MECHANISM OF ATP SYNTHESIS BY RESPIRATORY CHAIN

呼吸链合成 ATP 的机制

• 批准号: 3271511
• 负责人: RICHARD L CROSS
• 金额: $ 20.08万
• 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 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271511
• 关键词: adenosine diphosphate; adenosine triphosphate; adenosinetriphosphatase; affinity labeling; bioenergetics; chemical binding; chemical structure function; crosslink; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; guanosine triphosphate; high performance liquid chromatography; hydrolysis; inosine; membrane; mitochondria; nucleotide analog; nucleotide metabolism; oxidative phosphorylation; phosphatase inhibitor; photochemistry; radiotracer

The catalytic site for ATP synthesis by mitochondrial oxidative phosphorylation is on a membrane-bound enzyme, MF1, that can be readily prepared in a soluble form. The structure mechanism, and regulation of MF1 is the subject of this proposal. MF1 contains three catalytic and three noncatalytic nucleotide binding sites. We find diadenosine-5'-polyphosphates, such as AP5A and AP6A, to inhibit F1. Inhibition requires the presence of at least one vacant noncatalytic site in addition to vacant catalytic sites; suggesting that the bifunctional ligand spans a catalytic and a noncatalytic site. To test this, we will determine the effect of AP6A binding on the number of remaining accessible catalytic and noncatalytic sites. We will also synthesize the bifunctional 2-azido analog of AP6A and use it to photolabel MF1. We ill determine whether the crosslinked sites are on the same or adjacent subunits and whether the modified residues re known to contribute to the catalytic and noncatalytic sites. Addition studies of the structure of nucleotide sites on MF1 will include determining whether structural asymmetry affects the photolabeling of residues at the noncatalytic site. We will also synthesize the 6-azido analog of ATP and attempt to identify additional regions of the nucleotide binding domains. MF1 has been reported to show triphasic kinetics in the micromolar concentration range for ATP. We plan to test the possibility that one of the breaks in the kinetic plots comes from filling a vacant noncatalytic sites. Using a hydrolysis substrate such as GTP, we will test the effect of low concentrations of 2-azido-ADP on the activity. If the rate is altered, we will photolyze and identify the site involved. We will assay for a slow transphosphorylation reaction between adjacent catalytic and noncatalytic sites that may be involved in regulation. These experiments will include testing for phosphoryl transfer between nucleotide at the catalytic site and 2-azido nucleotide that has been covalently tethered to a noncatalytic site. We will attempt to assess the effect of structural asymmetry on the mechanism of MF1 and MF0F1. We will determine whether the permanent structural asymmetry evident for noncatalytic sites on MF1 causes pre-existing functional asymmetry among the catalytic sites. We will also determine whether the unique properties of the noncatalytic sites are randomized during ATP hydrolysis by MF0F1. This approach should provide a test a notary mechanism that has been proposed.

通过线粒体氧化的ATP合成的催化位点 磷酸化在膜结合的酶MF1上，可以是 容易以可溶性形式制备。 结构机制，以及 MF1的调节是该提案的主题。 MF1包含三个催化和三个非催化核苷酸 绑定位点。 我们发现多丁胺-5'-聚球磷酸盐，例如AP5A 和AP6A，以抑制F1。 抑制需要AT 除空催化外，至少一个空置的非催化位点 站点；提示双功能配体跨越催化 和一个非催化部位。 为了测试这一点，我们将确定 AP6A绑定对剩余可访问次数的影响 催化和非催化位点。 我们还将合成 双功能的2-齐多AP6A类似物，并将其用于光标记MF1。 我们确定交联的站点是在相同的还是 相邻亚基以及是否已知修饰的残基已知 有助于催化和非催化位点。 MF1上核苷酸位点结构的加法研究将 包括确定结构不对称是否影响 在非催化部位的残基的光标记。 我们也会 合成ATP的6-齐多类似物，并尝试识别 核苷酸结合结构域的其他区域。 据报道，MF1在微摩尔中显示三倍体动力学 ATP的浓度范围。 我们计划测试 动力学图中的休息之一来自空缺 非催化位点。 使用水解底物，例如GTP，我们 将测试低浓度2-齐达多-ADP对 活动。 如果速率变化，我们将光水解并识别 涉及的网站。 我们将测定在缓慢的磷酸化反应之间 可能参与的相邻催化和非催化位点 规定。 这些实验将包括测试磷酸 在催化位点和2-氮杂的核苷酸之间转移 核苷酸已共价绑在非催化 地点。 我们将尝试评估结构不对称对 MF1和MF0F1的机制。 我们将确定是否 对于非催化位点的永久性结构不对称性 MF1导致催化中现有的功能不对称性 站点。 我们还将确定是否是 在MF0F1的ATP水解过程中，非催化位点是随机的。 这种方法应提供一种测试的公证机制 已提出。