Conformational Dynamics in Glutathione S-Transferase

描述（由申请人提供）：谷胱甘肽S-转移酶（GST）是 代谢环境异生物质的解毒酶家族 和药物，包括抗癌剂， 谷胱甘肽（GSH）。GST还通过代谢脂质调节氧化应激 氢过氧化物和脂质羟基烯醛。GST可能会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敏感。作为 结构相关蛋白质的典型家族，GST提供了一个模型， 了解基质多样性的演变，这显然与 一些GST中蛋白质动力学的进化。GSTA 1 -1同种型具有 两个不寻常的特征，可能独特地有助于其催化多样性， 解毒酶一个特征是催化Tyr具有异常低的 pKa，其可能提供静电力并增加溶剂化。 活动现场。该Tyr的电离状态在过程中不改变。 催化循环的化学步骤，和不寻常的功能， 电离特性仍然未知。第二个特点是动态的 C-末端螺旋，其经历配体依赖性的重新分布， “开放”和“闭合”构象。高度相关，几乎在结构上 相同，GST具有“静态”的C-末端螺旋， “开”或“关”。“这项提议探讨了Tyr的催化功能， 电离性能和C-末端螺旋，特别是， 假设这两个特征作为进化桥梁共同进化 原始GST和高度进化的底物特异性同种型之间的差异。为了 为了了解GST家族的结构、功能和动力学， 本建议的具体目标是：1）确定GSTA的阶段1 -1 C-末端关闭的催化; 2）确定C-末端的功能。 活性位点Tyr的不寻常的电离性质; 3）探索 通过GSTA 1 -1的定向进化的底物多样性的分子决定因素 和GSTA 4 -4的定向去进化。使用的技术包括X射线 晶体学、NMR和模型三元复合物的荧光，以监测 C-末端结构和动力学。为了确定是否 C-末端必须在化学步骤的过渡态中闭合，线性 自由能关系将被利用。之间的关系，如果有的话， 将探讨C-末端动力学和催化Tyr的电离 用停流动力学方法和稳态荧光， 工程化Trp报告基因。

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.