SUBSTRATE SPECIFICITY OF GLUTATHIONE TRANSFERASES

这项研究计划的长远目标是理解分子 谷胱甘肽转移酶底物专一性的基础。这些 酶是一类解毒酶，广泛存在于 种类繁多，包括植物、昆虫和哺乳动物。在人类身上， 谷胱甘肽转移酶在抗癌物质中的作用 以及肿瘤对化疗药物耐药性的发展 毒品。 这些酶的一个耐人寻味的和重要的功能特性是 它们对疏水化合物的广泛底物专一性。单人间 谷胱甘肽转移酶在几种不同的 底物和不同的谷胱甘肽转移酶表现不同 底物特性。底物的分子机制 特异性将通过测试三个来调查，而不是必须的 独家的、可行的假设： 广泛的底物特异性可能是由于存在几个 活性部位内包含的功能性疏水结合部位 区域。为了检验这一假设残留物与不同的 疏水性衬底将通过磁化转移来识别 实验。底物中某些残留物的潜在参与 结合和后续催化将通过定点定向进行测试 诱变。 不同的谷胱甘肽转移酶可以利用 底物结合改变不同位置的自由能 这种反应是相互协调的。自由能在不同介质中的储存 通过测量配体结合对酶的影响来评估酶 酰胺交换动力学。此信息将与 决定自由能关系的动力学速率常数 储存和催化。 蛋白质动力学可能在底物结合和产物中发挥作用 通过限制对活动站点的访问来释放。蛋白质动力学将是 通过计算机模拟研究，测量N-15核弛豫 速率和二硫键交联法。动态变化的蛋白质 属性将通过遗传和化学手段产生，以确认 蛋白质动力学与催化的关系。 这些实验将提供一个全面的分子描述 这些酶的结构与其能力之间的关系 在结构不同的底物上发挥作用。此信息将 在设计化疗药物时必不可少，而不是 不被这些酶激活的。

The long range of this research program is to understand the molecular basis of substrate specificity of glutathione transferases. These enzymes are a family of detoxification enzymes which are found in a wide range of species, including plants, insects, and mammals. In humans, glutathione transferases play a role in the resistance toward carcinogens and the development of drug resistance of tumors to chemotherapeutic drugs. An intriguing and functionally important property of these enzymes is their broad substrate specificity toward hydrophobic compounds. A single glutathione transferase is catalytically active on several different substrates and different glutathione transferase display different substrate specificities. The molecular mechanism of substrate specificity will be investigated by testing three, not necessarily exclusive, working hypotheses: Broad substrate specificity may result from the existence of several functional hydrophobic binding sites contained within the active site region. To test this hypothesis residues in contact with different hydrophobic substrates will be identified by magnetization transfer experiments. The potential involvement of certain residues in substrates binding and subsequent catalysis will be tested by site-directed mutagenesis. Different glutathione transferases may utilize the free energy of substrate binding to alter the free energy of different positions along the reaction co-ordinate. The storage of free energy in different enzymes will be assessed by measuring the effect of ligand binding on amide exchange kinetics. This information will be correlated with kinetic rate constants to determine the relationship between free-energy storage and catalysis. Protein dynamics may play a role in substrate binding and product release by gating access to the active site. Protein dynamics will be investigated by computer modeling, measurement of N-15 nuclear relaxation rates, and by disulfide cross-linking. Protein with altered dynamic properties will be generated by genetic and chemical means to confirm the relationship between protein dynamics and catalysis. These experiments will provide a comprehensive molecular description of the relationship between the structure of these enzymes and their ability to function on structurally diverse substrates. This information will be essential in the design of chemotherapeutic drugs that are not inactive by these enzymes.