GLUTATHIONE TRANSFERASES-DEFINING STRUCTURE & FUNCTION

描述（由申请人提供）：谷胱甘肽转移酶催化谷胱甘肽（GSH）硫的亲核加成到各种具有亲电官能团的内源性和外源性底物上。它们是哺乳动物代谢和解毒烷基化剂中最重要的酶。此外，它们还代表了一个具有多种未开发功能的蛋白质超家族。了解谷胱甘肽转移酶及其同源物在原核生物和真核生物生物学中的作用是本项目的目标。这一目标将通过阐明分子结构与功能之间的关系来实现。研究将集中在一些鲜为人知的哺乳动物和细菌酶的结构和催化机制方面。将追求四个具体目标。首先，研究谷胱甘肽转移酶的结构、动力学和催化作用；(i)使用酰胺-质子交换质谱法探测控制配体（产物）从活性位点释放速度的酶类中特定结构元素的动力学；（ii）将酰胺-质子交换质谱法扩展到毫秒时间域；(3)通过串联质谱法绘制质子交换动力学到单个残基的图谱。其次，将研究大肠杆菌蛋白组中选定的gsi - 1转移酶同源物，以确定其独特的生物学作用。GSH转移酶超家族的三个成员EGST， b2989和yibF的功能将通过；（一）基因产物的克隆、表达和机理表征；（ii）利用二维差示凝胶电泳和质谱分析不同环境条件下大肠杆菌蛋白质组中同源物的表达；（iii）删除选定的基因以确定表型反应；（四）基因产物的结构测定。第三，将通过(i)测定大鼠kappa类酶的三维结构，（ii）分析该酶的底物特异性，以及（iii）密切相关的2-羟色胺-2-羧酸异构酶在恶臭假单胞菌萘分解代谢途径中的机制分析来研究kappa类和相关的GSI-I转移酶。最后，微粒体酶的机制和结构研究将通过；(i)活化酶独特的机理行为分析；（ii）利用酰胺-质子交换质谱法探索洗涤剂溶液中天然和活化形式的酶的结构和动力学；(3)二维晶体酰胺-质子交换质谱分析。

DESCRIPTION (provided by applicant): The glutathione transferases catalyze the nucleophilic addition of the sulfur of glutathione (GSH) to a wide variety of endogenous and xenobiotic substrates bearing electrophilic functional groups. They are the single most important enzyme in the metabolism and detoxication of alkylating agents in mammals. In addition, they represent a superfamily of proteins with diverse and unexplored functions. It is the goal of this project to understand the role of glutathione transferases and their homologues in the biology of both prokaryotes and eukaryotes. This goal will be accomplished through the elucidation of the relationship between molecular structure and function. The investigations will focus on poorly understood aspects of the structure and catalytic mechanism of selected mammalian and bacterial enzymes. Four specific aims will be pursued. First, the structure, dynamics and catalysis of glutathione transferases will be elucidated through; (i) the use amide-proton exchange mass spectrometry to probe the dynamics of specific structural elements in the class mu enzymes that control the rate of release of ligands (products) from the active site; (ii) extension of amide-proton exchange mass spectrometry into the millisecond time domain; and (iii) mapping of proton exchange kinetics to individual residues by tandem mass spectrometry. Second, selected GSI-I transferase homologues in the Escherichia coil proteome will be investigated to determine their unique biological roles. The functions of three members of the GSH transferase superfamily EGST, b2989 and yibF will be explored by; (i) cloning, expression and mechanistic characterization of the gene products; (ii) profiling the expression of the homologues in the E. coli proteome under different environmental conditions by two-dimensional difference gel electrophoresis and mass spectrometry; (iii) deletion of selected genes to determine phenotypic responses; and (iv) structure determination of the gene products. Third, class kappa and related GSI-I transferases will be investigated through (i) determination of the three dimensional structure of the class kappa enzyme from rat, (ii) analysis of the substrate specificity of the enzyme, and (iii) mechanistic analysis of the closely related 2-hydroxychromene-2-carboxylate isomerase in the naphthalene catabolic pathway of Pseudomonas putida. Finally, mechanistic and structural investigations of the microsomal enzyme will be extended by; (i) analysis of the unique mechanistic behavior of the activated enzyme; (ii) an exploration of the structure and dynamics of the native and activated forms of the enzyme in detergent solution by amide-proton exchange mass spectrometry; and (iii) amide-proton exchange analysis of two-dimensional crystals by mass spectrometry.