PROTEIN/LIPID INTERACTIONS

蛋白质/脂质相互作用

• 批准号: 7001262
• 负责人: ROBERT B GENNIS
• 金额: $ 48.55万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-01-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7001262
• 关键词: Escherichia coli; Rhodospirillales; Vibrio cholerae; X ray crystallography; active sites; bacterial proteins; cytochrome oxidase; enzyme activity; enzyme structure; infrared spectrometry; interferometry; intermolecular interaction; mutant; nuclear magnetic resonance spectroscopy; respiratory burst oxidase; site directed mutagenesis; subtilisins; virulence

DESCRIPTION (provided by applicant): The proposed research is focused on the catalytic mechanisms and structures of respiratory oxidases. These membrane enzymes catalyze the reduction of O2 to H2O, and conserve the considerable free energy liberated by this reaction as a proton motive force. All respiratory oxidases belong to either the proton pumping "heme-copper oxidase" superfamily or the nonpumping "tri-heme oxidase" superfamily. The heme-copper oxidases include all the mitochondrial cytochrome oxidases. As a crucial enzyme in cellular bioenergetics, human cytochrome oxidase is of medical significance, and has been studied as the locus of a set of genetic diseases. The prokaryotic enzyme we are studying from Rhodobacter sphaeroides is an excellent model system for understanding how this enzyme functions. The cbb3-type oxidase from Vibrio cholerae is a second member of the home-copper oxidase superfamily that will be studied. Learning how the same functions are accomplished in these two distantly related enzymes should be revealing. Interest in cytochrome oxidase research is primarily directed towards understanding the proton pump mechanism. For each O2 reduced to H2O, eight charges are driven across the membrane, generating a substantial transmembrane voltage. Research on the aa3-type oxidase from R. sphaeroides utilizes site-directed mutagenesis in combination with a several spectroscopic techniques, including solid state NMR and FTIR difference spectroscopy. The X-ray structure of this enzyme is known and serves as a guide to planning and interpreting experiments. One mutant of particular interest is N139D. This mutation results in increasing the cytochrome oxidase activity of the enzyme by at least 50% over the wild type, but completely eliminates proton pumping. Proton uptake, proton release and intra-protein proton movements will be examined. Understanding how the proton pump is decoupled from the catalytic function will provide fundamental information about how the proton pump works. The tri-heme oxidases are unique to prokaryotes, but are also of medical significance as potential drug targets because of their apparent importance for the virulence of some pathogenic bacteria. Research on the tri-heme oxidases will focus on the cytochrome bd from E. coli and the cytochrome bb' from V. cholerae. The bb'-type enzyme has just recently been discovered and part of the research plan is to purify and characterize this enzyme for the first time. The tri-heme oxidases have two closely interacting heroes at the active site, and spectroscopic methods will be used to elucidate the details of the home-home interaction. Identifying residues involved in intra-protein proton movement from the bulk solution to the active site will also be a goal in the near future. Finally, it is essential to obtain a structure of this enzyme by X-ray crystallography, and efforts will be made to accomplish this.

描述（由申请人提供）：拟议的研究集中于呼吸氧化酶的催化机理和结构。这些膜酶催化了O2对H2O的还原，并保存了该反应作为质子动力的大量自由能。所有呼吸氧化酶都属于质子泵送的“血红素 - 波波氧化酶”超家族或非倾斜“三 - 血红素氧化酶”超家族。血红素 - 铜氧化酶包括所有线粒体细胞色素氧化酶。作为细胞生物能学的关键酶，人细胞色素氧化酶具有医学意义，并且已被研究为一组遗传疾病的基因座。我们正在从杜鹃杆菌中研究的原核酶是理解该酶如何功能的出色模型系统。来自Vibrio Cholerae的CBB3型氧化酶是将研究的家用氧化酶超家族的第二个成员。应了解这两个遥远相关的酶在这两个酶中如何实现相同的功能。 对细胞色素氧化酶研究的兴趣主要是针对理解质子泵机制的。对于减少到H2O的每个O2，八个电荷都在整个膜上驱动，从而产生大量的跨膜电压。对R. sphaeroides的AA3型氧化酶的研究利用了位置定向的诱变与多种光谱技术（包括固态NMR和FTIR差异光谱）结合使用。该酶的X射线结构已知，并作为计划和解释实验的指南。一个特别感兴趣的突变体是N139D。该突变导致酶的细胞色素氧化酶活性在野生型中至少增加了50％，但完全消除了质子泵送。将检查质子摄取，质子释放和蛋白质内质子运动。了解如何与催化功能分离质子泵将提供有关质子泵如何工作的基本信息。 三血红素氧化酶是原核生物独有的，但由于其对某些致病细菌的毒力的重要性而言，作为潜在药物靶标的医学意义。对三血红素氧化酶的研究将集中于大肠杆菌的细胞色素BD和V. Cholerae的细胞色素BB'。 BB'Type酶最近才被发现，研究计划的一部分是首次纯化和表征该酶。三血红素氧化酶在活性部位具有两个紧密相互作用的英雄，并且光谱方法将用于阐明家庭家庭相互作用的细节。在不久的将来，识别从散装溶液到活跃位点的蛋白质质子运动涉及的残基也将是一个目标。最后，必须通过X射线晶体学获得该酶的结构，并为实现这一目标而做出努力。