MECHANISTIC STUDIES ON A CO2+ DEPENDENT MAP FROM E COLI

大肠杆菌 CO2 相关图谱的机理研究

• 批准号: 6138579
• 负责人: RICHARD C HOLZ
• 金额: $ 16.54万
• 依托单位: UTAH STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6138579
• 关键词: Escherichia coli; X ray crystallography; active sites; aminopeptidase; bacterial proteins; carbon dioxide; cobalt; enzyme inhibitors; enzyme mechanism; enzyme substrate analog; histidine; iron; methionine; microorganism culture; nuclear magnetic resonance spectroscopy; threonine; tyrosine

Thee long range goals of this research are to define the reaction mechanism and substrate specificity of the cobalt(II)-dependent methionine aminopeptidase from Escherichia coli (MAP). In eukaryotic as well as in prokaryotic cells, methiomine aminopeptidases selectively cleave methionine residues from the N-termini of terminal polypeptide chains. In the cytosol of eukaryotes, all proteins are initiated with an N-terminal methionine. The composition of mature N-termini play important roles in the directed degradation and cellular targeting of proteins involved in signal transduction, protein trafficking, cancer cell growth, and viral infection. Recently, one of the two MAP's found in yeast was shown to be the target of the angiogenic chemotherapeutic agents fumagilln and its analog AGM 1470. Therefore, MAP's appear to play a critical role in the proliferation of endothelial cells and likely serve as important targets for inhibiting the growth and proliferation of tumors. As isolated from several bacterial and mammalian sources, MAPs are maximally stimulated by two g-atoms of Co(II). On the other hand, the addition of Zn(II) or Mg(II) to apo- MAP's do not provide active enzymes. The MAP from E. coli has been shown by X-ray crystallography to contain a dinuclear cobalt(II) active site. Therefore, MAP is a member of a new class of cobalt- containing enzymes that includes mammalian MAP' isolated from both porcine liver and humans. Our approach will be to utilize the X-ray crystal structures of the MAP's from E. coli and P. furiosus in conjunction with site mutagenesis, biochemical, and spectroscopic methods to gain fingerprints of each step in the catalytic mechanism. The MAP from E. coli is an ideal enzyme for a study of this type because it is small, highly soluble, readily available in large amounts, and is capable of being genetically manipulated. The specific aims of this proposal are to: 1) define the structural and magnetic properties of the dicobalt(II) an diiron(II) MAP enzymes, 2) characterize how substrate analog inhibitors interact with both the dicobalt(II) and diiron(II) MAP enzymes, 3) determine whether the nucleophilic hydroxide moiety, a key element in the proposed peptide bond cleavage mechanism, is coordinated to one or both metal ions in MAP, 4) prepare and purify variant enzymes with altered active site carboxylic acid, histidine, threonine, and tyrosine residues, 5) provide evidence for and propose a detailed mechanism for MAP. It is anticipated that the successful completion of the studies described in this proposal will provide new insights into the hydrolytic reaction catalyzed by MAP and will ultimately assist in the design and synthesis of new chemotherapeutic agents targeted specifically to MAP's.

这项研究的远距离目标是定义反应 钴（II）依赖性的机理和底物特异性 大肠杆菌的蛋氨酸氨基肽酶（地图）。 在真核生物中 以及在原核细胞中，甲基二氨氨酸氨基肽酶有选择地 从末端多肽的N末端切割蛋氨酸残基 链。 在真核生物的细胞质中，所有蛋白质均与 N末端蛋氨酸。 成熟的N-Termini播放的组成 在定向降解和细胞靶向中的重要作用 参与信号转导，蛋白质运输，癌症的蛋白质 细胞生长和病毒感染。 最近，发现了两个地图之一 在酵母中，证明是血管生成化学治疗的靶标 代理商Fumagilln及其模拟AGM 1470。因此，地图似乎是 在内皮细胞的增殖中起关键作用， 可能是抑制增长和的重要目标 肿瘤的扩散。从几个细菌和 哺乳动物来源，地图受到两个g原子的最大刺激 CO（II）。 另一方面，将Zn（ii）或mg（ii）添加到apo- 地图不提供活性酶。 大肠杆菌的地图已经 由X射线晶体学显示，含有二核钴（II）活性 地点。 因此，MAP是一类新的钴的成员 包含包括从两者中孤立的哺乳动物地图的酶 猪肝和人类。 我们的方法是利用X射线晶体结构 地图来自大肠杆菌和狂热疟原虫与位点诱变， 生化和光谱法以获取每个步骤的指纹 在催化机制中。 大肠杆菌的地图是理想的酶 这种类型的研究是因为它很小，高度可溶，很容易 大量可用，并且能够在遗传上获得 操纵。 该建议的具体目的是：1）定义 Dicobalt的结构和磁性（II）AN DIOIRN（II）地图 酶，2）表征底物类似物抑制剂如何与 Dicobalt（II）和Diiron（II）地图酶，3）确定是否是否 亲核氢氧化物部分，这是拟议肽的关键元素 粘结裂解机制，在一个或两个金属离子中协调 地图，4）准备和纯化变体酶，有效位点改变 羧酸，组氨酸，苏氨酸和酪氨酸残基5）提供 证据并提出了地图的详细机制。 预计成功完成了研究 该提案中描述的将为水解提供新的见解 反应由地图催化，最终将有助于设计和 专门针对地图的新化学治疗剂的合成。