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.
|Edmondson, Dale E (2014) Hydrogen peroxide produced by mitochondrial monoamine oxidase catalysis: biological implications. Curr Pharm Des 20:155-60|
|Orru, R; Aldeco, M; Edmondson, D E (2013) Do MAO A and MAO B utilize the same mechanism for the C-H bond cleavage step in catalysis? Evidence suggesting differing mechanisms. J Neural Transm (Vienna) 120:847-51|
|Binda, Claudia; Milczek, Erika M; Bonivento, Daniele et al. (2011) Lights and shadows on monoamine oxidase inhibition in neuroprotective pharmacological therapies. Curr Top Med Chem 11:2788-96|
|Wang, Jin; Edmondson, Dale E (2011) ²H kinetic isotope effects and pH dependence of catalysis as mechanistic probes of rat monoamine oxidase A: comparisons with the human enzyme. Biochemistry 50:7710-7|
|Aldeco, Milagros; Arslan, Betul Kacar; Edmondson, Dale E (2011) Catalytic and inhibitor binding properties of zebrafish monoamine oxidase (zMAO): comparisons with human MAO A and MAO B. Comp Biochem Physiol B Biochem Mol Biol 159:78-83|
|Milczek, Erika M; Binda, Claudia; Rovida, Stefano et al. (2011) The 'gating' residues Ile199 and Tyr326 in human monoamine oxidase B function in substrate and inhibitor recognition. FEBS J 278:4860-9|
|Wang, Jin; Edmondson, Dale E (2011) Topological probes of monoamine oxidases A and B in rat liver mitochondria: inhibition by TEMPO-substituted pargyline analogues and inactivation by proteolysis. Biochemistry 50:2499-505|
|Nucci, Nathaniel V; Pometun, Maxim S; Wand, A Joshua (2011) Mapping the hydration dynamics of ubiquitin. J Am Chem Soc 133:12326-9|
|Binda, Claudia; Mattevi, Andrea; Edmondson, Dale E (2011) Structural properties of human monoamine oxidases A and B. Int Rev Neurobiol 100:1-11|
|MacMillar, Susanna; Edmondson, Dale E; Matsson, Olle (2011) Nitrogen kinetic isotope effects for the monoamine oxidase B-catalyzed oxidation of benzylamine and (1,1-(2)H2)benzylamine: nitrogen rehybridization and CH bond cleavage are not concerted. J Am Chem Soc 133:12319-21|
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