ATP BINDING SITE PHOTOAFFINITY PROBES FOR F1-ATPASE

在一个分六个阶段的实验设计中，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 对牛心和大鼠肝脏线粒体F1-ATPase酶的影响。这个 我们的方法的新颖性是使用了新的、定点定向的光亲和标记 由我们合成的，每个都具有二苯甲酮作为光活性 官能团：BzATP/BzADP；BzAF(荧光体， 核苷酸位点选择性探针)和碘-BzATP(一种重原子修饰 BzATP)。这些光亲和探针的~3H、~(32)P和~(14)C-衍生物 提供灵敏的亚基位置检测手段和 光解诱导的共价结合的精氨酸-肽图谱 F1，以及评估低水平催化活性的能力 在光解之前与这些ATP底物中的几个类似物结合。两者都有 天然的和核苷酸耗尽的BHF1应根据 亚基定位、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”(即催化的)和 (2)“不可交换”(即假定无催化能力) 位点数/摩尔F1。试图独家地给“不可交换”贴上标签 将执行核苷酸位置，如果成功，将对ATP的影响 应对营业额进行评估。 新合成的pH敏感荧光探针BzAF(一种荧光素 衍生品)，应首次作为直接、 特定部位监测催化剂的潜在构象变化 F1的位置(S)，可能会因[H]的变化而发生 系统。分子的荧光寿命和偏振的测定 结合的荧光素部分可以提供关于即时的关键信息 腺嘌呤核苷酸结合位点的环境，以及产量信息 关于这类地点可能存在的环境异质性。 通过与L.M.Amzel的合作安排，标记的Iodo-BZ-核苷酸 大鼠肝F1代应进行结晶和X射线衍射 分析以确定至少其中之一的拓扑轨迹 这种无处不在的、真正支持生命的酶上的催化部位区域。

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.