Enzyme Dynamics During Catalysis

描述(由申请者提供)：对酶作用的完整理解需要关于结构、动力学和功能的详细知识。虽然蛋白质运动是酶功能的基础，但这是酶学中了解最少的部分。传统上，酶反应是通过检测底物周转率来研究的。我们的长期目标是通过使用动态核磁共振光谱来表征底物周转过程中酶的运动，以非经典的方式检查酶的催化作用。 这项资助集中在三种可逆酶作为模型系统：人类亲环素A(CypA)和PIN1的Pro顺式/反式异构酶，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，将表征催化过程中异构酶的内在蛋白质动力学，并将其与底物相互转化测量的动力学数据进行比较。虽然长期以来人们一直假设运动对酶的活性至关重要，但对催化过程中运动的实验检测将直接解决诸如活性部位的灵活性如何与催化有关以及集体运动在酶功能中扮演什么角色等问题。其次，将从机制上探索CypA与HIV-1衣壳蛋白之间的相互作用。这种相互作用是HIV-1复制所必需的，然而，CypA的作用尚不清楚。利用核磁共振和诱变技术，我们将研究这种酶/底物复合体的结构重排和动力学行为。最后，将对超嗜热性和中温性酶对进行比较研究，作为检验蛋白质动力学和催化之间关系的第二种方法。这个系统提供了独特的机会，可以使用温度作为一个强大的参数来调整催化功率和动力学。核自旋松弛、核磁共振线形分析和核磁共振交换光谱被用作主要方法，因为它们允许对蛋白质柔韧性进行特定残基的量化。基于结构的药物设计目前主要基于静态结构。然而，许多抑制剂以一种只有通过蛋白质构象动力学才能实现的方式与其靶标结合。这项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一般的理解，这将促进(加强)合理的、基于结构的药物设计方法的成功。

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.