ATP BINDING SITE PHOTOAFFINITY PROBES FOR F1-ATPASE

F1-ATPase 的 ATP 结合位点光亲和探针

• 批准号: 3290963
• 负责人: PETER S COLEMAN
• 金额: $ 12.91万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3290963
• 关键词: adenosine triphosphate; adenosinetriphosphatase; affinity labeling; chemical binding; chemical synthesis; conformation; enzyme structure; enzyme substrate analog; fluorescence polarization; fluorescent dye /probe; gel electrophoresis; heart; liver; mitochondria; photochemistry

In a six-stage experimental design, we shall study, in detail, the relative locations and catalytic functions of the adenine nucleotide binding sites on the beef heart and rat liver mitochondrial F1-ATPase enzymes. The novelty of our approach uses new, site-directed photoaffinity labels synthesized by us, each possessing benzophenone as the photoactive functional group: BzATP/BzADP; BzAF (a fluorescent, nucleotide-site-selective probe); and Iodo-BzATP (a heavy atom modification of BzATP). 3H-, 32P- and 14C-derivatives of these photoaffinity probes offer the means of sensitive detection of subunit location and arginine-peptide mapping upon photolytically-induced covalent binding to F1, and, as well, the ability to assess low levels of catalytic activity with several of these ATP substrate analogs prior to photolysis. Both native and nucleotide-depleted BHF1 shall be examined with regard to the subunit location, binding stoichiometry, and catalytic characteristics of Bz-nucleotide covalent binding to (1) "exchangeable" (i.e., catalytic) and (2) "non-exchangeable" (i.e., presumably non-catalytically competent) sites/mol F1. Attempts to label, exclusively, the "non-exchangeable" nucleotide sites will be performed, and, if successful, the effects on ATP turnover shall be assessed. The newly synthesized, pH-sensitive fluorescent probe, BzAF (a fluorescein derivative), shall be employed, for the first time, as a direct, site-specific monitor of potential conformational changes at the catalytic site(s) of F1 that may occur in response to shifts in the [H+] of the system. Determinations of fluorescence lifetimes and polarization of the bound fluorescein moiety can provide critical information on the immediate environment of the adenine nucleotide binding sites, and yield information on the possible environmental heterogeneity of such sites. Via a collaborative arrangement with L.M. Amzel, Iodo-Bz-nucleotide labeled rat liver F1 shall be subjected to crystallization and X-ray diffraction analysis in order to determine the topological locus of at least one of the catalytic site domains on this ubiquitous, truly life-supporting enzyme.

在一个六阶段的实验设计中，我们将详细研究， 腺嘌呤核苷酸结合位点的位置和催化功能 牛心脏和大鼠肝脏线粒体F1-ATP酶。 的 我们的方法的新奇使用新的，定点的光亲和标记 我们合成的，每一个都有二苯甲酮作为光活性 官能团：BzATP/BzADP; BzAF（荧光， 核苷酸位点选择性探针）和Iodo-BzATP（重原子修饰 BzATP）。 这些光亲和探针的3 H-、32 P-和14 C-衍生物 提供了灵敏检测子单元位置的手段， 光解诱导共价结合后的精氨酸-肽图谱 F1，以及评估低水平催化活性的能力 与这些ATP底物类似物中的几种在光解之前反应。 两 应检查天然和核苷酸缺失的BHF 1， 亚基位置，结合化学计量，和催化特性， Bz-核苷酸共价结合到（1）“可交换的”（即，催化剂）和 (2)“不可交换的”（即，可能无催化活性） 位点/mol F1。 试图专门标记“不可交换” 将进行核苷酸位点，如果成功，对ATP的影响 营业额将予以评估。 新合成的pH敏感的荧光探针BzAF（一种荧光素 第一次，作为一个直接的， 在催化剂上的潜在构象变化的位点特异性监测 F1的一个或多个位点，可能响应于F1的[H+]的变化而发生。 系统 荧光寿命和偏振态的测定 结合的荧光素部分可以提供关于直接 腺嘌呤核苷酸结合位点的环境和产量信息 这些地点可能存在的环境异质性。 通过与L.M.的合作安排，Amzel，碘-Bz-核苷酸标记 大鼠肝脏F1应进行结晶和X射线衍射 分析，以确定至少一个拓扑轨迹， 这种无处不在的真正支持生命的酶上的催化位点域。