Probing Metalloenzymes with Sensitizer-Tethered Substrates

用敏化剂束缚的底物探测金属酶

• 批准号: 9807150
• 负责人: Harry Gray
• 金额: $ 52万
• 依托单位: California Institute of Technology
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-10-01 至 2001-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9807150&HistoricalAwards=false
• 关键词: Probing; Metalloenzymes; Sensitizer; Tethered; Substrates

This award in the Inorganic, Bioinorganic, and Organometallic Chemistry Program supports metalloenzyme research by Drs. Harry B. Gray and John H. Richards of the Beckman Institute, California Institute of Technology. The goal of the work is to develop a new method for probing the properties and reactivities of redox metalloenzymes. The prototypical heme oxygenase, cytochrome P450, will be the main system studied. This enzyme stereospecifically hydroxlates hydrocarbons using dioxygen as the oxidant. In many metalloenzymes, rate-limiting electron transfer (ET) steps preclude observation of other critical activation steps. To attack this problem, natural redox partners will be replaced with new reagents that are able to exchange electrons at much faster rates. Laser-initiated electron transfer reactions will be used to drive enzymatic reactions. In order to deliver the laser-generated redox equivalents, a direct covalent or hydrogen-bonded pathway has been shown to be essential. Here a direct link between a redox sensitizer and the enzyme active site will be formed by taking advantage of the affinity of the enzyme for its active site. Ruthenium-diimine sensitizers will be covalently tethered to cytochrome P450 substrates to provide a conduit for delivering oxiding and reducing equivalents to the heme cofactor. The initial goal is to prepare and characterize the oxo-iron (ferryl) intermediates that have been suggested as the active hydroxylating agents. Enzyme for this work will be expressed in E. coli, and site-directed mutagenesis will be employed to study the importance of specific residues in ferryl-formation reactions. The reactive intermediates will be studied using a variety of photochemical methods. Metalloenzymes catalyze many reactions involving small-molecule activation. For example, enzymes promote reduction of oxygen gas to water, reduction of nitrogen gas to ammonia, and use oxygen gas to add oxygen atoms to carbon chains. These reactions are important not only biologically but also commercially. This research aims at understanding the enzyme that promotes addition of oxygen to hydrocarbons by oxygen gas. The reactive enzyme intermediate has never been observed, so a stategy for replacing the reagents from the natural system with ones that can react more readily will be used to enable photochemical observation of reactions at the active site.

该奖项在无机，生物无机和有机化学 项目支持Harry B博士的金属酶研究。作者声明：John H. 加州理工学院贝克曼研究所的理查兹说。 这项工作的目标是发展一种新的方法来探测 氧化还原金属酶的性质和反应性。 原型 血红素加氧酶，细胞色素P450，将是研究的主要系统。 这 酶立体特异性地使用分子氧作为羟基， 氧化剂 在许多金属酶中，限速电子转移（ET） 步骤排除了其它关键激活步骤的观察。 攻击 为了解决这个问题，天然氧化还原伙伴将被新试剂取代 能够以更快的速度交换电子。 激光引发的电子转移反应将用于驱动 酶促反应 为了将激光产生的氧化还原 等价物，已经显示了直接共价或氢键键合途径 是必不可少的。 在此，氧化还原敏化剂与氧化还原反应之间的直接联系是通过氧化还原反应来实现的。 利用酶的亲和力形成酶活性位点 酶的活性部位。 钌-二亚胺敏化剂将是 与细胞色素P450底物共价连接以提供导管 用于将氧化和还原等价物传递给血红素辅因子。 最初的目标是制备和表征氧代铁（ferryl） 已被认为是活性羟基化的中间体 剂. 本工作的酶将以E.杆菌和 定点突变将被用来研究的重要性， 铁基形成反应中的特定残基。 反应性 将使用各种光化学方法研究中间体。 金属酶催化许多涉及小分子的反应 activation. 例如，酶促进氧气的还原， 水，氮气还原为氨，并使用氧气添加 氧原子变成碳链 这些反应不仅重要， 不仅是生物学上的，也是商业上的。 本研究旨在了解 促进氧与烃类化合物反应的酶 气体 反应性酶中间体从未被观察到，所以 用天然试剂替代天然试剂的策略， 可以更容易地反应，将用于使光化学观察 在活性部位的反应。