GLUTATHIONE S TRANSFERASES--SUBSTRATE AND SUBUNIT SITES

谷胱甘肽S-转移酶（GST）在外源性物质的解毒过程中起重要作用，催化谷胱甘肽巯基对外源性物质底物的亲核攻击。因为它们能催化几种已知致癌物的失活。由于它们催化几种已知致癌物的失活，这些酶可以提供对致癌作用的防御。另一方面，实体瘤中GST水平的升高似乎是对细胞毒性药物治疗产生耐药性的主要因素。基于序列相似性和底物特异性，GST被分为至少六个不同的基因家族;并且这些同工酶在赋予对特定抗癌药物的抗性的能力方面不同。已知主要二聚体哺乳动物GST的氨基酸序列，并且已经确定了pi类晶体、mu类303同工酶晶体和alpha类1-1同工酶晶体的三维结构。然而，重要的问题仍然存在：（1）在各种GST同工酶中，哪些氨基酸有助于特异性结合异源底物;（2）给定的酶是否具有一种以上类型的异源底物位点和其他非底物位点（可能是监管）场所;（3）哪些酶氨基酸是亚基相互作用的最重要决定因素，以及它们的二聚体结构在酶的功能中起什么作用。我们将研究大鼠同工酶1-1作为GST α类的代表，大鼠酶3-3作为mu类的代表，猪肺酶作为pi类的一个例子。这些同工酶在底物特异性上不同，并且它们的序列的比较揭示了79-89%相同加上一类内的相似残基，但类之间仅约40%。我们对这些酶在溶液中的活性位点的研究将与使用X射线坐标的蛋白质晶体结构的计算机建模进行互补和比较。我们计划使用亲和标记来实现异生物质底物和非底物位点中氨基酸的特异性修饰和鉴定。我们将使用定点突变来替换参与亚基相互作用的氨基酸，并评估通过亲和标记鉴定的氨基酸的功能。突变酶将被表达和纯化，其单体-二聚体分布，催化和结合特性将被检查。本研究的目的是提供知识基础的合理设计的抑制剂的具体异生物质底物网站GST用于新的组合化疗，以提高疗效的烷基化癌症药物。

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. 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 six different gene families based on sequence similarity and substrate specificity; and these isozymes differ in their ability to confer resistance to particular anti-cancer drugs. The amino acid sequences are known for the major dimeric mammalian GSTs and three dimensional structures have been determined for crystals of the pi-class, of the 303 isozyme of the mu-class and of the 1-1 isozyme of the alpha-class. However, important questions remain: (1) which amino acids contribute to the specificity of binding of xenobiotic substrates in the various GST isozymes; (2) does a given enzyme have more than one type of xenobiotic substrate site plus other non-substrate (possibly regulatory) sites; (3) which enzymic amino acids are the most important determinants of subunit interaction, as well as what role in the function of the enzymes i played by their dimeric structure. We will examine rat isozyme 1-1 as representative of the alpha-class of GSTs, rat enzyme 3-3 as representative of the mu-class, and pig lung enzyme as an example of the pi-class. These isozymes differ in substrate specificity and comparison of their sequences reveals 79-89% identical plus similar residues within a class, but only about 40% between classes. 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 plan to use affinity labeling to effect specific modification and identification of amino acids in the xenobiotic substrate and non-substrate sites. We will use site-directed mutagenesis to replace amino acids proposed as participating in subunit interaction, as well as to evaluate the function of amino acids identified by affinity labeling. Mutant enzymes will be expressed and purified, and their monomer-dimer distribution, catalytic and binding characteristics will be examined. 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 for alkylating cancer drugs.