STRUCTURE AND REACTIVITY OF COPPER AMINE OXIDASES

含铜胺氧化酶广泛分布于自然界 并参与生物伯胺的代谢， 它在心血管系统中有多种功能， 胃肠道和神经系统;更一般地，原发性 胺与细胞增殖的控制有关， 分化 胺氧化酶也负责 结缔组织结构蛋白（弹性蛋白和 胶原蛋白）。 主要目标 主要研究内容有：（1）阐明 含铜胺氧化酶的结构，特别是 活性中心;（2）确定铜的作用，有机 辅因子，以及催化中的任何铜-辅因子相互作用 胺氧化酶的作用机制;（3）开发详细的 胺氧化酶之间的结构/反应性/功能相关性。 将采用几种实验方法。 协调一致 计划努力获得X射线晶体结构，至少 一种胺氧化酶。 同时，从活性肽衍生的肽 几种酶的位点，并含有辅因子的衍生物 将被制备和结构表征。 共振拉曼， EPR，脉冲EPR和ENODR光谱将用于探测 各种氧化态的有机辅因子的结构， 它与铜离子可能的相互作用。 也是 提出利用Cu（II）的性质作为内置探针 通过使用几种不同的光谱 技术，包括共振拉曼、EPR（CW和脉冲）、光学 （吸收，圆二色性，磁性圆二色性） 光谱学和溶剂核磁弛豫色散 测量. 我们打算深入研究静止的，还原的， 几种胺的贫铜和金属取代形式 氧化酶 停流和低温动力学研究将 以进一步界定机制作用， 铜 旨在捕获或稳定催化剂的实验 提出了中间体，以结构表征 这些中间体。 底物还原形式的酶 将受到特别关注。 光谱和动力学 胺氧化酶与小分子相互作用的研究 （抑制剂、底物类似物、产品）。 总的来说，这些实验的结果应该提供 关于结构和功能的重要新信息 这些不同的酶。

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; more generally, primary amines are implicated in the control of cell proliferation and differentiation. Amine oxidases are also responsible for the crosslinking of connective tissue structural proteins (elastin and collagen) in aorta, lung, and cartilage. The principal objectives of the proposed research are: (1) to elucidate the molecular structure(s) of copper-containing amine oxidases, particularly of the active sites; (2) to determine the role of copper, the organic cofactor, and any copper-cofactor interactions in catalytic mechanisms of amine oxidases; (3) to develop detailed structure/reactivity/function correlations among amine oxidases. Several experimental approaches will be employed. A concerted effort is planned to obtain the X-ray crystal structure of at least one amine oxidase. Concurrently, peptides derived from the active sites of several enzymes and containing derivatives of the cofactor will be prepared and structurally characterized. Resonance Raman, EPR, pulsed EPR, and ENODR spectroscopy will be used to probe the structure of the organic cofactor in various oxidation states and its possible interactions with the copper ions. It is also proposed to exploit the properties of Cu(II) as a built-in probe of the active site by using several different spectroscopic techniques, including resonance Raman, EPR (CW and pulsed), optical (absorption, circular dichroism, magnetic circular dichroism) spectroscopy and solvent nuclear magnetic relaxation dispersion measurements. We intend to intensively study the resting, reduced, copper-depleted, and metal-substituted forms of several amine oxidases. Stopped-flow and low-temperature kinetics studies will be carried out in order to further define mechanistic role(s) for copper. Experiments designed to trap or stabilize catalytic intermediates are proposed in order to structurally characterize any such intermediates. Substrate-reduced forms of the enzymes will receive particular attention. Spectroscopic and kinetics studies of the interactions of amine oxidases with small molecules (inhibitors, substrate analogs, products) are planned. Collectively, the results of these experiments should provide significant new information concerning the structure and function of this diverse group of enzymes.