LINKING FUNCTIONS OF THE F0F1 ATP SYNTHASE

F0F1 ATP 合成酶的连接功能

• 批准号: 2415249
• 负责人: ROBERT K. NAKAMOTO
• 金额: $ 17.13万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415249
• 关键词: X ray crystallography; catalyst; conformation; disulfide bond; electron transport; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; hydrogen transport; hydrogen transporting ATP synthase; mutant; protein purification; protein sequence; suppressor mutations; transfection /expression vector

The FoF1 ATP synthase of mitochondria, chloroplasts and bacteria couples the energy of the proton motive force to the synthesis of ATP. In the case of bacteria such as Escherichia coli, the enzyme also acts as an ATP-driven proton pump. Studies of the multiple subunit complex have shown that catalysis occurs in the soluble F1 sector while transport is carried out in the membranous Fo; however, very little is known about the how these two types of activities are linked. The long-range goal of this proposal is to understand the molecular mechanism of coupling between transport functions. To do this, the main tool will be the genetic technique of second-site reversion. Recently, several second- site mutations near the carboxyl terminus of the E. coli gamma subunit were found to suppress the effect of an amino terminal mutation, gammaMet-23-Lys, which caused extremely inefficient coupling. Because the second-site mutations were identified based on a functional selection, the suppressors in turn became implicated in coupling. Furthermore, because linkage between catalysis and transport must involve several subunits, regions of the gamma subunit defined by the suppressors should interact with others subunits. The first specific aim will seek to identify residues in those subunits by isolating a series of suppressor mutations. Taken together, a chain of interacting residues will define segments of the complex that participate in coupling. The second specific aim will take advantage of transport and coupling mutations to characterize how transport regulates catalysis. For the first time, kinetic parameters of partial reactions of these mutant enzymes will be measured to elucidate the catalytic steps which are coupled to transport. The third specific aim will obtain structural information which is critical for understanding the mechanism of coupling. The proximal position of residues suggested by the suppressor studies will be tested by replacing them with cysteine and assayed for formation of disulfide bonds. The cysteine mutations will also be useful for specific placement of spectroscopic probes to detect changes in conformation during enzyme activity. Finally, long-range plans include crystallization of F1 subunits for X-ray diffraction and structure determination so that the perturbations of mutations affecting coupling can be assessed within the protein structure. Each specific aim will contribute equally towards understanding how catalysis is coupled to transport.

线粒体，叶绿体和细菌夫妇的FOF1 ATP合酶 质子动力与ATP合成的能量。 在 细菌（例如大肠杆菌）的病例，该酶也充当 ATP驱动的质子泵。 多个亚基综合体的研究 表明催化发生在可溶性F1扇区，而运输是 在膜的fo中进行；但是，关于 这两种类型的活动如何链接。 远程目标 该建议是了解耦合的分子机制 在运输功能之间。 为此，主要工具将是 第二站点归还的遗传技术。 最近，几个 大肠杆菌伽马亚基的羧基末端附近的位点突变 发现抑制氨基末端突变的作用， Gammamet-23-lys，导致极其效率的耦合。 因为 基于功能鉴定了第二位点突变 选择，抑制器反过来涉及耦合。 此外，因为催化与运输之间的连锁必须涉及 抑制器定义的几个亚基，伽马亚基的区域 应该与其他亚基互动。 第一个具体目标将寻求 通过隔离一系列亚基中的残留物 抑制突变。 两者一起，一系列相互作用的残基 将定义参与耦合的综合体的细分。 这 第二个特定目标将利用运输和耦合 突变以表征转运如何调节催化。 为了 第一次，这些突变体的部分反应的动力学参数 将测量酶以阐明催化步骤 连续运输。 第三个特定目标将获得结构 对于理解机制至关重要的信息 耦合。 抑制器建议的残基的近端位置 研究将通过用半胱氨酸代替并进行测定来测试研究 二硫键的形成。 半胱氨酸突变也将很有用 用于特定放置光谱探针以检测变化 酶活性期间的构象。 最后，远程计划包括 F1亚基的结晶用于X射线衍射和结构 决心，以便影响耦合的突变的扰动 可以在蛋白质结构中进行评估。 每个特定目标将 同等地理解催化如何与 运输。