DNA STRUCTURE IN PRESENCE OF MUTAGENIC ADDUCTS & PROTEIN ADDUCTED DNA STRUCTURE

这项研究计划的长期目标是了解 谷胱甘肽转移酶底物专一性的分子基础。 这些酶是一个解毒酶家族，被发现 在广泛的物种中，包括植物、昆虫和哺乳动物。 在人类中，谷胱甘肽转移酶在抗药性中发挥作用。 致癌物与肿瘤耐药的发展 到化疗药物。一个耐人寻味且功能重要的 这些酶的性质是它们广泛的底物专一性 疏水化合物。单一的谷胱甘肽转移酶是 在几种不同的底物和不同的 谷胱甘肽转移酶表现出不同的底物特异性。 底物专一性的分子机制将被研究。 通过测试三个不一定是排他性的工作假设：1) 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，2)不同的谷胱甘肽转移酶可以利用游离的 底物结合能改变不同分子的自由能 沿反应坐标的位置。自由能的储存 将通过测量不同酶的作用来评估 配体结合对酰胺交换动力学的影响。这一信息将是 与动力学速率常数相关联以确定关系 在自由能储存和催化之间，3)蛋白质动力学可能 通过门控通路在底物结合和产品释放中发挥作用 到活动站点。蛋白质动力学将由计算机进行研究 ~(15)N核弛豫率和二硫键的模拟、测量 交联剂。具有改变动力学性质的蛋白质将是 由遗传和化学手段产生的关系得到确认 蛋白质动力学和催化之间的关系。这些实验将 提供对这种关系的全面的分子描述 在这些酶的结构和它们的功能能力之间 在结构不同的衬底上。这一信息将是 在化学治疗药物的设计中必不可少的 被这些酶灭活的。上述研究的一个关键组成部分是 使用核磁共振方法来评估这些化合物的结构和动力学 溶液中的分子。

The long-range goal 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 towards 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 towards hydrophobic compounds. A single glutathione transferase is catalytically active on several different substrates and different glutathione transferases display different substrate specificities. The molecular mechanism of substrate specificity will be investigated by testing three, not necessarily exclusive, working hypotheses: 1) broad substrate specificity may result from the existence of several functional hydrophobic binding sites contained within the active site regions, 2) different glutathione transferases may utilize the free energy of substrate binding to alter the free energy of different positions along the reaction coordinate. 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, 3) 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 15N nuclear relaxation rates and by disulfide crosslinking. Proteins 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 inactivated by these enzymes. A key component of the above studies is the use of NMR methods to assess the structure and dynamics of these molecules in solution.