Subunit Assembly and Folding of Glutathione Transferases

EXCEED提供的空间。谷胱甘肽(GSH)转移酶催化GSH加成到带有亲电官能团的分子上。在哺乳动物中常见的典型GSH转移酶具有广泛的底物特异性，为烷化剂的代谢和解毒提供了一条主要途径。这些酶以相同或密切相关的亚基二聚体的形式存在。典型GSH转移酶的三维结构表明，THACH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。以前：这个项目的结果提供了对基本折叠途径和二聚体组装的洞察。尽管存在Thl，但人们对特定分子相互作用对亚单位或二聚体稳定性的影响知之甚少。此外，这里没有关于底物和配体与活性中心结合的基本能量学的信息。最近，一种细菌GSH转移酶(FOSA)被鉴定为：它催化GSH与抗生素磷霉素的加成反应。这种酶对磷霉素非常特异，并对抗生素产生抗药性。它是目前已知的唯一一种金属酶3SH转移酶。金属离子(Mn2)通过在反应中提供亲电助剂而直接参与催化反应。虽然FOSA与典型的nzyme相比有一个完全不同的折叠，但它也是一个二聚体。有趣的是，初步证据表明，金属离子结合在二聚体中的：WO亚基之间。因此，FOSA为研究金属离子3n蛋白折叠和二聚体组装的影响提供了一个独特的机会。本应用的目的是定义突变的典型GSH转移酶和磷霉素抗性蛋白FOSA的热力学稳定性，阐明与二聚体稳定性相关的特定分子相互作用，并建立底物和配体与这些酶结合的能量学。该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类MU 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)建立FOSA的量热酶分析方法，以确定其反应能。这项研究将由Heini Dirr教授和他的研究小组在威特沃特斯兰德大学生物化学系的蛋白质结构-功能研究计划中进行，作为NIH#R01GM30910-18号拨款的延伸。表演网站========================================Section End===========================================

EXCEEDTHE SPACE PROVIDED. 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 9rovide a major route for the metabolism and detoxification of alkylating agents. The enzymes exist as dimers o] identical or closely related subunits. The three-dimensional structures of canonical GSH transferases reveal thai _ach subunit is divided into two domains (I and II) that interact in a head-to-tail fashion to form a dimer. Previous :esults of this project have provided insight into the basic folding pathways and dimer assembly. In spite of thL, Little is known about the influence of specific molecular interactions on subunit or dimer stability. In addition, :here is no information on the basic energetics of substrate and ligand binding to the active sites. In very recent _,ork a bacterial GSH transferase (FosA) that catalyzes the addition of GSH to the antibiotic fosfomycin has been :haracterized. The enzyme is very specific toward fosfomycin and confers resistance to the antibiotic. It is the only 3SH transferase known to be a metalloenzyme. The metal ion (Mn 2¿) is directly involved in catalysis by providing zlectrophilic assistance in the reaction. Although FosA has a completely different fold as compared to the canonical _nzymes it is also a dimer. Interestingly, preliminary evidence suggests that the metal ions are bound between the :wo subunits in the dimer. Thus, FosA provides a unique opportunity to investigate the influence of the metal ion 3n 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 fol mutant class mu GSH transferases and the FosA protein; (ii) the characterization of the protein-ligand moleculm 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. PERFORMANCE SITE ========================================Section End===========================================