Exploiting the distinctive catalysis of chemically modified enzymes

酶是大自然用来加速化学反应的催化剂。由于酶是在室温下在水中操作的非常有效的催化剂，因此它们在药物分子等化学品的“绿色”制造中具有巨大的潜力。然而，在化学合成中应用的一个主要限制是酶对特定的起始材料具有高度选择性，并且并不总是可能找到适合于特定应用的自然进化的酶。我们建议修饰一种特定的酶，使其可以催化更广泛的有用的化学转化。酶是由20个氨基酸组成的蛋白质。从本质上讲，蛋白质中的氨基酸序列类似于项链中的彩色珠子序列。虽然自然界利用了一些非常有用的氨基酸构建模块，但酶的功能仍然受到通常用于构建蛋白质的氨基酸只有20个的事实的限制。在这里，我们建议利用新的方法来制备具有更广泛的氨基酸结构单元的蛋白质。我们已经证明了将这些新的氨基酸（称为非规范氨基酸）定位在酶的整个活性位点的能力，并且可以通过使用非规范氨基酸以使用“天然”构建块无法实现的方式改变酶的特异性。在这个提议中，我们将首先通过将新氨基酸定位在一系列位置来扩大酶的特异性范围，然后使用已建立的测定法，我们将发现只有当新氨基酸存在时才能发生的新反应。在工作的第二部分，我们将开发新的测定方法，使我们能够进一步扩展可能性。首先，我们将利用核磁共振（NMR）跟踪使用标记底物的反应，然后我们将利用药物发现领域的NMR技术（称为基于片段的药物设计）来寻找更广泛的底物，当我们在酶中有非典型氨基酸时。最后，也是最令人兴奋的是，插入“非天然”氨基酸的能力也为构建全新的酶化学开辟了道路--可能使用插入的"非天然“氨基酸之一，但不可能使用20种天然氨基酸中的任何一种。为此，我们从一个称为有机催化的化学领域获得灵感。在这里，相对简单的有机分子可以催化反应，这一领域与酶的特异性和催化能力的结合将被用来产生具有巨大潜力的酶，用于一系列重要化学品的新的，更绿色的路线。我们将发现的修饰酶将能够催化目前酶无法催化的反应。这种酶在复杂的生物活性分子如药物的“绿色”合成中具有巨大的价值。

Enzymes are the catalysts that nature uses to accelerate chemical reactions. Because enzymes are spectacularly efficient catalysts that operate in water at room temperature, they have tremendous potential to be exploited in the 'green' manufacture of chemicals such as drug molecules. However, a major limitation for application in chemical synthesis is that enzymes are highly selective for a particular starting material, and it is not always possible to find an enzyme that nature has evolved that is suitable for a specific application. We propose to modify a specific enzyme such that it may be exploited in the catalysis of a much wider range of useful chemical transformations.Enzymes are proteins that are constructed from 20 amino acid building blocks. Essentially, the sequence of amino acids in a protein is analogous to a sequence of coloured beads in a necklace. Although nature exploits some very useful amino acid building blocks, the functions of enzymes are nonetheless limited by the fact that there are only 20 amino acids that are usually used to construct proteins. Here, we propose to exploit new methods to prepare proteins with a much wider range of amino acid building blocks. We have demonstrated the ability to position these new amino acids, termed non-canonical amino acids, throughout the active site of an enzyme and that the specificity of the enzyme may be changed by using the non-canonical amino acids in ways that cannot be achieved using the 'natural' building blocks. In this proposal we will first expand the range of specificity of the enzyme by positioning the new amino acids at a range of positions and then using an established assay we will find new reactions that can occur only when the new amino acid is present. In the second part of the work we will develop new assays to allow us to extend the possibilities even further. Firstly we will make use of nuclear magnetic resonance (NMR) to follow the reaction using labelled substrates, and then we will make use of NMR techniques from the realm of drug discovery (named fragment-based drug design) to search for a wider range of substrates used when we have non-canonical amino acids in the enzyme. We will also extend the chemical method for incorporation of the non-canonical amino acid.Finally, and most excitingly, the ability to insert 'unnatural' amino acid building blocks also opens the way to building completely novel enzyme chemistries - possible using one of the inserted 'unnatural' amino acids, but not possible using any of the 20 natural amino acids. For this we have taken inspiration from a field of chemistry termed organocatalysis. Here relatively simple organic molecules can catalyse reactions, and the coupling of this area with the specificity and catalytic power of enzymes will be used to generate enzymes with huge potential for new, greener routes to a range of important chemicals.The modified enzymes that we will discover will be able to catalyse reactions for which enzymes are not currently available. Such enzymes would have tremendous value in the 'green' synthesis of complex biologically active molecules such as drugs.