Functional Dynamics during Induced-fit Enzyme Turnover

描述（由申请人提供）：运动是许多蛋白质作用的关键。人们对动力学知之甚少，特别是在微秒和更慢的区域，这往往限制了周转.它们超越了大多数实验和计算方法。我们的长期目标是从根本上了解构象动力学如何支撑机制。这将通过表征单个氨基酸的结构和动力学运动及其与诱导适合酶的周转周期中的功能的关系来实现。最近开发的核磁共振弛豫分散分析方法将被应用到一个模型酶，以确定构象交换率。这些将与高分辨率晶体结构和NMR残留偶极耦合表征更快的动力学集成。精氨酸激酶呈现了底物诱导的结构域和环运动以及固有的协同运动的例子。精氨酸激酶的NMR光谱和X-射线衍射对于这种大小（42 kDa）的代表性酶具有异常高的质量。这提供了一个独特的机会来阐明那些在功能上对许多蛋白质很重要的运动代表的结构-动力学-功能相关性。重点将是映射每个残基的骨架构象交换率，特别是在微秒/毫秒制度。将在酶周转期间在各种平衡底物/产物浓度下测量NMR弛豫交换速率，以确定哪些运动与结合、解离或反应步骤相关。将在随机顺序双-双机制内确定稳定酶复合物的晶体结构，并且将使用NMR弛豫滴定来确定与结合或解离相关的构象变化的交换速率。因此，结构变化和运动的时间常数将通过周转周期的步骤为每个氨基酸进行解剖。运动的功能作用将通过对温度、突变和酶的其他扰动后的动力学和动力学的联合分析来确定。健康相关性：对分子运动的基本理解对于理解人类蛋白质如何实现其正常功能以及遗传突变可能导致疾病的机制至关重要。

DESCRIPTION (provided by applicant): Motions are key to the action of many proteins. Dynamics are poorly understood, especially, in the micro- second and slower regimes that often limit turnover. They lie beyond most experimental and computational methods. Our long term objective is a fundamental understanding of how conformational dynamics underpin mechanism. This will be achieved by characterizing structurally and kinetically the motions of individual amino acids and their relation to function in the turnover cycle of an induced-fit enzyme. Recently developed methods of NMR relaxation dispersion analysis will be applied to a model enzyme to determine conformational exchange rates. These will be integrated with high resolution crystal structures and NMR residual dipolar coupling characterization of faster dynamics. Arginine kinase presents examples of domain and loop movements that are substrate-induced, as well as inherent concerted motions. Both the NMR spectra and x-ray diffraction of arginine kinase are of unusually high quality for a representative enzyme of this size (42 kDa). This presents a unique opportunity to elucidate the structure-dynamics- function correlates of motions representative of those that are functionally important for many proteins. The emphasis will be on mapping the backbone conformational exchange rates for each residue, particularly in the micro-/milli-second regime. NMR relaxation exchange rates will be measured during enzyme turnover at various equilibrium substrate/product concentrations to determine which motions are associated with binding, dissociation or reaction steps. Crystal structures will be determined for the stable enzyme complexes within the random sequential bi-bi mechanism, and NMR relaxation titration will be used to determine the exchange rates for conformational changes associated with binding or dissociation. Thus, the structural changes and time constants of motion will be dissected for each amino acid through the steps of the turnover cycle. The functional role of the motions will be established through joint analysis of kinetics and dynamics following temperature, mutational and other perturbations of the enzyme. Health Relevance: A fundamental understanding of molecular motion is essential to understanding how human proteins achieve their normal function and the mechanisms through which disease may result from genetic mutations.