SUBSTRATE SPECIFICITY OF GLUTATHIONE TRANSFERASES

这项研究计划的长期目标是了解 谷胱甘肽转移酶底物特异性的基础。 这些 酶是解毒酶的一个家族，在广泛的 一系列物种，包括植物、昆虫和哺乳动物。 在人类中， 谷胱甘肽转移酶在抵抗致癌物中起作用 以及肿瘤对化疗药物耐药性的发展 毒品 这些酶的一个有趣的和功能上重要的特性是 它们对疏水化合物的广泛底物特异性。 单个 谷胱甘肽转移酶对几种不同的 底物和不同的谷胱甘肽转移酶显示不同的 底物特异性 底物的分子机理 将通过测试三个来研究特异性，不一定 排他性的工作假设： 广泛的底物特异性可能是由于存在几个 活性位点内含有的功能性疏水结合位点 地区 为了验证这一假设， 疏水基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 结合和随后的催化作用将通过定点 诱变 不同的谷胱甘肽转移酶可以利用谷胱甘肽的自由能。 衬底结合以改变沿着不同位置的自由能 反应坐标。 自由能的储存在不同的 将通过测量配体结合对酶活性的影响来评估酶活性。 酰胺交换动力学 这些信息将与 动力学速率常数，以确定自由能 存储和催化。 蛋白质动力学可能在底物结合和产物生成中起作用。 通过门控访问活动站点来释放。 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，和通过二硫化物交联。 动态改变的蛋白质 将通过遗传和化学手段产生特性，以确认 蛋白质动力学和催化作用之间的关系。 这些实验将提供一个全面的分子描述， 这些酶的结构和它们的能力之间的关系 在结构多样的基质上发挥作用。 这些信息将 在设计化疗药物中是必不可少的， 不受这些酶的影响

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.