Structural and thermodynamic features which govern enzymatic nitric oxide detoxif

说明（由申请人提供）：意义。我们的目的是确定基本的结构和热力学特征，控制酶解毒一氧化氮。我们使用了计算设计和生化分析的循环，人工一氧化氮双加氧酶（NOD）是由人工血红素基氧结合蛋白结构域和源自自然界的黄素蛋白还原酶结构域结合而成的。使用这样一种强大的、简单的蛋白质使得对蛋白质进行小规模和大规模的改变变得更加容易，并积极地识别酶功能所必需的关键特征。一氧化氮在人类生物学的许多信号传导过程中起着核心作用，但由于其化学反应性的程度，它也与许多严重疾病如卢伽雷氏病和缺血性脑损伤有关。因此，一种优良的一氧化氮双加氧酶有望在许多病理条件的未来治疗中发挥作用。相反，在以血红蛋白为基础的血液替代品中，不必要的NOD活性会产生严重的并发症，因此了解如何在不影响氧结合的情况下降低或消除这些治疗方法中的NOD活性是很重要的。创新。这种催化结构代表了下一代蛋白质设计，将设计技术从目前的重点从单一辅助因子的简单蛋白质结构域转移到更具有挑战性和更复杂的多结构域结构，更接近于自然界中看到的复杂组装。这个项目有能力极大地推进两项重要技术：基于血红蛋白的血液替代品和酶疗法。首先，从这个项目中吸取的经验教训有望重振以血红蛋白为基础的血液替代品领域，既可以重组天然血红蛋白，又可以创造出全新的氧转运蛋白，与一氧化氮的反应最小，同时仍能携带氧气。其次，合成酶有可能改变酶治疗领域，因为设计的酶比天然酶具有许多优势，最重要的是利用非天然辅因子的能力

DESCRIPTION (provided by applicant): Significance. We aim to determine the essential structural and thermodynamic features which govern enzymatic nitric oxide detoxification. We use a cycle of computational design and biochemical analysis of an artificial nitric oxide dioxygenase (NOD) formed by combining an artificial heme-based oxygen binding protein domain with a flavoprotein reductase domain derived from nature. Use of such a robust, simple protein makes it significantly easier to make both small- and large-scale changes to the protein and positively identify critical features necessary for enzyme function. Nitric oxide plays a central role in many signaling process in human biology, yet due to its degree of chemical reactivity it has also been implicated in a surprising number of serious disorders such as Lou Gehrig's Disease and ischemic brain injury. A superior nitric oxide dioxygenase thus promises to be useful in future treatments of many pathological conditions. Conversely, unwanted NOD activity has produced severe complications in hemoglobin-based blood substitutes, and it is important to learn how to reduce or eliminate NOD activity in these therapeutics without adversely affecting oxygen binding. Innovation. This catalytic construct represents the next generation in protein design, moving design technology from the current focus on simple protein domains with single cofactors to significantly more challenging and sophisticated multidomain structures that more closely resemble the complex assemblies seen in nature. This project has the capacity to dramatically advance two important technologies: hemoglobin-based blood substitutes and enzyme therapeutics. First, lessons learned in this project promise to revitalize the field of hemoglobin-based blood substitutes, enabling both the reengineering of native hemoglobins and the creation of entirely new oxygen transport proteins minimally reactive with nitric oxide while still carrying oxygen. Second, a synthetic enzyme has the potential to transform the field of enzyme therapy because of the many advantages designed enzymes have over their natural counterparts, most importantly the ability to utilize non-natural cofactors better optimized for the target activity and their greatly increased stability over natural protein (53). This project thus represents a new direction in enzyme therapy, and our design pathway is an enabling technology which will be used by us and others in the creation of future enzyme therapeutics. Specific Aims. This work will allow us to answer some important questions about this enzyme: Aim 1. What role does the heme reduction potential play in the nitric oxide dioxygenase reaction? Aim 2. How important are electron transfer dynamics and thermodynamics in this reaction? Aim 3. How do protein dynamics and structure govern NOD function?