Subunit Assembly and Folding of Glutathione Transferases

描述（由申请人提供） 谷胱甘肽（GSH）转移酶催化GSH与带有亲电官能团的分子的加成。典型的GSH转移酶通常存在于哺乳动物中，具有广泛的底物特异性，并为烷化剂的代谢和解毒提供了主要途径。这些酶以相同或密切相关的亚基的二聚体形式存在。典型的GSH转移酶的三维结构显示，每个亚基被分成两个结构域（I和II），它们以头对尾的方式相互作用形成二聚体。该项目的先前结果提供了对基本折叠途径和二聚体组装的深入了解。尽管如此，很少有人知道特定的分子相互作用对亚基或二聚体稳定性的影响。此外，没有关于底物和配体与活性位点结合的基本能量学的信息。在最近的工作中，细菌谷胱甘肽转移酶（FosA），催化添加谷胱甘肽的抗生素磷霉素已被确定。这种酶对磷霉素有很强的特异性，并能产生抗药性。它是唯一已知的GSH转移酶是一种金属酶。金属离子（Mn 2+）通过在反应中提供亲电辅助而直接参与催化。虽然FosA与经典酶相比具有完全不同的折叠，但它也是二聚体。有趣的是，初步证据表明，金属离子之间的两个亚基的二聚体的约束。因此，FosA提供了一个独特的机会来研究金属离子对蛋白质折叠和二聚体组装的影响。本申请的目的是定义热力学稳定性和阐明与突变型典型GSH转移酶和磷霉素抗性蛋白FosA的二聚体稳定性相关的特定分子相互作用，并建立与这些酶结合的底物和配体的能量学。研究计划的目标将通过完成以下具体目标来实现：（i）阐明FosA的热力学稳定性以及突变型mu类GSH转移酶和FosA蛋白的折叠途径;（ii）通过热力学分析表征mu类GSH转移酶和FosA的蛋白质-配体分子识别过程;（iii）发展FosA的量热酶分析法，以确定其反应能。这项研究将由威特沃特斯兰德大学生物化学系蛋白质结构-功能研究计划的Heini Dirr教授和他的研究小组进行，作为NIH资助#R 01 GM 30910 -18的延伸。

DESCRIPTION (provided by applicant) The glutathione GSH) transferases catalyze the addition of GSH to-molecules bearing electrophilic functional groups. The canonical GSH transferases commonly found in mammals exhibit broad substrate specificity and provide 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 canonical GSH transferases reveal that each subunit is divided into two domains (I and II) that interact in a head-to-tail fashion to form a dimer. Previous results of this project have provided insight into the basic folding pathways and dimer assembly. In spite of this, little is known about the influence of specific molecular interactions on subunit or dimer stability. In addition, there is no information on the basic energetics of substrate and ligand binding to the active sites. In very recent work a bacterial GSH transferase (FosA) that catalyzes the addition of GSH to the antibiotic fosfomycin has been characterized. The enzyme is very specific toward fosfomycin and confers resistance to the antibiotic. It is the only GSH transferase known to be a metalloenzyme. The metal ion (Mn 2+) is directly involved in catalysis by providing electrophilic assistance in the reaction. Although FosA has a completely different fold as compared to the canonical enzymes it is also a dimer. Interestingly, preliminary evidence suggests that the metal ions are bound between the two subunits in the dimer. Thus, FosA provides a unique opportunity to investigate the influence of the metal ion on protein folding and dimer assembly. The objectives of this application are to define the thermodynamic stabilities and elucidate specific molecular interactions associated with dimer stability of mutant canonical GSH transferases and the fosfomycin resistance protein, FosA, and to establish the energetics of substrate and ligand binding to these enzymes. The objectives of the research plan will be realized through completion of the following specific aims: (i) the elucidation of the thermodynamic stability of FosA and the folding pathways for mutant class mu GSH transferases and the FosA protein; (ii) the characterization of the protein-ligand molecular recognition processes of class mu GSH transferase and FosA by means of a thermodynamic analysis and; (iii) the development of a calorimetric enzyme assay for FosA for determining its reaction energetics. The research will be carried out by Prof. Heini Dirr and his research group in the Protein Structure-Function Research Programme al the Department of Biochemistry, University of Witwatersrand as an extension of NIH grant # R01GM30910-18.