Flavoenzyme Mechanisms: Redox and Non-redox Reactions

描述（由申请人提供）：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是胺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是土壤细菌中常见的一种重要的分解代谢酶，广泛应用于临床肾功能评价。MSOX超家族包含几种具有重要生物医学意义的人类酶，包括肌氨酸脱氢酶（一种肌素血症患者存在缺陷的酶）和单胺氧化酶（一种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酶催化的机制，酶特异性的分子基础以及蛋白质环境对黄素固有化学反应性的调节。我们将在获得l -脯氨酸E-S配合物的初步2.0 a分辨率结构的基础上，在研究中确定各种msox -底物配合物的晶体结构。胺氧化通常需要一个未质子化的氨基，但MSOX的底物是在中性pH下以不反应的两性离子形式存在于溶液中的氨基酸。我们将评估底物被MSOX氧化激活的机制，该机制涉及酶结合的pKa与游离氨基酸的显著降低。底物氧化的机制将在基于机制的抑制剂、替代底物、氘同位素效应、立体化学分析、诱变和分子动力学模拟等方面进行研究。我们将探讨共价黄素连锁在重组和结构研究中的作用，MSOX载脂蛋白制备中含有允许非共价黄素结合但阻止共价附着的突变。共价黄素附着的机制将在野生型脱酶或适当突变体的体外黄素化研究中得到表征。这些研究代表了一种多学科的方法，包括生物化学、结构和建模的结合。

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.