Structure's Influence on Reactivity in Metalloenzymes

结构对金属酶反应性的影响

• 批准号: 8048332
• 负责人: Julia A Kovacs
• 金额: $ 13.8万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-15 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8048332
• 关键词: Active Sites; Alkenes; Benchmarking; Binding; Cardiac; Chemistry; Cytochrome P450; Dependence; Disease; Distal; Drug Metabolic Detoxication; Electron Nuclear Double Resonance; Enzymes; Funding; Heme; Heme Iron; Hydrocarbons; Hydrogen Bonding; Length; Ligands; Malignant Neoplasms; Modeling; Modification; Myocardial Infarction; NBL1 gene; Nitriles; Nitrogen; Oxygen; PRTN3 gene; Parkinson Disease; Pathway interactions; Peroxides; Positioning Attribute; Post-Translational Protein Processing; Protons; Reaction; Reporting; Research; Research Personnel; Spectrum Analysis; Structure; Sulfenic Acids; Sulfur; Superoxides; Techniques; Testing; Work; biological systems; cold temperature; cysteinesulfenic acid; design; metalloenzyme; nitrile hydratase; oxidation; programs; protonation; superoxide reductase; wasting

Our research program is aimed at determining how cysteinates influence function in non-heme iron enzymes. Cysteinate-ligated non-heme iron containing SOR and NHase are involved in the detoxification of superoxide radicals, and the detoxification of nitrile wastes, respectively. Superoxide has been implicated in a number of disease states, including cancer, Alzheimers, Parkinsons, and cardiac damage following a heart attack. The SOR active site closely resembles that of the heme enzyme P450 which oxidizes unactivated hydrocarbons. During this past funding period, we reported the first functional model for SOR, the first example of a thiolate-ligated Felll-OOH, and a model for the unmodified form of NHase. Neither the mechanism of superoxide reduction by SOR, nor the function and mechanism of post-translational NHase cysteinate modification, are well understood. During this funding period we plan to: ¿ investigate the mechanism of formation of our cis and trans Felll-peroxos in order to understand the proton-dependence of these reactions, and determine how the positioning of the thiolate (cis vs trans) influences the available mechanistic pathways. ¿ compare the reactivity of our cis vs. trans thiolate-ligated Felll-OOH with electrophilic and nucleophilic substrates, and H-atom donors, in order to see if an SOR model can promote P450 chemistry. ¿ determine whether thiolate ligands create favorable reaction pathways affording FelV=O, or perhaps even FeV=O species. And, determine whether a trans-thiolate is more efficient than a cis-thiolate at promoting oxidation chemistry by creating a more basic high valent FelV=0 (as was recently proposed for P450). ¿ synthesize a new thiolate-ligated (NSSPy) Fe-peroxide structurally-related to the extensively characterized nitrogen-ligated N4Py Fe-peroxides so that we can determine how thiolates influence function. ¿ synthesize a new trans thiolate-ligand that incorporates steric bulk and H-bonding residues designed to stabilize an Felll-OOH or FelV=O, and/or direct proton delivery to the distal peroxo oxygen. ¿ explore alternative functions of SOR involving SO42-, NO3-, or NO2- reduction. ¿ examine the possibility that post-translational modification of the NHase cysteinates occurs via a mechanism involving an Felll-OOH. ¿ determine how the post-translational oxygenation of two cis NHase cysteinates influences function by examining the reactivity of our unmodified NHase model with oxo-atom and proton donors.

我们的研究计划旨在确定半胱氨酸群如何影响非血红素铁的功​​能 酶。半胱氨酸绑扎的非血红素含有SOR和NHASE参与排毒 超氧化物自由基和硝酸废物的排毒。超氧已与 许多疾病状态，包括癌症，阿尔茨海默氏症，帕金森氏症和心脏损害 攻击。 SOR活性位点与血红素酶P450相似，该酶P450氧化物未激活 碳氢化合物。在过去的资金期间，我们报道了SOR的第一个功能模型，第一个 硫醇矿石绑扎的felll-ooh的示例，以及未修饰形式的NHase的模型。也不是 SOR减少超氧化物的机理，也不是翻译后NHASE的功能和机制 半胱氨酸的修饰是有充分理解的。在此资助期间，我们计划：»调查 我们的顺式和反式逆变的机理，以了解质子的依赖性 这些反应，并确定硫醇酸盐（顺式vs trans）的定位如何影响可用的 机械途径。比较我们的顺式反应性与反式硫代硫酸盐绑扎的Felll-ooh与 亲电和核系统性底物以及H原子供体，以查看SOR模型是否可以促进 P450化学。 �确定硫醇酸盐配体是否会产生有利的反应途径，使FELV = O， 也许甚至是fev = o种。并且，确定thr-硫醇是否比A更有效 顺式硫醇酸盐通过创建更碱性的高瓦伦特felv = 0（如最近，促进氧化化学 提议用于P450）。合成新的硫醇酯绑扎（NSSPY）fe-过氧于结构上与 广泛表征了氮粘合的N4Py Fe-过氧化物，以便我们可以确定硫醇盐 影响功能。 �合成了一种新的硫代硫酸盐，该配体结合了空间散装和H键 旨在稳定跌倒或felv = o的残留物和/或直接质子递送到远端的peroxo 氧。 »探索涉及SO42-，NO3-或NO2-还原的SOR的替代功能。考试 Nhase半胱氨酸酯的翻译后修饰的可能性是通过涉及的机制发生的 Felll-ooh。 �确定两个顺式半胱氨酸酯的翻译后氧化如何影响 通过检查我们未修饰的NHASE模型与Oxo-Atom和质子供体的反应性的功能。