Flavoenzyme Mechanisms: Redox and Non-redox Reactions

描述(申请人提供)：这项拟议的研究涉及对单体肌氨酸氧化酶(MSOX)结构与功能关系的全面调查，MSOX是氨氧化酶超家族的典型成员，含有维生素核黄素的共价结合衍生物。MSOX是一种重要的分解代谢酶，广泛存在于土壤细菌中，广泛应用于临床肾功能评价。MSOX超家族包含几种具有生物医学意义的人类酶，包括肌氨酸脱氢酶和单胺氧化酶，肌氨酸脱氢酶是肌氨酸血症患者的缺陷酶，单胺氧化酶是治疗帕金森病和抑郁症的药物靶点。这些研究的总体目标是更深入地了解黄素酶的催化机制、酶专一性的分子基础以及蛋白质环境对黄素内在化学反应的调节。我们将在研究中确定各种MSOX底物络合物的晶体结构，这些研究是在我们成功获得E-S与L-脯氨酸络合物的2.0A分辨结构的基础上进行的。胺氧化一般需要一个未质子化的氨基，但MSOX的底物是以非活性两性离子形式存在于中性pH溶液中的氨基酸。我们将评估底物被MSOX氧化激活的提议，其机制涉及酶结合氨基酸相对于游离氨基酸的PKA的大幅降低。底物氧化的机理将在基于机理的缓蚀剂、交替底物、氚同位素效应、立体化学分析、诱变和分子动力学模拟等研究中进行。我们将用一种含有突变的MSOX载脂蛋白制剂来探索共价黄素连接在重组和结构研究中的作用，该突变允许非共价黄素结合但阻止共价结合。共价黄素结合的机制将在用野生型脱辅酶或适当的突变体进行的体外黄素化研究中表征。这些研究代表了一种多学科的方法，涉及生化、结构和建模工作的组合。

DESCRIPTION (provided by applicant): The proposed research involves a comprehensive investigation of the relationship of structure to function in monomeric sarcosine oxidase (MSOX), a prototypical member of a superfamily of amine-oxidizing enzymes that contain covalently bound derivatives of the vitamin riboflavin. MSOX is an important catabolic enzyme commonly found in soil bacteria and widely used in the clinical evaluation of renal function. The MSOX superfamily contains several biomedically significant human enzymes, including sarcosine dehydrogenase, an enzyme defective in sarcosinemic patients and monoamine oxidase, a drug target in the treatment of Parkinson's disease and depression. The overall goal of these studies is to gain a deeper understanding of the mechanism of flavoenzyme catalysis, the molecular basis for enzyme specificity and the modulation of the inherent chemical reactivity of flavins by the protein environment. We will determine the crystal structure for various MSOX-substrate complexes in studies that build on our success in obtaining a preliminary 2.0 A resolution structure for the E-S complex with L-proline. Amine oxidation generally requires an unprotonated amino group but the substrates for MSOX are amino acids that exist in solution at neutral pH as unreactive zwitterions. We will evaluate the proposal that substrates are activated for oxidation by MSOX via a mechanism that involves a substantial decrease in the pKa of the enzyme-bound versus the free amino acid. The mechanism of substrate oxidation will be investigated in studies involving a mechanism-based inhibitor, alternate substrates, deuterium isotope effects, stereochemical analysis, mutagenesis and molecular dynamic simulations. We will probe the role of the covalent flavin linkage in reconstitution and structural studies with a MSOX apoprotein preparation containing a mutation that allows noncovalent flavin binding but blocks covalent attachment. The mechanism of covalent flavin attachment will be characterized in in vitro flavinylation studies with wild type apoenzyme or appropriate mutants. These studies represent a multidisciplinary approach, involving a combination of biochemical, structural and modeling efforts.