Structure's Influence on Reactivity in Metalloenzymes

结构对金属酶反应活性的影响

• 批准号: 7218626
• 负责人: Julia A Kovacs
• 金额: $ 31.35万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7218626
• 关键词: Active Sites; Alkenes; Benchmarking; Binding; Biological; Cardiac; Chemistry; Class; Cytochrome P450; Dependence; Disease; Distal; Docosahexaenoic Acids; Drug Metabolic Detoxication; Electron Nuclear Double Resonance; Enzymes; Funding; Heme; Heme Iron; Hydrocarbons; Length; Ligands; Malignant Neoplasms; Modeling; Modification; Myocardial Infarction; NBL1 gene; Nitriles; Nitrogen; Numbers; Object Attachment; 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; System; Techniques; Testing; Work; cold temperature; cysteinesulfenic acid; design; metalloenzyme; nitrile hydratase; oxidation; programs; protonation; superoxide reductase; wasting

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：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酶的功能。含有含SOR和NHASE的半胱氨酸绑扎的非血红素铁参与超氧化物自由基的排毒和硝酸废物的排毒。心脏病发作后，超氧已经与许多疾病状态有关，包括癌症，阿尔茨海默氏症，帕金森氏症和心脏损伤。 SOR活性位点与血红素酶P450相似，该酶P450氧化未激活的烃。在过去的资金期间，我们报道了SOR的第一个功能模型，是硫醇矿石绑扎的Felll-ooh的第一个示例，以及未修饰的NHASE形式的模型。尚未众所周知，SOR减少超氧化物的机制，也不是翻译后NHASE半胱氨酸修饰的功能和机制。在此资金期间，我们计划： - 研究我们的顺式和反式逆变 - 过敏的形成机理，以了解这些反应的质子依赖性，并确定硫醇酸盐（顺式vs vs trans）的定位如何影响可用机械途径。 - 比较我们的顺式与反硫代硫酸盐绑扎的Felll-ooh与亲电和亲核基质的反应性，以及H原子供体，以查看SOR模型是否可以促进P450化学。 - 确定是否会产生有利的反应途径，使FELV = O，甚至FEV = O种。并且，通过创建更碱性的高瓦伦特FELV = 0（如最近提出的P450提出），确定反硫醇是否比顺式硫醇盐更有效地促进氧化化学。 - 合成新的硫醇酯绑扎（NSSPY）fe-过氧于结构上与广泛表征的氮粘合的N4Py Fe-过氧化物，以便我们可以确定硫醇酸盐如何影响功能。 - 合成一种新的反式硫代盐配体，该配体融合了旨在稳定felll-ooh或felv = o的空间块和H键残基，和/或将质子直接递送到远端过氧氧。 - 探索涉及SO42-，NO3-或NO2-还原的SOR的替代功能。 - 检查Nhase半胱氨酸群的翻译后修饰的可能性是通过涉及Fell-ooh的机制发生的。 - 确定两种顺式半胱氨酸酯的翻译后氧合如何通过检查未修饰的NHASE模型与氧气原子和质子供体的反应性来影响功能。