MECHANISM OF ATP SYNTHESIS BY THE RESPIRATORY CHAIN

呼吸链合成 ATP 的机制

• 批准号: 3271515
• 负责人: RICHARD L CROSS
• 金额: $ 12.63万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-05-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271515
• 关键词: adenosine diphosphate; adenosine triphosphate; affinity chromatography; arginine; bioenergetics; cellular respiration; chemical structure function; cofactor; cow; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; enzyme substrate complex; guanosine triphosphate; heart; hydrolysis; inosine; lysine; membrane; mitochondria; nucleotide metabolism; oxidative phosphorylation; photochemistry; protein engineering; protein sequence; radiotracer; stoichiometry

The catalytic site for ATP synthesis by mitochondrial and bacterial oxidative phosphorylation is on a membrane-bound enzyme, F1, that can be readily prepared in a soluble form. The structure, mechanism, and regulation of F1 is the subject of this proposal. Rate constants for several elementary steps in catalysis by F1 will be determine by direct measurements employing rapid kinetic techniques, oxygen exchange reactions, isotope trap procedures, and Sephadex centrifuge column binding assays. The effects that enzyme modification and purification of monomeric catalytic subunit have on the apparent strong cooperative interactions between multiple catalytic sites will be studied. The results may also provide information regarding the number of functional sites and their location within or between subunits. The adenine nucleotide sites on F1 show asymmetric behavior. Experiments are designed to determine whether this is due to ligand-induced site-site cooperativity or to permanent structural asymmetry. Some of the nucleotide sites appear to have an regulatory role. We will prepare F1 with different known compositions of adenine nucleotide at these sites and measure transient and steady-state kinetic properties of soluble and membrane-bound enzyme. It has been proposed that F1 has separate catalytic sites for ATP synthesis and ATP hydrolysis. We will examine possible alternative explanations for several of the observations cited in support of this idea. Energized release of 125I-labeled mitochondrial inhibitor protein will be used as a measure of the fraction of F1 that participates in ATP synthesis of following modification by "uni-directional" reagents. Also, measurements of rate constants for GTP and ITP cleavage at a single site may explain the failure of these substrates to drive energy-requiring membrane processes. F1 binds two anionic substrates and has essential lysine and arginine residues that may participate in change interactions at the catalytic site. We have designed and synthesized a new affinity probe for lysine at adenine nucleotide sites. Using this reagent and phenylglyoxal we will attempt to modify, isolate, and sequence peptides containing essential residues.

线粒体和细菌合成ATP合成的催化位点 氧化磷酸化在膜结合的酶F1上，可以是 容易以可溶性形式制备。 结构，机制和 F1的监管是该提案的主题。 F1催化中几个基本步骤的速率常数为 通过使用快速动力学技术的直接测量来确定氧气 交换反应，同位素陷阱程序和Sephadex离心柱 绑定测定。 酶修饰和纯化的影响 单体催化亚基具有明显强的合作社 将研究多个催化位点之间的相互作用。 结果 还可以提供有关功能站点数量和 它们在亚基内或之间的位置。 F1上的腺嘌呤核苷酸位点显示不对称行为。 实验 旨在确定这是否是由于配体诱导的位点 合作或永久性结构不对称。 一些核苷酸 站点似乎具有调节作用。 我们将准备不同的F1 腺嘌呤核苷酸在这些位点的已知组成并测量 可溶性和膜结合的瞬态和稳态动力学特性 酶。 已经提出F1具有单独的ATP合成催化位点 和ATP水解。 我们将研究可能的替代解释 引用的几项观察是为了支持这一想法。 充满活力 释放125i标记的线粒体抑制剂蛋白将被用作 参与ATP合成的F1分数的度量 通过“单向”试剂进行修饰。 另外，测量 在单个站点上GTP和ITP裂解的速率常数可以解释 这些底物无法驱动能量征收膜过程。 F1结合两个阴离子底物，并具有必需的赖氨酸和精氨酸 可能参与催化时变化相互作用的残留物 地点。 我们已经设计并合成了赖氨酸的新亲和力探针 腺嘌呤核苷酸位点。 使用该试剂和苯基乙二醇，我们将 尝试修改，隔离和序列含有必需的肽 残留物。