Conformational Dynamics in Glutathione S-Transferase

说明(申请人提供)：谷胱甘肽S转移酶(GST)为 代谢环境外源物质的解毒酶家族 以及药物，包括抗癌药物，通过将它们与三肽结合 谷胱甘肽(GSH)GSTS还通过代谢脂质来调节氧化应激 过氧化氢和脂类羟基烯类。GST很可能在以下方面发挥作用 对动脉粥样硬化、白内障和神经退行性疾病的敏感性。作为一名 结构相关蛋白质的典型家族，GSTs为 理解底物多样性的演变，这显然与 随着一些GST中蛋白质动力学的进化。GSTA1-1亚型有 可能为其催化多样性做出独特贡献的两个不同寻常的特征 一种解毒酶。其中一个特点是催化轮胎具有异常低的 PKA，它可能提供静电力和增加溶剂化 活动站点。该轮胎的电离状态在以下过程中不变 催化循环的化学步骤，以及不寻常的功能 电离性质尚不清楚。第二个特征是动态的 C-末端螺旋，它经历配体依赖的重新分配 “开放”和“封闭”构象。高度相关，几乎在结构上 相同的，GST具有C-末端螺旋，这些螺旋是静态的，并保持在 “打开”或“关闭”。本提案探讨了轮胎的催化作用 C-末端螺旋的电离性质，特别是 假设这两个特征作为进化的桥梁而共同进化 在原始GST和高度进化的底物特异性亚型之间。按顺序 为了了解GST家族的结构、功能和动态， 该提案的具体目标是：1)确定GSTA1-1的阶段 C末端关闭时的催化作用；2)确定 活性中心TYR的异常电离性质；3)探索 GSTA1-1定向进化底物多样性的分子决定因素 和GSTA4-4的定向去进化。将使用的技术包括x射线 模型三元络合物的结晶学、核磁共振和荧光监测 C-末端的结构和动力学。为了确定是否 对于化学步骤，C末端必须在过渡态闭合，线性 自由能关系将被利用。之间的关系(如果有的话) 本课程将探讨催化酪氨酸的C末端动力学和电离 与停流动力学方法和稳态荧光与 工程设计的TRP记者。

DESCRIPTION (provided by applicant): The glutathione S-transferases (GSTs) are a family of detoxification enzymes that metabolize environmental xenobiotics and drugs, including anti-cancer agents, by conjugating them to the tripeptide glutathione (GSH). GSTs also modulate oxidative stress by metabolizing lipid hydroperoxides and lipid hydroxy-enals. GSTs are likely to play a role in sensitivity to atherosclerosis, cataracts, and neurodegenerative diseases. As a canonical family of structurally related proteins, the GSTs provide a model for understanding the evolution of substrate diversity, which apparently correlates with the evolution of protein dynamics in some GSTs. The GSTA1-1 isoform has two unusual features that may uniquely contribute to its catalytic diversity as a detoxification enzyme. One feature is a catalytic Tyr with an unusually low pKa, which, possibly, provides electrostatic forces and increases solvation of the active site. The ionization state of this Tyr does not change during chemical steps of the catalytic cycle, and the function of the unusual ionization properties remains unknown. The second feature is a dynamic C-terminal helix, which undergoes ligand-dependent redistribution between 'open' and 'closed' conformations. Highly related, nearly structurally identical, GSTs possess C-terminal helices that are 'static' and remain either 'open' or 'closed.' This proposal explores the catalytic function of the Tyr ionization properties and of the C-terminal helix and, in particular, the hypothesis that the two features have co-evolved as an evolutionary bridge between primitive GSTs and highly evolved substrate specific isoforms. In order to understand the structure, function, and dynamics of the GST family, the specific aims of this proposal are: 1) to determine the stage of GSTA1-1 catalysis at which the C-terminus closes; 2) to determine the function of the unusual ionization properties of the active site Tyr; 3) to explore the molecular determinants of substrate diversity by directed evolution of GSTA1-1 and directed de-evolution of GSTA4-4. The techniques to be used include x-ray crystallography, NMR, and fluorescence of model ternary complexes, to monitor the C-terminal structure and dynamics. In order to determine whether the C-terminus must be closed in the transition state for the chemical step, linear free energy relationships will be exploited. The relationship, if any, between C-terminal dynamics and the ionization of the catalytic Tyr will be explored with stopped-flow kinetic approaches and steady state fluorescence with an engineered Trp reporter.