FLAVOENZYME MECHANISMS: REDOX AND NON-REDOX REACTIONS

描述(申请人的描述)：拟议的研究涉及 结构与功能关系的综合研究 单体肌氨酸氧化酶(MSOX)和异四聚体肌氨酸氧化酶 (TSOX)，细菌酶用于临床评估肾功能。 总体目标是更深入地了解黄素是如何 酶催化胺氧化和1-碳转移为四氢叶酸， 具有相当重要生理意义的反应。MSOX和TSOX是成员 一个主要的胺氧化还原酶超家族，它包含许多 临床上重要的酶，如单胺氧化酶和肌氨酸 脱氢酶，肌氨酸血症患者体内的一种酶缺陷。这些胺 氧化还原酶都表现出对黄素共价掺入的要求 (FAD或FMN)。MSOX和TSOX催化肌氨酸氧化脱甲基化 (N-甲基甘氨酸)，但表现出显著的结构和功能差异。 MSOX是一种含有共价结合FAD的单体蛋白。TSOX是一种 含有三种辅酶(FAD、NAD+和共价结合)的多聚酶 FMN)，并催化肌氨酸氧化和合成 5，10-亚甲基四氢叶酸；β亚基似乎是结构 MSOX的同源基因。拟议的研究是在PI研究小组以前工作的基础上进行的 使用MSOX，由自由的高分辨率结构(1.3-2.0 A)引导 酶及其与底物类似物的络合物具有以下原理 目的：确定肌氨酸氧化的机制；评价 共价黄素键在催化中的作用.测定 共价黄素结合的机理；影响黄素结合的因素的评估 调节黄素的氧化还原特性。与TSOX的研究建立在我们在 获得衍射质量(2.8A)的晶体有两个主要目标： 阐明多重的结构和功能组织 这种复合双功能酶中的亚基和辅酶.测定 肌氨酸氧化与肌氨酸高效偶联的机理 5，10-亚甲基四氢叶酸的合成，与反应相关的化学 由肌氨酸脱氢酶催化。这些研究代表了一种 多学科方法，涉及快速反应动力学、立体化学 分析、诱变、结晶学、基于机理的抑制剂的使用和 磁场效应。

DESCRIPTION (applicant's description): The proposed research involves a comprehensive investigation of the relationship of structure to function in monomeric sarcosine oxidase (MSOX) and heterotetrameric sarcosine oxidase (TSOX), bacterial enzymes used in the clinical evaluation of renal function. The overall goal is to gain a deeper understanding of how flavin-containing enzymes catalyze amine oxidation and 1-carbon transfer to tetrahydrofolate, reactions of considerable physiological importance. MSOX and TSOX are members of a major superfamily of amine oxidoreductases that contains a number of clinically important enzymes like monoamine oxidase and sarcosine dehydrogenase, an enzyme defective in patients with sarcosinemia. These amine oxidoreductases all exhibit a requirement for covalent incorporation of flavin (FAD or FMN). MSOX and TSOX catalyze the oxidative demethylation of sarcosine (N-methylglycine) but exhibit notable structural and functional differences. MSOX is a monomeric protein containing covalently bound FAD. TSOX is a multimeric enzyme that contains three coenzymes (FAD, NAD+ and covalently bound FMN) and catalyzes both sarcosine oxidation and synthesis of 5,10-methylenetetrahydrofolate; the beta subunit appears to be the structural homolog of MSOX. The proposed studies build on previous work by the PI Studies with MSOX, guided by high resolution structures (1.3 - 2.0 A) of the free enzyme and its complexes with substrate analogs, have the following principal objectives: Determination of the mechanism of sarcosine oxidation; evaluation of the role of the covalent flavin linkage in catalysis; determination of the mechanism of covalent flavin attachment; evaluation of the factors that modulate flavin redox properties. Studies with TSOX build on our success in obtaining diffraction quality (2.8 A) crystals and have two major objectives: Elucidation of the structural and functional organization of the multiple subunits and coenzymes in this complex bifunctional enzyme; determination of the mechanism for efficient coupling of sarcosine oxidation with 5,10-methylenetetrahydrofolate synthesis, chemistry relevant to the reactions catalyzed by sarcosine dehydrogenase. These studies represent a multidisciplinary approach, involving rapid reaction kinetics, stereochemical analysis, mutagenesis, crystallography, use of mechanism-based inhibitors and magnetic field effects.