STRUCTURE AND REACTIVITY OF COPPER AMINE OXIDASES

含铜胺氧化酶广泛分布于自然界和 参与生物源伯胺的新陈代谢，这些伯胺 心血管、胃肠和心脏的多种功能 神经系统。胺氧化酶也是导致 结缔组织结构蛋白(弹性蛋白和 胶原蛋白)。最近，胺氧化酶成为第一个例子。 一种可能被证明是一种全新类别的酶，也就是说，其中一个 翻译后修饰的氨基酸侧链存在于 活性中心，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，特别是活性部位的分子结构， 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 氧化豌豆幼苗胺氧化酶的晶体结构及制备 还原的铜(I)形式的晶体样品及其与 X射线用底物或底物类似物和抑制剂 衍射测量；(2)详细定义了胺的作用机理 氧化物酶，包括铜的作用(S)，并严格测试 关于铜(I)-半喹酮态是催化剂的假设 与氧反应的中间体；(3)阐明电子 铜(II)晶位的结构和相关结构性质 氧化态和底物还原态，以及在铜(I)- 半喹酮状态。为了补充X射线分析，计划 分离活性部位多肽并测序。磁性圆盘 二向色性、X射线吸收(EXAFS)、共振拉曼、Endor和CW- 并将使用脉冲EPR光谱对铜进行表征 中心，包括铜(I)-半喹酮态。形成和形成了 铜(I)-半喹酮态的后续反应将是 通过温度跳跃和停流循环进行研究 二向色性。其他机械信息将来自 “慢”胺底物的光谱和动力学实验 与NO和过氧化氢的反应。平行光谱学实验 甲胺脱氢酶和半乳糖氧化酶是计划中的。 胺氧化酶、甲胺脱氢酶、 半乳糖氧化物酶将提供对结构的更多见解- 翻译后含酶之间的功能关系 修饰，氧化还原活性氨基酸在其活性部位。

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.