SUBUNIT ASSEMBLY AND FOLDING OF GLUTATHIONE TRANSFERASES

谷胱甘肽转移酶的亚基组装和折叠

• 批准号: 2873242
• 负责人: RICHARD N ARMSTRONG
• 金额: $ 1.55万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2873242
• 关键词: chemical stability; enzyme biosynthesis; enzyme structure; glutathione transferase; hydropathy; mutant; protein folding; protein structure function; site directed mutagenesis; thermodynamics

DESCRIPTION The glutathione (GSH) transferases catalyze the addition of GSH to endogenous and xenobiotic molecules bearing electrophilic functional groups. As such they represent a major route for the metabolism and detoxification of alkylating agents. The enzymes exist as dimers of identical or closely related subunits. The three-dimensional structures of representatives of each of the five known gene classes (alpha, mu, pi, sigma and theta) of GSH transferases reveal that each subunit is divided into two domains (I and II) that interact in a head-to-tail fashion to form the dimer. There appear to be two basic subunit interface types, an alpha/mu/pi type and a sigma/theta type, the former having a specific hydrophobic ball-and-socket interaction and the latter being more hydrophilic. In spite of the extensive structural data little is known about the influence of the domain interactions on subunit or domain stability. The role of dimer interactions in the conformational stability and catalytic activity of GSH transferases remains largely unexplored. The objectives of this application are to define the thermodynamic stabilities and folding pathways of class sigma and class mu GSH transferases which have distinctly different dimer interfaces and to establish the contributions of specific intersubunit interactions identified by crystallography in determining the conformational stability of the homodimeric structures and the specificity of subunit-subunit associations. The objectives of the research plan will be realized through completion of the following four specific aims. (1) The thermodynamic stabilities and folding pathways of native class mu enzymes (i.e., the rat M1-1 and M2-2 isoenzymes) and a class sigma enzyme (S1-1) from squid will be determined in order to establish the influence of the very different dimer interfaces on the energetics of the subunit-subunit interactions. (2) The importance of a key hydrophobic interaction involving Phe56 in the class mu isoenzyme and a specific hydrophilic interaction involving Arg77(mu)/Arg68 (sigma) will be determined by site-specific mutagenesis. (3) Domain-domain interactions at the dimer interface will be investigated with chimeric homodimeric class mu enzymes in which domains I and II of the M1-1 and M2-2 isoenzymes have been switched. (4) Crystal structures of the mutant and chimeric enzymes will be determined. The research will be carried out in collaboration with the Protein Structure Function Research Programme in the Department of Biochemistry, University of Witwatersrand. Glutathione S-transferases exist as either catalytically inactive unfolded monomers or as catalytically active domain 1 : domain 2 head-to-tail dimers. The folding and unfolding is reversible and follows a two state mechanism interconverting folded catalytically active dimer with unfolded catalytically inactive monomer. The subunit:subunit interactions in the folded dimeric structure are thus important for both the stabilization of the association (dimerization) of the subunits and the stabilization of the tertiary structures of the folded subunits of the dimer. Preliminary data on a sigma class enzyme from squid suggests that there may be other detectable intermediates in the folding pathway, not just the unfolded monomer and folded dimer. The proposed research for the Dirr group will examine both folding and unfolding reactions. Functional probes to detect and quantitate the folded dimeric native structure are catalytic activity and binding of fluorescent ligands. Structural probes include a variety of spectroscopic, chemical, electrophoresis and proteolysis assays. The thermodynamics of folding/unfolding will be measured using differential scanning calorimetry.

描述 谷胱甘肽（GSH）转移酶催化GSH的加成， 携带亲电官能团的内源性和外源性分子。 因此，它们代表了新陈代谢和解毒的主要途径 烷基化试剂。 酶以相同或相近的二聚体形式存在， 相关子单位。 的代表性的三维结构 GSH的五个已知基因类别（α、μ、π、σ和θ）中的每一个 转移酶揭示每个亚基分为两个结构域（I和II） 以首尾相连的方式相互作用形成二聚体。 看来 是两种基本的亚基界面类型，α/μ/π型和σ/θ型 类型，前者具有特定的疏水球窝相互作用 而后者更亲水。 尽管有广泛的结构性 关于域相互作用的影响， 亚基或结构域稳定性。 二聚体相互作用在 GSH转移酶的构象稳定性和催化活性仍然存在 大部分未开发。 此应用程序的目标是定义 热力学稳定性和sigma类和mu类的折叠途径 GSH转移酶具有明显不同的二聚体界面， 确定特定亚基间相互作用的贡献 通过晶体学确定的构象稳定性 同源二聚体结构和亚基-亚基缔合的特异性。 研究计划的目标将通过完成 以下四个具体目标。 (1)热力学稳定性和 天然μ类酶的折叠途径（即，大鼠M1-1和M2-2 同工酶）和sigma类酶（S1-1）将在 为了建立非常不同的二聚体界面对 亚基间相互作用的能量学。 (2)的重要性 涉及μ类同工酶中Phe 56的关键疏水相互作用和 涉及Arg 77（mu）/Arg 68（sigma）特异性亲水相互作用将 通过位点特异性诱变确定。 (3)域-域相互作用. 将用嵌合同型二聚体μ类研究二聚体界面 酶，其中M1-1和M2-2同工酶的结构域I和II已被 交换。 (4)突变体和嵌合酶的晶体结构将被分析。 测定 这项研究将与 蛋白质结构与功能研究计划 威特沃特斯兰德大学生物化学系。 存在谷胱甘肽S-转移酶 作为无催化活性的未折叠单体或作为催化活性的未折叠单体， 活性结构域1：结构域2头-尾二聚体。 折叠和展开 是可逆的，并遵循两个状态的机制相互转换折叠 催化活性二聚体与未折叠的催化非活性单体。 因此，折叠的二聚体结构中的亚基：亚基相互作用是 重要的是稳定的缔合（二聚化） 亚基和折叠的三级结构的稳定性 二聚体的亚基。 鱿鱼中一种sigma类酶的初步研究 这表明在折叠过程中可能存在其他可检测的中间体 途径，而不仅仅是未折叠的单体和折叠的二聚体。 对Dirr群的拟议研究将检查折叠和 展开反应 检测和定量折叠的 二聚体天然结构是催化活性和荧光结合 配体。 结构探针包括各种光谱，化学， 电泳和蛋白水解测定。 的热力学 使用差示扫描量热法测量折叠/解折叠。