also want to explain it in ways that everyone can understand.This is important, because until we understand how enzymes really work, we are to a large extent groping around in the dark, and we are unlikely to be able to build an enzyme that works well. Many scientists think that because we know the structural details of enzymes, we understand them already. This is sadly not true. Science has shown that real progress comes from a proper understanding of the problem, which is what this research aims to produce.

酶是进行自然界所有反应的催化剂。100多年来，我们已经知道酶的结构必须与反应分子（“底物”）的结构紧密匹配，x射线和核磁共振结构已经详细展示了许多酶是如何实现这一目标的（锁和钥匙假说）。然而，到目前为止，与真正的酶令人印象深刻的催化能力相比，我们设计新酶的尝试相当可怜。最好的合理设计的酶比真正的酶慢至少一百万倍。部分原因是结构必须非常精确。然而，另一个我们才刚刚开始了解的原因是，酶不仅仅是一个静态的框架，它还在不断地运动，主要是由于溶剂分子的持续轰击。这就为酶提供了大量的动能，似乎这些随机的热动能被引导到一些非常具体的运动中来帮助酶完成它的催化作用。实现这一目标的主要方法之一是酶的“正常”或静息状态是“开放”状态，在这种状态下，活性位点（反应发生的地方）不在其最佳配置中。酶内部的运动非常特别地关闭了活性位点，并且被精确地调整，因此在任何时候只有少数酶分子处于这种活性或“关闭”状态。底物在封闭状态下比在开放状态下结合得更紧密，因此底物的存在将几乎所有的酶分子拉到更活跃的封闭状态。这个模型是对诱导拟合假说的改进，称为构象选择。目前还不清楚为什么酶需要这样做。在某些情况下，这是因为底物不能进入封闭状态，但更普遍的原因可能是进化不希望酶活跃，除非有底物结合，以避免不必要的反应。这项提议旨在了解一种被称为藤蔓酶的模型酶的这些运动，这种酶可以消化RNA。我们已经证明，在藤壶中，有两种不同的运动需要形成闭合状态。其中一种是酶的简单弯曲，就像铰链关闭一样，这是一种低能量和常见的运动。另一种方法需要活性位点周围的几个环闭合在一起，就像手的手指闭合一样，除非铰链已经闭合，否则无法有效地发生。我们有很好的证据证明这种情况会发生，但我们需要更多的细节来正确地理解它：我们需要知道速率、能量和结构，以及这些运动是如何由藤本酶的结构决定的。它到底是做什么的，又是怎么做的？第一个动作很容易理解，第二个动作就不容易理解了。一旦我们理解了它，我们也想用每个人都能理解的方式来解释它。这很重要，因为在我们了解酶的真正工作原理之前，我们在很大程度上是在黑暗中摸索，我们不太可能制造出一种功能良好的酶。许多科学家认为，因为我们知道酶的结构细节，我们已经了解它们了。遗憾的是，事实并非如此。科学表明，真正的进步来自对问题的正确理解，而这正是本研究的目的。

项目成果

Pressure-Dependent Chemical Shifts in the R3 Domain of Talin Show that It Is Thermodynamically Poised for Binding to Either Vinculin or RIAM

• DOI: 10.1016/j.str.2017.10.008
• 发表时间: 2017-12-05
• 期刊: STRUCTURE
• 影响因子: 5.7
• 作者: Baxter, Nicola J.;Zacharchenko, Thomas;Williamson, Mike P.
• 通讯作者: Williamson, Mike P.