Structural and thermodynamic features which govern enzymatic nitric oxide detoxif

控制一氧化氮酶解毒的结构和热力学特征

• 批准号: 8767796
• 负责人: Ronald Koder
• 金额: $ 27.23万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8767796
• 关键词: Active Sites; Affect; Affinity; Amyotrophic Lateral Sclerosis; Binding; Biochemical; Blood Substitutes; Carrier Proteins; Chemicals; Chimera organism; Complex; Cytochrome P450; Dioxygenases; Disease; Distal; Drug Metabolic Detoxication; Electron Transport; Electrons; Electrostatics; Enzymes; Equilibrium; Face; Flavins; Flavoproteins; Future; Heart Diseases; Heme; Heme Iron; Hemoglobin; Histidine; Human; Human Biology; Ions; Ischemic Brain Injury; Learning; Left; Ligand Binding; Ligands; Ligation; Malignant Neoplasms; Medicine; Metabolic; Methods; Modification; Molecular; Nature; Nitrates; Nitric Oxide; Oxides; Oxidoreductase; Oxygen; Pathway interactions; Penetration; Play; Poison; Property; Protein Binding Domain; Protein Dynamics; Proteins; Reaction; Role; Rotation; Scientist; Side; Site; Stroke; Structure; Surface; Technology; Tertiary Protein Structure; Testing; Therapeutic; Thermodynamics; Water; Work; alpha helix; base; cofactor; design; driving force; effective therapy; enzyme therapy; innovation; molecular dynamics; next generation; oxygen transport; phthalate 4,5-dioxygenase; protein structure; public health relevance; semiquinone; signal processing; synthetic enzyme; therapeutic enzyme

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?

描述（由申请人提供）：意义。我们的目标是确定控制酶促一氧化氮解毒的基本结构和热力学特征。我们对人工一氧化氮双加氧酶（NOD）进行计算设计和生化分析，该酶是通过将人工血红素氧结合蛋白结构域与源自自然的黄素蛋白还原酶结构域相结合而形成的。使用这种强大、简单的蛋白质可以更轻松地对蛋白质进行小规模和大规模的改变，并积极识别酶功能所需的关键特征。一氧化氮在人类生物学的许多信号传导过程中发挥着核心作用，但由于其化学反应程度，它也与大量严重疾病有关，例如卢伽雷氏病和缺血性脑损伤。因此，优良的一氧化氮双加氧酶有望用于未来治疗许多病理状况。相反，不需要的 NOD 活性在基于血红蛋白的血液代用品中产生了严重的并发症，因此了解如何减少或消除这些疗法中的 NOD 活性而不会对氧结合产生不利影响非常重要。创新。这种催化结构代表了下一代蛋白质设计，将设计技术从当前对具有单一辅因子的简单蛋白质结构域的关注转向更具挑战性和复杂性的多结构域结构，这些结构更类似于自然界中看到的复杂组装体。该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，该项目中吸取的经验教训有望重振基于血红蛋白的血液替代品领域，从而能够重新设计天然血红蛋白，并创建全新的氧运输蛋白，其与一氧化氮的反应最小，同时仍携带氧气。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 对目标活性进行了更好的优化，并且其稳定性比天然蛋白质大大提高 (53)。因此，该项目代表了酶疗法的新方向，我们的设计途径是一种使能技术，我们和其他人将在创建未来酶疗法时使用该技术。具体目标。这项工作将使我们能够回答有关该酶的一些重要问题： 目标 1. 血红素还原电位在一氧化氮双加氧酶反应中起什么作用？目标 2. 电子转移动力学和热力学在此反应中有多重要？目标 3. 蛋白质动力学和结构如何控制 NOD 功能？