Covalent Flavin Coenzymes in Flavoenzyme Catalysis

描述（由申请方提供）：需要继续支持以扩展正在进行的靶酶特异性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）。这项拟定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人群。提出了三个具体的目标，这将提供深入了解这些膜结合酶的活性位点空腔，这将导致高度特异性抑制剂的发展：1。将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，通过晶体学分析和结合研究的组合来确定MAO B和MAO A中I2咪唑啉结合位点的分子基础。将创建并检查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2。为了探测与MAO B入口腔结合的特异性抑制剂;将通过单和双Ala突变以及与WT酶相比确定这些突变酶形式的催化和抑制剂结合特性来确定门控残基Ile 199和Tyr 326在人MAO B中的功能作用。将对荧光抑制剂与MAO B和MAO A结合进行快速反应动力学研究，以研究其相对结合机制中涉及的动力学。利用苄胺和氨甲基吡啶类似物在不同pH值下的快速反应动力学，探讨MAO B和MAO A还原半反应的C-H键断裂机理。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。通过快速扫描MAO B的wt和lys 296突变体的停止流动来研究MAO A和MAO B的氧化半反应的机制，以测试该残基的功能作用。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+供体发挥作用。

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.