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。动力学很少被理解，特别是在微秒和更慢的制度，往往限制周转。它们超越了大多数实验和计算方法。我们的长期目标是对构象动力学如何支撑机制的基本理解。这将通过在结构和动力学上表征单个氨基酸的运动及其在诱导拟合酶的周转周期中的功能关系来实现。最近发展的核磁共振弛豫色散分析方法将应用于模型酶来确定构象交换率。这些将集成高分辨率晶体结构和NMR残留偶极耦合表征更快的动力学。精氨酸激酶展示了结构域和环运动的例子，这些运动是底物诱导的，以及固有的协调运动。精氨酸激酶的核磁共振光谱和x射线衍射对这种大小（42 kDa）的代表性酶来说都是异常高的质量。这提供了一个独特的机会来阐明那些对许多蛋白质在功能上很重要的运动代表的结构-动力学-功能相关。重点将是绘制每个残基的主链构象交换率，特别是在微/毫秒范围内。在各种平衡底物/产物浓度的酶周转过程中，将测量核磁共振弛豫交换率，以确定哪些运动与结合、解离或反应步骤有关。在随机顺序的bi-bi机制下，稳定的酶复合物的晶体结构将被确定，核磁共振弛豫滴定法将被用来确定与结合或解离相关的构象变化的交换速率。因此，结构的变化和运动的时间常数将剖析每个氨基酸通过周转周期的步骤。这些运动的功能作用将通过对酶在温度、突变和其他扰动下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基因突变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.