Mechanisms of Copper Transport and Catalysis

项目总结 铜是真核细胞利用的一种必需微量元素，是一种酶促氧化还原辅因子。在高处 浓度的金属是有毒的，复杂的动态平衡机制已经开发出来，以确保 它被维持在正确的生理水平。原核生物，如革兰氏阴性病原体，具有 降低了对金属的要求，并对铜表现出很好的敏感性 哺乳动物细胞。出于这个原因，巨噬细胞利用致病性铜中毒作为其 防御入侵者，而后者通过提升其出口商来对抗宿主防御 机械设备。我们的研究主要集中在铜的传输和催化机制上，分为两个部分 分成三个独立但重叠的项目。项目1考察了反应机理 以氨基甘氨酸单加氧酶(PHM)为代表的单核单加氧酶是唯一的酶 能够将关键的翻译后酰胺安装到多肽激素中。我们已经确认了三个 尚未解答的重要问题--(一)电子转移途径(二)化学形态 反应中间体和(Iii)底物触发的机理，并设计了一种方法 审问每一个人。项目2通过对致病铜出口的研究，重点研究致病铜出口的机制。 CusCBAF的大肠杆菌出口商。在这里，我们使用一种新的标记方法来跟踪金属离子的通量 它们在泵中移动。我们建立在成功确定角色的最新结果的基础上 CusF和CusB的金属化，铜从CusF到CusB的转移速率，以及 CusF-CusB络合物作为共享的配体中间体。未回答的问题包括 CusF对CusA金属交换的机理，金属结合残基在选择性和选择性中的作用 效率，以及在CUSS调节子中新的His2Phe铜(I)/银(I)结合位点的作用。项目3 通过调查新发现的铜的结构/功能，将我们的计划带入新的领域- 依赖蛋白激酶MEK1/2和ULK1/2参与细胞信号转导和肿瘤发生。集成的 所有这三个项目的方法都利用了PI在开发可以询问 铜的化学，尤指与氧反应的铜(I)态，其形式为 其中的铜在牢房里被贩卖。这项努力的两个方面特别有用：(1)发展 利用X射线吸收和发射的铜(I)配位结构的金属定向探针 光谱学和(Ii)利用硒蛋氨酸取代偶联的标记策略的发展 以Se K EXAFS。这项研究的预期结果是更好地理解生物学如何 利用铜的基本化学成分实现功能，认识到功能需要 在选择性金属化的要求、催化的结构决定因素和 由强健的细菌输出泵诱导的致病毒力。

PROJECT SUMMARY Copper is an essential trace element utilized by eukaryotic cells as an enzymatic redox cofactor. At high concentrations the metal is toxic, and complex homeostatic mechanisms have developed to ensure that it is maintained at the correct physiological level. Prokaryotes such as gram negative pathogens have a diminished requirement for the metal, and show sensitivity to copper at levels well tolerated by mammalian cells. For this reason, macrophages utilize pathogenic copper toxicity as part of their defense against invaders while the latter combat the host-defense by upregulating their exporter machinery. Our research is focused on the mechanisms of copper transport and catalysis and is divided into three separate but overlapping projects. Project 1 examines the reaction mechanism of mononuclear monooxygenases exemplified by peptidylglycine monooxygenase (PHM), the only enzyme capable of installing the critical post-translational amide into peptide hormones. We have identified three important unanswered questions – (i) the pathway for electron transfer (ii) the chemical speciation of reaction intermediates and (iii) the mechanism of substrate triggering, and have designed an approach for interrogating each. Project 2 focuses on the mechanism of pathogenic copper export via a study of the CusCBAF exporter of E. coli. Here we use a novel labeling approach to track the flux of metal ions as they move through the pump. We build on recent results that have successfully determined the role CusF and CusB in metalating CusA, the rates of copper transfer from CusF to CusB, and the identity of the CusF-CusB complex as a shared ligand intermediate. Unanswered questions include the mechanism of CusF to CusA metal exchange, the role of metal-binding residues in selectivity and efficiency, and the function of the novel His2Phe Cu(I)/Ag(I) binding site in the CusS regulator. Project 3 takes our program into new territory by investigating structure/function of the newly discovered copper- dependent kinases MEK1/2 and ULK1/2 involved in cell signaling and tumorogenesis. The integrated approach to all three projects leverages the Pi's track record in developing toolsets that can interrogate the chemistry of copper, but particularly the Cu(I) state which reacts with oxygen, and is the form in which copper is trafficked in the cell. Two aspects of this effort are of particular utility: (i) development of metal-directed probes of Cu(I) coordinate structure utilizing x-ray absorption and emission spectroscopies and (ii) development of labeling strategies utilizing selenomethionine substitution coupled to Se K EXAFS. The expected outcome of the research is a better understanding of how biology leverages the fundamental chemistry of copper to achieve functionality, recognizing that function requires balance between the requirements for selective metalation, the structural determinants of catalysis, and pathogenic virulence induced by robust bacterial export pumps.