Subunit Assembly and Folding of Glutathione Transferases

超出规定的空间。谷胱甘肽（GSH）转移酶催化GSH与亲电官能团分子的加成。哺乳动物中常见的典型谷胱甘肽转移酶具有广泛的底物特异性，并为烷基化剂的代谢和解毒提供了主要途径。酶以二聚体形式存在于相同或密切相关的亚基中。典型谷胱甘肽转移酶的三维结构表明，每个亚基分为两个结构域（I和II），它们以头对尾的方式相互作用形成二聚体。上一篇：该项目的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，关于特定分子相互作用对亚基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位点结合的基本能量学的信息。在最近的研究中，一种细菌谷胱甘肽转移酶（FosA）催化谷胱甘肽加入抗生素磷霉素中。这种酶对磷霉素有很强的特异性，并对抗生素产生耐药性。它是唯一已知的3SH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性辅助直接参与催化。虽然FosA与典型的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，金属离子在二聚体的两个亚基之间结合。因此，FosA提供了一个独特的机会来研究金属离子3n蛋白折叠和二聚体组装的影响。本应用程序的目的是定义热力学稳定性，阐明与突变型典型谷胱甘肽转移酶和磷霉素抗性蛋白FosA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。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）利用热力学分析方法表征了GSH转移酶和FosA类蛋白-配体分子识别过程；（iii）开发一种用于测定FosA反应能量的量热酶测定法。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-功能研究项目的Heini Dirr教授和他的研究小组进行，作为NIH资助# R01GM30910-18的延伸。网站性能 ======================================== 节结束 ===========================================

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===========================================