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: 7541816
• 负责人: THOMAS L POULOS
• 金额: $ 21.81万
• 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/7541816
• 关键词: Active Sites; Area; Binding; CYP3A4 gene; Camphor 5-Monooxygenase; Catalysis; Characteristics; Collaborations; Complex; Cytochrome P450; Data; Development; Dioxygen; Distal; Drug Delivery Systems; Drug Design; Drug toxicity; Electronics; Enzymes; Future; Heme; Hemeproteins; Hemoglobin; Hour; Human; Hydrogen Bonding; Hydroxylation; Kinetics; Leghemoglobin; Life; Ligands; Location; Malignant Neoplasms; Maps; Measurement; Methodology; Methods; Molecular Conformation; Mycoses; Myoglobin; N-palmitoylglycine; Nitric Oxide; Nitric Oxide Synthase; Oxygenases; Pharmaceutical Preparations; Physiological; Planet Mars; Positioning Attribute; Principal Investigator; Property; Proteins; Protons; Reaction; Route; Series; Signal Transduction; Site; Solutions; Source; Specificity; Steroids; Structure; Substrate Interaction; Therapeutic Agents; Toxin; Water; adduct; analog; base; drug metabolism; farmer; inhibitor/antagonist; interest; nitroxyl; programs; protein expression; protein structure; research study; solid state nuclear magnetic resonance

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)：基于血红素的加氧酶(如各种细胞色素P450(Cyt P450)、一氧化氮合酶(NOS)和血红素加氧酶(HO))的结构早已被研究，希望这些数据可以用于预测产物和抑制剂的特异性。但大多数结构数据已经获得了酶的非生产状态，因此可能与活性物种无关。在这份R21中，我们建议将这些加氧酶的不寻常的亚铁氮氧基加合物(HNO-FeII)作为直接在周转之前的二氧化亚铁加合物(O2-FeII)的结构类似物。HNO与O2是等电子的，并被证明与肌红蛋白、血红蛋白和豆红蛋白等O2载体紧密且不可逆转地结合。HNO-FeII加合物和O2-FeII加合物一样是抗磁性的，但寿命要长得多。最重要的是，亚硝酰氢化物在活性口袋的正中心提供了一个光谱柄；1HNMR中的共振发生在大约15ppm，远离其他基于蛋白质的信号，因此可以用来表征口袋内底物结合的状态，类似于正在进行的周转。结合的HNO还在口袋内形成氢键相互作用，这在确定加氧酶活性方面是重要的。两种血红素加氧酶都有稳定的HNO加合物，核磁共振氢谱的特征峰约为15ppm。在有底物和没有底物的P450BM3和P450cam中已经产生了类似的加合物；并且提出了获得这种nNOS加合物的类似方法。一个关键的建议实验将是使用抗磁氢化物共振通过2D NOE方法绘制出P450BM3中的底物位置，因为以前的核磁共振和结晶学研究给出了截然不同的距离。这一探索性建议的一个关键重点是开发此类加合物的合成方法，跟踪形成动力学和产率，评估寿命和可能的反应活性，并确定关键性质，如H/D交换率，这些性质在将其用作结构探针时将是重要的。最终，这些加合物可能用于评估底物和缓蚀剂在口袋中的流动性。我们处于一个独特的位置来进行这些研究：Pi Farmer的团队首先发现了肌红蛋白的这种HNO加合物，并表征了它的结构和反应活性，同时还发现了几种合成这种加合物的方法。Copi Poulos长期以来一直对血红素蛋白质结构和配体和抑制剂结合的特征感兴趣；他在所有目标蛋白质(HO、P450和NOS)的蛋白质表达和晶体特征方面具有专长。项目叙事基于血红素的加氧酶在药物新陈代谢、一氧化氮和类固醇等生理效应物的合成以及毒素(如亚铁血红素本身)的营养化方面发挥着关键作用。它们是药物寿命和作用的关键决定因素，本身就是治疗剂(例如真菌感染、癌症)的重要靶标，是造成广泛药物毒性的原因。它们活性的一个重要方面是结合和羟化特定部位的靶标的能力。预测P450底物和抑制物特异性的能力是所有这些涉及P450的区域的核心。

项目成果

Nitrosyl hydride (HNO) as an O2 analogue: long-lived HNO adducts of ferrous globins.

• DOI: 10.1021/bi900122r
• 发表时间: 2009-06-09
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Kumar MR;Pervitsky D;Chen L;Poulos T;Kundu S;Hargrove MS;Rivera EJ;Diaz A;Colón JL;Farmer PJ
• 通讯作者: Farmer PJ