Covalent Flavin Coenzyme in Flavoenzyme Catalysis

描述（由申请人提供）：本申请寻求继续资助，以研究人单胺氧化酶A和B（MAO A和MAO B）的结构和功能，其位于线粒体外膜中并催化胺神经递质和异生物胺的氧化。在过去的支持期间，每种酶的三维结构的阐明导致了新的问题，这将需要一种综合的方法来研究它们的膜结构和功能，这些结构和功能由五个具体目标来解决。扩展的结构研究将寻求与临床使用的抑制剂复合的MAO A的更高分辨率结构以及模型膜纳米盘中两种酶的结构，以测试当前的MAO-膜相互作用模型。将研究作为人类选择性突变的MAO A Glu 151 Lys突变的结构/功能作用，以了解该突变的功能作用及其在改变人类MAO A寡聚状态中的拟定作用。关于抑制剂功能和产生的过氧化氢对MAO催化的降解作用的相关性，将在人和其他哺乳动物线粒体中确定外线粒体膜中的MAO A和MAO B的拓扑定位。机制研究将调查的作用，酪氨酸“芳香笼”位于活性位点前的共价黄素和他们的耦合偶极胺活化的影响。从模型计算的预测，Lys 296在底物还原的黄素与氧的反应中的作用，催化营业额将确定使用诱变方法。荧光和自旋标记EPR研究将被用作探针的寡聚状态和结构的膜结合形式的每种酶和结果与现有的结构数据进行比较。机理研究将扩展到测试炔属和环丙胺不可逆抑制剂对MAO A和MAO B的基于机理的抑制的可能模型。这项研究的结果应该提供新的见解的分子基础的发展，新的和高度特异性的单胺氧化酶A和单胺氧化酶B抑制剂，将有临床效益。MAO B抑制剂将作为神经保护剂用于预防和治疗与年龄相关的神经障碍。MAO A抑制剂作为新型抗抑郁药可能具有临床益处。

DESCRIPTION (provided by applicant): This application seeks continued funding to investigate the structure and function of human Monoamine Oxidases A and B (MAO A and MAO B) which are located in the outer mitochondrial membrane and catalyze the oxidation of amine neuro-transmitters and xenobiotic amines. The elucidation of the 3- dimensional structure of each enzyme during the past period of support has resulted in new questions which will require an integrated approach of investigation of their membrane structures and functions which are addressed by five specific aims. Extended structural studies will seek a higher resolution structure of MAO A complexed with clinically used inhibitors as well as the structures of both enzymes in model membrane Nanodiscs to test current models of MAO-membrane interactions. The structural/functional role of a MAO A Glu151Lys mutation which is a selection mutation for the human will be investigated to understand the functional role of this mutation and its proposed role in altering the oligomeric state of human MAO A. The topological localization of both MAO A and MAO B in the outer mitochondrial membrane will be determined in human and other mammalian mitochondria with regard to correlations of inhibitor function and the degradative effect of hydrogen peroxide produced on MAO catalysis. Mechanistic studies will investigate the role of a tyrosine "aromatic cage" situated in the active site in front of the covalent flavin and the proposed influence of their coupled dipoles in amine activation. From predictions of model calculations, the role of Lys296 in the reaction of substrate-reduced flavin with oxygen in catalytic turnover will be determined using mutagenesis approaches. Fluorescence and spin label EPR studies will be used as probes of the oligomeric states and structures of the membrane-bound forms of each enzyme and the results compared with available structural data. Mechanistic studies will be extended to test possible models for the mechanism- based inhibition of MAO A and MAO B by the acetylenic and cyclopropyl amine irreversible inhibitors. The results of this investigation should provide new insights into the molecular basis for the development of new and highly specific MAO A and MAO B inhibitors that would have clinical benefit. The MAO B inhibitors would function as neuroprotective agents for the prevention of and treatment of age-related neurological disorders. The MAO A inhibitors could have clinical benefit as new types of anti-depressants.