AFFINITY LABELING OF GLUTATHIONE S TRANSFERASES

谷胱甘肽 S 转移酶的亲和标记

• 批准号: 2654163
• 负责人: ROBERTA Fishman COLMAN
• 金额: $ 13.88万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2654163
• 关键词: X ray crystallography; active sites; affinity labeling; computer simulation; enzyme structure; glutathione transferase; isozymes; mathematical model; protein structure function; radiotracer; site directed mutagenesis

Glutathione S.transferases (GST) are important in the detoxification of xenobiotics, catalyzing the nucleophilic attack by the thiol group of glutathione on the xenobiotic substrate. Since they catalyze the inactivation of several known carcinogens, these enzymes can provide a defense against carcinogenesis. On the other hand, the elevation of GST levels in solid tumors appears to be a major factor in the development of resistance to treatment with cytotoxic agents. The GSTs are grouped into at least five different gene families based on sequence similarity and substrate specificity; e.g., the 1-1 isozyme confers the greatest cellular resistance to the anti-cancer drugs, chlorambucil and melphalan, while the 3-3 isozyme confers the most increase in resistance to cisplatin. The complete amino acid sequences have been determined for the major GSTs of mammalian liver, and three-dimensional structures have recently been reported for crystals of the 3-3 isozyme of the mu class, of two pi class enzymes and of the 1-1 isozyme of the alpha class. However, important questions remain about which amino acid residues contribute to the binding of the xenobiotic substrate, how these residues determine the substrate specificities of the various isozymes of GST, and whether a given enzyme has more than one type of xenobiotic substrate site. We will examine isozyme 1-1 of rat liver as representative of the alpha family, and isozyme 3-3 of rat liver as an example of the mu family of GSTs. These isozymes differ in substrate specificity and comparison of their sequences reveals 87% identical plus similar residues within the mu family but only about 30% between the alpha and mu families. Our studies of the active sites of these enzymes while in solution will be complementary to and will be compared by computer modeling to structures of the protein crystals using the X-ray coordinates. We propose initially to use affinity labeling to effect specific chemical modification and identification of amino acid residues in the region of the catalytic sites of these isozymes. A series of novel reagents will be synthesized based either on the structure of selected xenobiotic substrates or on that of glutathione and featuring reactive electrophilic or photoreactive functional groups capable of covalently labeling amino acid side chains once the reagent binds at the active site. Radioactive precursors are available to synthesize labeled reagents to facilitate isolation of the modified amino acids. Once the modified amino acids have been identified, we will use site-directed mutagenesis to construct appropriate mutant 1-1 and 3-3 enzymes, which will be expressed and characterized to test the function of the target amino acids. This study aims to provide the knowledge base for rational design of inhibitors specific for particular xenobiotic substrate sites for GST for use in novel combination chemotherapy to enhance the efficacy of alkylating cancer drugs.

谷胱甘肽 S.转移酶 (GST) 在解毒中很重要 异生素，催化硫醇基团的亲核攻击 外源性底物上的谷胱甘肽。 由于它们催化 灭活几种已知的致癌物质，这些酶可以提供 防御癌变。 另一方面，GST的上涨 实体瘤中的水平似乎是发展的主要因素 对细胞毒性药物治疗的耐药性。 GST 已分组 根据序列相似性分为至少五个不同的基因家族 和底物特异性；例如，1-1 同工酶具有最大的 细胞对抗癌药物苯丁酸氮芥和美法仑的耐药性， 而 3-3 同工酶可最大程度地提高抗性 顺铂。 完整的氨基酸序列已确定 哺乳动物肝脏的主要 GST 和三维结构 最近报道了 mu 类 3-3 同工酶的晶体， 两种 pi 类酶和 α 类 1-1 同工酶。 然而，关于哪些氨基酸残基仍然存在重要问题 有助于外源底物的结合，这些残留物如何 确定 GST 各种同工酶的底物特异性，以及 给定的酶是否具有不止一种类型的外源底物 地点。我们将检查大鼠肝脏的同工酶 1-1 作为代表 α 家族，以及大鼠肝脏的同工酶 3-3（以 mu 家族为例） 商品及服务税。 这些同工酶在底物特异性和比较方面有所不同 它们的序列显示 87% 相同且残基相似 mu家族，但alpha家族和mu家族之间的比例只有30%左右。 我们的 对这些酶在溶液中的活性位点的研究将 与结构互补，并将通过计算机建模与结构进行比较 使用 X 射线坐标对蛋白质晶体进行分析。 我们建议 最初使用亲和标记来影响特定的化学物质 区域中氨基酸残基的修饰和鉴定 这些同工酶的催化位点。 一系列新型试剂将 根据选定的外源物质的结构合成 底物或谷胱甘肽的底物上，并具有反应性亲电性 或能够共价标记氨基的光反应性官能团 一旦试剂在活性位点结合，就会形成酸性侧链。放射性 前体可用于合成标记试剂，以促进 分离修饰的氨基酸。 一旦修饰氨基酸 已经确定，我们将使用定点诱变来构建 适当的突变体 1-1 和 3-3 酶，它们将被表达并 旨在测试目标氨基酸的功能。 这项研究 旨在为抑制剂的合理设计提供知识基础 特定于 GST 的特定外源底物位点，用于 新型联合化疗增强烷化疗效 癌症药物。