AFFINITY LABELING OF GLUTATHIONE S TRANSFERASES

谷胱甘肽S-转移酶(GST)在解毒中起重要作用 外源生物，催化硫醇基团的亲核攻击 异种生物底物上的谷胱甘肽。因为它们催化了 几种已知致癌物的失活，这些酶可以提供一种 预防癌变。另一方面，商品及服务税的上升 实体瘤中的水平似乎是发展的一个主要因素。 对细胞毒剂治疗的抗药性。对GST进行分组 根据序列相似性划分为至少五个不同的基因家族 和底物专一性；例如，1-1同工酶赋予最大 细胞对抗癌药物扑热息痛和马法兰的耐药性， 而3-3同工酶的抗性增加幅度最大 顺铂。完整的氨基酸序列已经确定 哺乳动物肝脏的主要GST和三维结构有 最近报道了Mu类3-3同工酶的晶体， 两个pi类酶和阿尔法类的1-1同工酶。 然而，重要的问题仍然是哪些氨基酸残基 有助于结合外源物质的底物，这些残基如何 确定GST各种同工酶的底物特异性，以及 一种特定的酶是否有一种以上的异种底物 地点。我们将检测大鼠肝脏的同工酶1-1作为代表 Alpha家族和大鼠肝脏同工酶3-3作为Mu家族的例子 商品及服务税。这些同工酶在底物专一性和比较性上有所不同 它们的序列显示87%的相同加上相似的残基在 但在阿尔法和穆氏家族之间只有30%左右。我们的 这些酶在溶液中的活性部位的研究将是 对结构的计算机建模进行了补充和比较 利用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 氨基酸残基的修饰与鉴定 这些同工酶的催化部位。一系列新型试剂将 根据所选的外源化合物的结构进行合成 底物或谷胱甘肽的底物，具有反应性亲电特性 或能够共价标记氨基的光反应官能团 一旦试剂结合在活性部位，就会形成酸性侧链。放射性 前体可用于合成标记试剂，以便于 修饰氨基酸的分离。一旦修饰的氨基酸 已经确定，我们将使用定点突变构建 适当的突变体1-1和3-3酶，将表达和 其特征是测试目标氨基酸的功能。本研究 旨在为合理设计缓蚀剂提供知识库 用于GST的特定异种底物部位 提高烷化疗效的新型联合化疗 抗癌药物。

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.