Spectroscopic and Computational Investigation of Copper Monooxygenases

铜单加氧酶的光谱和计算研究

• 批准号: 8456583
• 负责人: Ryan Cowley
• 金额: $ 4.92万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8456583
• 关键词: Active Sites; Alzheimer' s Disease; Biochemistry; Biological Models; Breast; Calibration; Catalysis; Catechol Oxidase; Complement; Computing Methodologies; Copper; Coupled; Coupling; Data; Distant; Dopamine; Electron Spin Resonance Spectroscopy; Electron Transport; Electronics; Electrons; Enzymes; Eukaryota; Exhibits; Family; Goals; Health; Hormones; Hydrogen; Hydroxylation; Indium; Investigation; Kinetics; Ligands; Lung; Magnetism; Malignant Neoplasms; Maps; Methods; Mixed Function Oxygenases; Modeling; Molecular; Mononuclear; Monophenol Monooxygenase; Neuropeptides; Oxygen; Oxygenases; Peroxides; Play; Reaction; Research; Resolution; Role; Site; Spectrum Analysis; Structure; Superoxides; Surveys; Synchrotrons; System; Techniques; Time; Training; Tyramine; base; circular magnetic dichroism; density; design; electronic structure; experience; improved; insight; mutant; neurochemistry; peptidylglycine alpha-amidating monooxygenase; public health relevance; research study; theories; tumor

DESCRIPTION (provided by applicant): Binuclear copper enzymes perform O2 activation for the monooxygenation of neuropeptides and hormones, reactions that are essential for neurochemistry for all higher eukaryotes. Binuclear copper monoogygenases can be classified into "coupled" and "non-coupled" based on the magnetic interaction between the two copper centers. The coupled binuclear enzymes (for example, tyrosinase (Ty) and catechol oxidase (CaO)) have two strongly coupled copper sites that reduce O2 to a dicopper peroxide intermediate that effects electrophilic aromatic substitution (EAS). Non-coupled binuclear copper monooxygenases (peptidylglycine ¿-hydroxylating monooxygenase (PHM), dopamine ¿-monooxygenase (D¿M), and tyramine ¿-monooxygenase (T¿M)) feature two copper centers that are distant, and exhibit no magnetic exchange interaction. These non-coupled enzymes generate a reactive monocopper-O2 species that reacts through hydrogen-atom abstraction (HAA). However, the means by which electronic structure and exchange coupling influence binuclear copper sites towards either EAS or HAA reactivity are not known. In contrast to the more well-studied coupled binuclear enzymes, direct spectroscopic probes of reaction intermediates in the non-coupled binuclear copper monooxygenase family have not provided sufficient information to explain the HAA and subsequent hydroxylation mechanisms. Recent advances in PHM and T¿M expression systems provide the opportunity to prepare active-site mutants for the first time which have the potential to allow kinetic trapping of key reaction intermediates for spectroscopic analysis. These studies require application of advanced spectroscopies such as resonance Raman, electron paramagnetic resonance, magnetic circular dichroism, and synchrotron-based methods, and in combination with computational methods, particularly density functional theory, the results will be related to enzymatic catalysis and provide a rationale for active site structural elements in the non-coupled binuclear copper oxygenases. Combined with previous results in the coupled binuclear family, an electronic structure/function model will be developed to explain the differences in reactivity between coupled and non- coupled binuclear copper enzymes. These studies will yield new details concerning the activation of O2 by copper enzymes, insight that is useful for understanding the biochemistry of copper on a molecular level.

描述（由申请人提供）：双核铜酶对神经肽和激素的单氧化进行O2活化，这些反应是所有高等真核生物神经化学所必需的。双核铜单加合酶根据两个铜中心之间的磁性相互作用可分为“偶联”和“非偶联”。偶联的双核酶（例如酪氨酸酶（Ty）和儿茶酚氧化酶（CaO））具有两个强偶联的铜位点，其将O2还原成过氧化二铜中间体，所述过氧化二铜中间体影响亲电芳香取代（EAS）。非偶联双核铜单加氧酶（肽基甘氨酸<$-羟基化单加氧酶（PHM）、多巴胺<$-单加氧酶（D <$M）和酪胺<$-单加氧酶（T <$M））的特征在于两个远离的铜中心，并且不表现出磁交换相互作用。这些非偶联酶产生反应性的单铜-O2物质，其通过氢原子提取（HAA）反应。然而，电子结构和交换耦合影响双核铜网站对EAS或HAA反应性的手段是未知的。与更好地研究耦合双核酶，直接光谱探针的反应中间体在非耦合双核铜单加氧酶家族没有提供足够的信息来解释HAA和随后的羟基化机制。PHM和T <$M表达系统的最新进展首次提供了制备活性位点突变体的机会，这些突变体具有允许动力学捕获关键反应中间体用于光谱分析的潜力。这些研究需要应用先进的光谱学，如共振拉曼，电子顺磁共振，磁圆二色性，和同步加速器为基础的方法，并结合计算方法，特别是密度泛函理论，结果将与酶催化，并提供了一个合理的非耦合双核铜加氧酶的活性位点的结构元素。结合偶联双核家族中以前的结果，将发展一个电子结构/功能模型来解释偶联和非偶联双核铜酶反应性的差异。这些研究将产生关于铜酶激活O2的新细节，这对于在分子水平上理解铜的生物化学是有用的。