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. ? ? ?
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