Covalent Flavin Coenzyme in Flavoenzyme Catalysis

描述（由申请人提供）：本申请寻求持续资助，以研究人类单胺氧化酶A和B （MAO A和MAO B）的结构和功能，它们位于线粒体外膜，催化胺类神经递质和外源胺的氧化。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，这是由五个具体目标解决的。进一步的结构研究将寻求更高分辨率的MAO a与临床使用的抑制剂配合的结构，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。毛的结构/功能角色Glu151Lys突变为人类选择突变将调查了解这种突变的功能作用及其提出的角色在改变人类的低聚物的状态毛a两毛毛a和B的拓扑定位在线粒体外膜将决定在人类和其他哺乳动物线粒体抑制函数的相关性和过氧化氢的降解效果由MAO催化产生。机制研究将探讨位于共价黄素前活性位点的酪氨酸“芳香笼”的作用，以及它们的偶极子偶极子对胺活化的影响。根据模型计算的预测，Lys296在底物还原黄素与氧催化转化反应中的作用将通过诱变方法确定。荧光和自旋标记EPR研究将被用作每种酶的膜结合形式的寡聚态和结构的探针，并将结果与现有的结构数据进行比较。机理研究将进一步扩展，以测试乙炔和环丙胺不可逆抑制剂对MAO A和MAO B的抑制机制的可能模型。该研究结果将为开发具有临床效益的新型高特异性MAO A和MAO B抑制剂的分子基础提供新的见解。MAO B抑制剂可作为神经保护剂预防和治疗与年龄相关的神经系统疾病。MAO A抑制剂作为新型抗抑郁药具有临床应用价值。

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.