Nitroxyl adducts as structural probes of oxygenase/substrate interactions

硝酰加合物作为加氧酶/底物相互作用的结构探针

• 批准号: 7363745
• 负责人: PATRICK J FARMER
• 金额: $ 18.47万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7363745
• 关键词: Active Sites; Area; Binding; CYP3A4 gene; Camphor 5-Monooxygenase; Catalysis; Characteristics; Collaborations; Complex; Condition; Cytochrome P450; Data; Development; Dioxygen; Distal; Drug Delivery Systems; Drug Design; Drug toxicity; Electronics; Enzymes; Future; Heme; Hemeproteins; Hemoglobin; Hour; Human; Hydroxylation; Kinetics; Leghemoglobin; Life; Ligands; Location; Malignant Neoplasms; Maps; Measurement; Methodology; Methods; Molecular Conformation; Mycoses; Myoglobin; N-palmitoylglycine; Nitric Oxide; Nitric Oxide Synthase; Object Attachment; Oxygenases; Pharmaceutical Preparations; Physiological; Planet Mars; Positioning Attribute; Principal Investigator; Property; Proteins; Protons; Range; Rate; Reaction; Route; Series; Signal Transduction; Site; Solutions; Source; Specificity; Steroids; Structure; Substrate Interaction; Therapeutic Agents; Toxin; Water; adduct; analog; base; concept; drug metabolism; farmer; inhibitor/antagonist; interest; nitroxyl; nuclear Overhauser enhancement; programs; protein expression; protein structure; research study; solid state

DESCRIPTION (provided by applicant): The structures of heme-based oxygenases (e.g. various cytochrome P450 (cyt P450), nitric oxide synthase (NOS) and heme oxygenase (HO)) have been long studied with the hope that such data can be used to predict product and inhibitor specificity. But most structural data has been obtained for non-productive states of the enzymes, and thus may not be relevant to the active species. In this R21, we propose the application of unusual ferrous nitroxyl adducts (HNO-FeII) of these oxygenases as structural analogues of the ferrous dioxygen adducts (O2-FeII) that directly precedes turnover. HNO is isoelectronic to O2, and is shown to bind tightly and irreversibly to O2 carriers like myoglobin, hemoglobin and leghemoglobin. The HNO-FeII adducts are diamagnetic, like the O2-FeII adducts, but much longer lived. Most importantly, the nitrosyl hydride provides a spectroscopic handle at the very center of the active pocket; the resonance in 1H NMR occurs ca. 15 ppm, well away from other protein-based signals, and therefore may be used to characterize substrate binding within the pocket in a state analogous to that proceeding turnover. The bound HNO also forms H-bonding interactions within the pocket, which are known to be important in determining oxygenase activity. Stable HNO adducts are demonstrated for two heme oxygenases, the characteristic peak ca 15 ppm seen by 1H NMR. Similar adducts have been generated in P450BM3 and P450cam with and without substrates; and similar methods are proposed to obtain such adducts of nNOS. A key proposed experiment will be to use the diamagnetic hydride resonance to map out the substrate position in P450BM3 by 2D NOE methods, as previous NMR and crystallographic studies give widely different distances. A key focus of this exploratory proposal is the development of synthetic methodologies to such adducts, following kinetics and yields of formation, assessing lifetime and possible reactivity, and determining key properties such as H/D exchange rates that would be important in their use as structural probes. Ultimately, these adducts may be of use in assessing mobility of substrate and inhibitors within the pocket. We are in a unique position to perform these studies: the PI Farmer's group first identified such an HNO adduct of myoglobin, and has characterized its structure and reactivity, as well uncovering several synthetic routes to such adducts. CoPI Poulos has longstanding interest in heme protein structure and in the characterizations of ligand and inhibitor binding; he has expertise in protein expression and crystallographic characteristics of all the target proteins (HO, P450 and NOS).Project Narrative Heme-based oxygenases are key players in the metabolism of drugs, in the synthesis of physiological effectors like nitric oxide and steroids, and in the nuetralization of toxins such as the hemes themselves. They are the key determinant of drug lifetime and action, are themselves important targets for therapeutic agents (e.g., fungal infections, cancer), are responsible for a broad range of drug toxicities. An important aspect of their activity is the ability to bind and hydroxylate targets at specific sites. The ability to predict P450 substrate and inhibitor specificity is central to all of these areas of P450 involvement.

描述（由申请人提供）：血红素基氧酶（例如各种细胞色素P450（Cyt P450），一氧化氮合酶（NOS）和血红素氧酶（HO））的结构已长期研究，希望可以使用此类数据来预测产物和抑制剂特异性。但是，大多数结构数据是针对酶的非生产状态获得的，因此可能与活性物种无关。在此R21中，我们提出了这些氧化酶的异常亚硝氧加合物（HNO-FEII）的应用，作为二氧化物加合物（O2-FEII）的结构类似物，这些类似物直接在流动率之前。 HNO对O2是等电原理，并且显示出与肌红蛋白，血红蛋白和leghemoglobin等O2载体紧密而不可逆转的结合。 HNO-FEII加合物是磁性的，例如O2-FEII加合物，但寿命更长。最重要的是，亚硝基氢化物在活动口袋的中心提供了光谱手柄。 1H NMR中的共振发生CA。 15 ppm，远离其他基于蛋白质的信号，因此可以用来表征口袋内的底物结合，状态类似于该程序营业额。绑定的HNO还形成了口袋内的H键相互作用，这对于确定氧合酶活性很重要。证明了两种血红素氧酶的稳定HNO加合物，即1H NMR观察到的特征峰Ca 15 ppm。在P450BM3和P450CAM中也产生了类似的加合物，带有和没有底物。并提出了类似的方法来获得这种NNO的加合物。一个关键的实验将是使用Dimagnetic Hydride共振通过2D NOE方法在P450BM3中绘制底物位置，因为先前的NMR和晶体学研究给出了很大的距离。该探索性建议的一个重点是，遵循形成的动力学和产量，评估生命周期和可能的反应性，并确定关键特性，例如H/D汇率率，在其用作结构探针中很重要。最终，这些加合物可能用于评估口袋内底物和抑制剂的迁移率。我们处于执行这些研究的独特位置：PI农民小组首先确定了这种肌红蛋白的HNO加合物，并表征了其结构和反应性，并发现了几种与此类合并的合成路线。 Copi Poulos对血红素蛋白结构以及配体和抑制剂结合的特征感兴趣。他在所有靶蛋白（HO，P450和NOS）的蛋白质表达和晶体学特征方面具有专业知识。基于叙事的叙事血红素氧酶是药物代谢的关键参与者，在生理氧化物和类固醇以及类固醇等生理效应子的合成中，以及像toxins of Toxins of Hemes hemes hemess hemess hemens the Hemes hemes hemes hemes hemes nuetralization。它们是药物寿命和作用的关键决定因素，它本身是治疗剂（例如真菌感染，癌症）的重要靶标，负责广泛的药物毒性。他们活性的一个重要方面是在特定位点结合和羟基靶标的能力。预测P450底物和抑制剂特异性的能力对于P450参与的所有这些领域都是至关重要的。