STRUCTURE AND REACTIVITY OF COPPER AMINE OXIDASES

含铜胺氧化酶广泛分布于自然界， 参与生物源伯胺的代谢， 心血管、胃肠道和 神经系统 胺氧化酶也负责 结缔组织结构蛋白（弹性蛋白和 胶原蛋白）。 最近，胺氧化酶作为第一个例子出现 一种可能被证明是一种全新的酶，也就是说， 后修饰的氨基酸侧链存在于 活性位点，并在催化中具有氧化还原作用。 主要目标是 为了阐明分子结构，特别是活性位点， 以及含铜胺氧化酶的催化机理。 协调的结构，光谱和机械实验将 同时进行。 具体目标是：（1）完成 氧化豌豆苗胺氧化酶的晶体结构及制备 还原的Cu（I）形式的结晶样品和与 X射线用底物或底物类似物及抑制剂 衍射测量;（2）详细定义胺的机理 氧化酶，包括铜的作用，并严格测试 假设Cu（I）-半醌态是催化剂， 与氧反应的中间体;（3）阐明电子 结构和相关的结构性质的Cu（II）网站， 氧化态和底物还原态，以及Cu（I）- 半醌态 为了补充X射线分析，计划 分离并测序活性位点肽。 磁性圆 二向色性、X射线吸收（EXAFS）、共振拉曼、ENDOR和cw- 脉冲EPR光谱将用于表征铜 位点，包括Cu（I）-半醌状态。 形成和 Cu（I）-半醌状态的后续反应将是 温度跃变和停流循环试验研究 二色性 更多的机械信息将来自 使用“慢”胺底物的光谱和动力学实验， 与NO和H2 O2的反应。 平行光谱实验 甲胺脱氢酶和半乳糖氧化酶。 胺氧化酶，甲胺氧化酶， 和半乳糖氧化酶将提供更多关于结构的信息 酶之间的功能关系， 在其活性位点上的修饰的氧化还原活性氨基酸。

Copper-containing amine oxidases are widely distributed in nature and are involved in the metabolism of biogenic primary amines, which have a variety of functions in the cardiovascular, gastrointestinal, and nervous systems. Amine oxidases are also responsible for the crosslinking of connective tissue structural proteins (elastin and collagen). Recently, amine oxidases have emerged as the first examples of what may prove to be a wholly new class enzyme, that is, where a post-translationally modified amino acid side chain is present in the active site and has a redox role in catalysis. The principal goals are to elucidate the molecular structures, especially of the active sites, and the catalytic mechanisms of copper-containing amine oxidases. Coordinated structural, spectroscopic, and mechanistic experiments will be carried out concurrently. Specific goals are to: (1) complete the crystal structure of oxidized pea seedling amine oxidase and prepare crystalline samples of the reduced Cu(I) form and complexes with substrates, or substrate analogues, and inhibitors, for X-ray diffraction measurements; (2) define in detail the mechanism of amine oxidases, including the role(s) of copper, and rigorously test the hypothesis that the Cu(I)- semiquinone state is the catalytic intermediate that reacts with oxygen; (3) elucidate the electronic structures and related structural properties of the Cu(II) sites in both the oxidized and substrate-reduced states, and in the Cu(I)- semiquinone state. To complement the X-ray analysis, it is planned to isolate and sequence active-site peptides. Magnetic circular dichroism, X-ray absorption (EXAFS), resonance Raman, ENDOR, and cw- and pulsed-EPR spectroscopy will be used to characterize the copper sites, including the Cu(I)-semiquinone state. The formation and subsequent reactions of the Cu(I)-semiquinone state will be investigated by both temperature-jump and stopped-flow circular dichroism. Additional mechanistic information will come from spectroscopic and kinetics experiments with "slow" amine substrates and the reactions with NO and H2O2. Parallel spectroscopic experiments on methylamine dehydrogenase and galactose oxidase are planned. Comparative studies among amine oxidases, methylamine dehydrogenases, and galactose oxidase will provide additional insights into structure- function relationships among enzymes containing post-translationally modified, redox-active amino acids in their active sites.