Physical Studies of Xanthine Oxidase and Trimethylamine Dehydrogenase

黄嘌呤氧化酶和三甲胺脱氢酶的物理研究

• 批准号: 9420185
• 负责人: Charles Hille
• 金额: $ 31万
• 依托单位: Ohio State University Research Foundation -DO NOT USE
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9420185&HistoricalAwards=false
• 关键词: Physical; Studies; Xanthine; Oxidase; Trimethylamine

Hille 9420185 A study of the biophysical properties of two oxidoreductases, xanthine oxidase and trimethylamine dehydrogenase, builds on past success in the PI's laboratory. The goal of this work is to fully characterize the kinetics of electron transfer within the two subject proteins and ascertain its role in catalysis. Xanthine oxidase from cow's milk catalyzes the oxidative hydroxylation of xanthine to form uric acid, a reaction that takes place at the molybdenum center of the enzyme. For a variety of reasons, the enzyme has become the prototypical molybdenum center of the enzyme. For a variety of reasons, the enzyme has become the prototypical molybdenum hydroxylation of substrate. Studies of electron transfer among the four redox-active centers of this enzyme will be pursued along two different lines: (1) pulse radiolysis studies in D2O and as a function of temperature will be performed with the aim of extending past work by the PI; and (2) use will be made of a ruthenium center that has been covalently attached near the molybdenum center of xanthine oxidase to very rapidly introduce reducing equivalents into the enzyme by flash photolysis. The advantage of this protocol is that the initiation of electron transfer is an intramolecular process, making possible studies at cryogenic temperatures. Other work will focus on several related aspects of trimethylamine dehydrogenase, an enzyme from the methylotrophic bacterium W3A1 that catalyzes the oxidative demethylation of trimethylamine to dimethylamine and formaldehyde. The enzyme possesses a covalently linked flavin mononucleotide cofactor and a 4Fe/4S (ferredoxin-type) center, and thus represents a useful system with which to extend past work on xanthine oxidase. The work includes: (1) studies of the rates of electron transfer between the two redox-active centers using pulse radiolysis; (2) characterization of the reaction of reduced trimethylamine dehydrogenase with its physiological oxidant, electron-transfe rring flavoprotein; and (3) development of a photolabile slow substrate for trimethylamine dehydrogenase that will be suitable for a Laue experiment in which the progress of the reaction at the active site is followed by timeresolved crystallography. %%% The experiments are designed to address two goals. The first has to do with how protonation/deprotonation events modulate electron transfer into and out of organic cofactors that are redoxactive (e.g., flavins and quinones), using xanthine oxidase and trimethylamine dehydrogenase as the experimental systems. The second involves a study of how the two cofactors of the enzyme trimethylamine dehydrogenase interact with one another, and the role of this interaction in catalytic turnover. This work involves a combination of kinetic and crystallographic approaches aimed at gaining a more complete understanding of how this enzyme functions mechanistically. ***

在PI实验室过去成功的基础上，对两种氧化还原酶，黄嘌呤氧化酶和三甲胺脱氢酶的生物物理特性进行了研究。 这项工作的目标是充分表征两个主题蛋白质内的电子转移的动力学，并确定其在催化中的作用。 牛奶中的黄嘌呤氧化酶催化黄嘌呤的氧化羟基化作用形成尿酸，这是一种发生在酶的钼中心的反应。 由于各种原因，该酶已成为该酶的原型钼中心。 由于多种原因，该酶已成为钼羟化反应的原型底物。 该酶的四个氧化还原活性中心之间的电子转移的研究将沿着沿着两条不同的路线进行：（1）将进行D2 O中的脉冲辐解研究和作为温度的函数的研究，目的是扩展PI过去的工作;以及（2）将使用已共价连接在黄嘌呤氧化酶的钼中心附近的钌中心，通过闪光光解将当量还原到酶中。 该协议的优点是，电子转移的启动是一个分子内的过程，使研究在低温下。 其他工作将集中在三甲基胺脱氢酶的几个相关方面，三甲基胺脱氢酶是一种来自甲基营养细菌W3 A1的酶，可以催化三甲基胺氧化脱甲基为二甲胺和甲醛。 该酶具有一个共价连接的黄素单甘肽辅因子和一个4Fe/4S（铁氧还蛋白型）中心，因此代表了一个有用的系统，以扩展过去的工作黄嘌呤氧化酶。 工作内容包括：（1）用脉冲辐解法研究了两个氧化还原活性中心之间的电子转移速率，（2）还原型三甲胺脱氢酶与其生理氧化剂电子转移黄素蛋白反应的特性;以及（3）开发了一种适合于劳厄实验的光不稳定的三甲基胺脱氢酶缓慢底物，在劳厄实验中，活性位点之后是时间分辨晶体学。 %实验旨在解决两个目标。 第一个与质子化/去质子化事件如何调节电子转移进出具有氧化还原活性的有机辅因子（例如，黄嘌呤氧化酶和三甲胺脱氢酶为实验体系。 第二个涉及的研究如何酶的两个辅因子的三甲基胺脱氢酶相互作用，这种相互作用在催化营业额的作用。 这项工作涉及动力学和结晶学方法的结合，旨在更全面地了解这种酶的功能机制。 ***