Catechol-O-methyltransferase (COMT): Resolving the mechanism of an archetypical methyl transferase with new experimental tools

使诸如多巴胺、左旋多巴和（去甲）肾上腺素的儿茶酚胺转移酶失活的主要手段通过称为儿茶酚-O-甲基转移酶（COMT）的酶的作用发生。因此，抑制COMT是治疗包括帕金森病、抑郁症和精神分裂症在内的一系列神经系统疾病的策略。除了作为药物靶标外，COMT还具有作为有用的生物催化剂的潜力，可以产生有价值的化学物质，如香草醛，香草豆提取物的主要成分。显然，详细了解COMT如何工作对一系列部门都有价值。然而，尽管有超过60年的研究，仍然有重大分歧的基本方面的物理机制的甲基转移酶，如COMT催化的反应。这项工作的重点是使用新的实验工具- NMR和光谱学，X射线晶体学-以及紧密集成的计算化学程序来研究COMT，重点是这种酶催化的物理机制。这项工作将利用我们最近的工作，产生了COMT的第一个NMR主链分配，这是必要的NMR数据映射到新的和/或现有的X射线晶体结构数据。我们将扩展这种方法来研究COMT如何结合一系列生理底物，如多巴胺。通过使用我们的NMR实验作为筛选方法，我们期望优化样品条件，以便我们可以解决COMT与结合的儿茶酚胺的第一个X射线晶体结构，从而更好地描述神经递质与COMT的结合。我们将扩展我们以前的NMR工作，以进行技术要求更高的高压和弛豫NMR实验，直接探测蛋白质的灵活性和动力学。除了光学荧光和振动实验，这些实验将允许识别任何特定的动态特征-氨基酸骨架和/或侧链运动-这可能有助于催化。将通过改变感兴趣的氨基酸（通过定点诱变）和通过比较缺乏感兴趣的氨基酸的野生型和突变酶的活性（酶周转）来研究这种行为的意义。这种方法允许直接评估COMT催化的动力学特征-这是更广泛的酶学社区中的一个主要争议领域。该提案的一个主要特点是将计算模拟与实验工作紧密结合，这将在曼彻斯特和亚利桑那大学的合作者进行。实验旨在提供可直接用作计算计算输入的数据。计算和模拟将根据实验数据直接进行测试/基准测试，以允许对计算参数进行连续改进，并指导后续实验的设计，例如在鉴定可能在催化中发挥主要作用的氨基酸时。总之，这种方法将允许新的见解COMT -并通过扩展，相关酶-工作。除了解决该领域长期存在的问题外，这项工作还可能导致旨在抑制COMT和相关酶的药物的改进，将提供基础数据，有助于合理（重新）设计用于生物催化的相关甲基转移酶应用，并将提供新的方法来解决广泛的其他酶中的类似机制问题。

The primary means of deactivation of catecholamine neutrotransmeters such as dopamine, levodopa and (nor)epinephrine occurs through the action of an enzyme called catechol-O-methyltransferase (COMT). Consequently, inhibition of COMT is a strategy for the treatment of a range of neurological disorders including Parkinson's disease, depression and schizophrenia. In addition to being a drug target, COMT has potential as a useful biocatalyst to create valuable chemicals such as vanillin, the primary component of the extract of the vanilla bean. Clearly a detailed understanding of how COMT works would be of value to a range of sectors. However, despite more than 60 years of study, there are still major disagreements as to fundamental aspects of the physical mechanism of the reactions catalysed by methyl transferases such as COMT. The focus of this work is to study COMT using new experimental tools - NMR and optical spectroscopies, X-ray crystallography - alongside a tightly integrated computational chemistry programme, with a focus on the physical mechanism of catalysis by this enzyme. This work will capitalise on our recent work, which produced the first NMR backbone assignments of COMT, which is necessary to map NMR data onto new and/or existing X-ray crystal structure data. We will extend this approach to study how COMT binds a range of physiological substrates such as dopamine. By using our NMR experiments as a screening method, we expect to optimise sample conditions so we can solve the first X-ray crystal structures of COMT with bound catecholamines, allowing better description of neurotransmitter binding to COMT. We will expand on our previous NMR work to perform more technically demanding high pressure and relaxation NMR experiments that directly probe protein flexibility and dynamics. Alongside optical fluorescence and vibrational experiments, these experiments will allow the identification of any specific dynamic features - amino acid backbone and/or side chain motions - that may contribute to catalysis. The significance of such behaviour will be investigated by altering amino acids of interesting (by site-directed mutagenesis) and by comparing the activity (enzyme turnover) of wild-type and mutant enzymes lacking amino acids of interest. This approach allows the direct assessment of dynamical features to COMT catalysis - a major area of controversy in the wider enzymology community.A major feature of the proposal is the close integration of computational simulations with experimental work, which will be performed both in Manchester and by collaborators at the University of Arizona. Experiments are designed to provide data that can be directly used as inputs to computational calculations. Calculations and simulations will be directly tested/benchmarked against experimental data to allow successive improvements to computational parameters and to guide the design of subsequent experiments, e.g. in the identification of amino acids that may play a major role in catalysis. Together, this approach will allow new insight into how COMT - and by extension, related enzymes - works. In addition to resolving long-standing questions in the field, this work could lead to improvements in drugs designed to inhibit COMT and related enzymes, will provide foundational data that will aid in the rational (re)design of related methyl transfer enzymes for use in biocatalysis applications, and will provide new methodology to tackle similar mechanistic questions in a wide range of other enzymes.