Continued support is requested to extend ongoing studies of the molecular basis for specific inhibitor design for the target enzymes: human mitochondrial Monoamine Oxidase A and B (MAO A and MAO B).This proposed work will extend current knowledge on the design of neuroprotectants and cardioprotectants that will serve a population that is susceptible to neurodegenerative and cardiovascular diseases on aging.
Three specific aims are proposed that will provide insights into the active site cavities of these membrane bound enzymes which should lead to the development of highly specific inhibitors: 1. The molecular basis of the I2 imidazoline binding site in MAO B and in MAO A will be determined by a combination of crystallographic analysis and binding studies using both purified and membrane bound preparations of both enzymes and testing a range of imidazoline ligands. Mutant forms of amino acid residues found to interact with the bound imidazoline ligands will be created and examined 2. To probe specific inhibitor binding to the entrance cavity of MAO B;The functional role of the gating residues Ile199 and Tyr326 in human MAO B will be determined by single and double Ala mutations and determination of the catalytic and inhibitor binding properties of these mutant enzyme forms in comparison with those of WT enzyme. Rapid reaction kinetic studies on fluorescent inhibitor binding to MAO B and to MAO A will be performed to investigate dynamics involved in their relative binding mechanisms 3. To probe the mechanism of C-H bond cleavage for the reductive half reaction of MAO B and MAO A using rapid reaction kinetics for benzylamine and aminomethylpyridine analogues at various pH values. Rapid scan kinetic spectral measurements will be used to detect the formation of any reaction intermediates. The question here is to test the validity of the proposed polar nucleophilic mechanism or, alternatively, a hydride mechanism. The mechanism of the oxidative half reactions of MAO A and MAO B will be investigated by rapid scan stopped flow of wt and lys296 mutants of MAO B to test the functional role of this residue. These proposed experiments will test a proposed mechanism for the oxidative half reaction that suggests this lys residue functions as an H+ donor.
Abnormalities in monoamine oxidases A and B levels are known to be involved in a number of disease states ranging from Parkinson's Disease to cardiovascular remodeling. The development of highly specific reversible inhibitors show promise as protectants as these enzyme levels increase with aging in the human.
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