Covalent Flavin Coenzymes in Flavoenzyme Catalysis

描述（由申请人提供）：要求继续支持扩展正在进行的针对目标酶的特定抑制剂设计的分子基础研究：人线粒体单胺氧化酶A和B （MAO A和MAO B）。这项建议的工作将扩展目前关于神经保护剂和心脏保护剂设计的知识，这些保护剂将服务于易患神经退行性疾病和心血管疾病的老年人。提出了三个特定的目标，将提供洞察这些膜结合酶的活性位点腔，这将导致高度特异性抑制剂的发展：1。MAO B和MAO A中I2咪唑啉结合位点的分子基础将通过结晶学分析和结合研究的结合来确定，结合研究使用两种酶的纯化和膜结合制剂，并测试一系列咪唑啉配体。发现与结合的咪唑啉配体相互作用的氨基酸残基的突变形式将被创建和检查2。探讨特异性抑制剂与MAO B入口腔的结合；通过单Ala和双Ala突变，以及与WT酶比较这些突变酶形式的催化和抑制剂结合特性，将确定门控残基Ile199和Tyr326在人MAO B中的功能作用。荧光抑制剂与MAO B和MAO A结合的快速反应动力学研究将探讨其相关结合机制的动力学。利用不同pH值下苄胺和氨基甲基吡啶类似物的快速反应动力学，探讨MAO B和MAO A还原半反应中C-H键的断裂机理。快速扫描动力学光谱测量将用于检测任何反应中间体的形成。这里的问题是测试所提出的极性亲核机制或氢化物机制的有效性。通过对MAO B的wt和lys296突变体进行快速扫描停流，研究MAO A和MAO B的氧化半反应机制，以检验该残基的功能作用。这些拟议的实验将测试氧化半反应的拟议机制，该机制表明该赖氨酸残基作为H+供体起作用。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.