Enzyme Dynamics During Catalysis

催化过程中的酶动力学

• 批准号: 7118705
• 负责人: DOROTHEE KERN
• 金额: $ 30.39万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118705
• 关键词: Archaea; Escherichia coli; active sites; adenylate kinase; chemical kinetics; conformation; enzyme activity; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate complex; gene mutation; molecular dynamics; nuclear magnetic resonance spectroscopy; peptidylprolyl isomerase; protein sequence; protein structure function; thermodynamics

DESCRIPTION (provided by applicant): A complete understanding of enzyme action requires a detailed knowledge about structure, dynamics and function. Although protein motions are the basis for enzyme function, this is the part of enzymology which is understood the least. Classically, enzyme reactions are studied by detecting substrate turnover. Our long-term goal is to examine enzyme catalysis in a nonclassical way, by characterizing motions in the enzyme during substrate turnover using dynamic NMR spectroscopy. This grant is focused on three reversible enzymes as model systems: the prolyl cis/trans isomerases human cyclophilin A (CypA) and Pin1, and adenylate kinase (Adk) from E.coli and Aquifex aeolicus. The static structures of these proteins are known. First, the intrinsic protein dynamics of the isomerases during catalysis will be characterized and compared to kinetic data measured for substrate interconversion. While it has been long hypothesized that motions are crucial for enzyme activity, the experimental detection of motions during catalysis will directly address questions, such as how the flexibility of the active site is linked to catalysis and what role collective motions play in enzyme function. Second, the interaction between CypA and the HIV-1 capsid protein will be explored mechanistically. This interaction is essential for HIV-1 replication, however, the role of CypA is not known. Using NMR and mutagenesis, structural rearrangements and the dynamic behavior of this enzyme/substrate complex will be studied. Finally, comparative studies on a hyper-thermophilic and a mesophilic enzyme pair will be performed as a second approach to examine the relation between protein dynamics and catalysis. This system provides the unique opportunity to use temperature as a powerful parameter to tune catalytic power and dynamics. Nuclear spin relaxation, NMR line-shape analysis, and NMR exchange spectroscopy are used as primary methods because they allow a residue-specific quantification of protein flexibility. Structure-based drug design is currently primarily based on static structures. However, many inhibitors bind to their targets in a way that can only be accomplished by conformational dynamics of the protein. The novel approach outlined in this proposal will lead to a deeper and more general understanding of protein dynamics, which should advance (enhance) the success of rational, structure based approaches for drug design.

描述（由申请人提供）：对酶作用的完整理解需要对结构，动力学和功能有详细的了解。 虽然蛋白质运动是酶功能的基础，但这是酶学中人们了解最少的部分。 传统上，酶反应通过检测底物周转来研究。 我们的长期目标是以非经典的方式研究酶催化，通过动态核磁共振光谱表征底物周转过程中酶的运动。 该资助主要集中在三种可逆酶作为模型系统：脯氨酰顺式/反式异构酶人类亲环素A（CypA）和Pin 1，以及来自大肠杆菌和Aquifex aeolicus的腺苷酸激酶（Adk）。 这些蛋白质的静态结构是已知的。 首先，在催化过程中的异构酶的内在蛋白质动力学的特点，并比较动力学数据测量底物相互转化。 虽然长期以来一直假设运动对酶活性至关重要，但催化过程中运动的实验检测将直接解决问题，例如活性位点的灵活性如何与催化作用联系在一起，以及集体运动在酶功能中发挥什么作用。 第二，CypA和HIV-1衣壳蛋白之间的相互作用将被探索机制。 这种相互作用对HIV-1复制至关重要，然而CypA的作用尚不清楚。 使用NMR和诱变，结构重排和这种酶/底物复合物的动态行为将进行研究。 最后，将对超嗜热酶和中温酶对进行比较研究，作为研究蛋白质动力学与催化之间关系的第二种方法。 该系统提供了独特的机会，使用温度作为一个强大的参数来调整催化功率和动力学。 核自旋弛豫、NMR线形分析和NMR交换光谱被用作主要方法，因为它们允许蛋白质柔性的残基特异性定量。 基于结构的药物设计目前主要基于静态结构。 然而，许多抑制剂以只能通过蛋白质的构象动力学来实现的方式与其靶结合。 本提案中概述的新方法将导致对蛋白质动力学的更深入和更普遍的理解，这将促进（增强）合理的基于结构的药物设计方法的成功。