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加成到 含有亲电官能团的内源和异源分子。 因此，它们是新陈代谢和解毒的主要途径。 烷基化剂。这些酶以相同或相近的二聚体形式存在。 相关亚基。代表人物的三维结构 谷胱甘肽的五个已知基因类别(α、u、pi、sigma和theta)中的每一个 转移酶显示，每个亚基分为两个结构域(I和II) 它们以头尾相接的方式相互作用，形成二聚体。似乎有一种 是两种基本子单元接口类型，即α/Mu/pi类型和sigma/theta类型 类型，前者具有特定的疏水球窝相互作用 而后者则更亲水。尽管有广泛的结构 数据鲜为人知的是域相互作用对 亚基或结构域的稳定性。二聚体相互作用在分子水平上的作用 谷胱甘肽转移酶残基的构象稳定性和催化活性 很大程度上是未被开发的。本应用程序的目标是定义 Sigma类和Mu类的热力学稳定性和折叠路径 具有明显不同二聚体界面的GSH转移酶 确定确定的特定亚基间相互作用的贡献 用结晶学方法测定化合物的构象稳定性 同源二聚体结构和亚基-亚基联合的专一性。 研究计划的目标将通过完成以下工作来实现 以下四个具体目标。(1)热力学稳定性和 天然类MU酶(即大鼠M1-1和M2-2)的折叠途径 同工酶)和鱿鱼中的一类sigma酶(S1-1)将在 以确定非常不同的二聚体界面对 亚基-亚基相互作用的能量学。(2) 涉及Mu同工酶中的Phe56和a的关键疏水相互作用 涉及Arg77(Mu)/Arg68(Sigma)的特定亲水相互作用将是 由定点突变决定。(3)域与域之间的交互 二聚体界面将用嵌合的同源二聚体类Mu进行研究 M1-1和M2-2同工酶的结构域I和II所在的酶 换了。(4)突变酶和嵌合酶的晶体结构 下定决心。这项研究将与 蛋白质结构功能研究计划 威特沃特斯兰德大学生物化学专业。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，并遵循两态机制相互转换折叠 催化活性二聚体与未折叠的催化非活性单体。 折叠二聚体结构中的亚基：亚基相互作用如下 对缔合(二聚化)的稳定很重要 褶皱分子的亚基及其三级结构的稳定化 二聚体的亚基。鱿鱼中一种sigma类酶的初步数据 这表明在折叠过程中可能还有其他可检测到的中间体 途径，而不仅仅是未折叠的单体和折叠的二聚体。 为Dirr小组拟议的研究将检查折叠和 展开反应。用于检测和定量折叠的功能探针 二聚体天然结构是荧光的催化活性和结合 配基。结构探针包括各种光谱的、化学的、 电泳法和蛋白水解法。从热力学的角度看 折叠/展开将使用差示扫描量热法进行测量。