Subunit Assembly and Folding of Glutathione Transferases

谷胱甘肽转移酶的亚基组装和折叠

• 批准号: 6826832
• 负责人: RICHARD N ARMSTRONG
• 金额: $ 4.03万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6826832
• 关键词: Subunit; Assembly; Folding; Glutathione; Transferases

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

超出提供的空间。谷胱甘肽(GSH)转移酶催化GSH添加到带有亲电官能团的分子上。哺乳动物中常见的经典 GSH 转移酶具有广泛的底物特异性，9 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 GSH 转移酶的三维结构揭示了 _ach 亚基分为两个结构域（I 和 II），它们以头尾相连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。尽管存在 thL，但人们对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。最近，一种细菌 GSH 转移酶 (FosA) 已被表征，该酶可催化 GSH 添加到抗生素磷霉素中。该酶对磷霉素具有很强的特异性，并赋予该抗生素耐药性。它是唯一已知的金属酶 3SH 转移酶。金属离子（Mn 2¿）通过在反应中提供亲电帮助而直接参与催化。尽管 FosA 与标准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结合在二聚体中的 :wo 亚基之间。因此，FosA 提供了一个独特的机会来研究金属离子 3n 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并阐明与突变型经典 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。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型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) 开发 FosA 量热酶测定法以确定其反应能量。该研究将由威特沃特斯兰德大学生物化学系的 Heini Dirr 教授及其研究小组在蛋白质结构功能研究项目中进行，作为 NIH 拨款 # R01GM30910-18 的延伸。表演网站==========================================章节结束===============================================

项目成果

Characterization of the binding of 8-anilinonaphthalene sulfonate to rat class Mu GST M1-1.

8-苯胺萘磺酸盐与大鼠 Mu GST M1-1 类结合的表征。

• DOI: 10.1016/j.bpc.2008.07.008
• 发表时间: 2008
• 期刊: Biophysical chemistry
• 影响因子: 3.8
• 作者: Kinsley,Nichole;Sayed,Yasien;Mosebi,Salerwe;Armstrong,RichardN;Dirr,HeiniW
• 通讯作者: Dirr,HeiniW

Class sigma glutathione transferase unfolds via a dimeric and a monomeric intermediate: impact of subunit interface on conformational stability in the superfamily.

西格玛谷胱甘肽转移酶通过二聚体和单体中间体展开：亚基界面对超家族构象稳定性的影响。

• DOI: 10.1021/bi981044b
• 发表时间: 1998
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Stevens,JM;Hornby,JA;Armstrong,RN;Dirr,HW
• 通讯作者: Dirr,HW