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特异性的分子基础。 这些酶是解毒酶家族， 包括植物、昆虫和哺乳动物在内的许多物种都有这种基因。 在人类中，谷胱甘肽转移酶在抵抗中发挥作用， 对致癌物和肿瘤耐药性的发展 化疗药物。 一个有趣的和功能重要的 这些酶的性质是它们对底物的广泛特异性， 疏水化合物。 单个谷胱甘肽转移酶是 在几种不同的基质上具有催化活性， 谷胱甘肽转移酶显示不同的底物特异性。 底物特异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种 活性位点内含有的功能性疏水结合位点 2）不同的谷胱甘肽转移酶可以利用游离的 底物结合的能量改变不同的自由能 沿反作用坐标沿着定位。 自由能的储存 在不同的酶将评估通过测量的影响， 酰胺交换动力学上的配体结合。 此信息将 与动力学速率常数相关，以确定 自由能储存和催化之间的关系，3）蛋白质动力学可能 通过门控通路在底物结合和产物释放中发挥作用 到活跃的网站。 蛋白质动力学将由计算机研究 建模，15N核弛豫速率的测量和二硫化物 交联 具有改变的动力学特性的蛋白质将被 通过遗传和化学手段产生的，以确认关系 蛋白质动力学和催化作用之间的联系 这些实验将 提供了一个全面的分子描述的关系， 这些酶的结构和它们的功能之间 在结构多样的基质上。 此信息将 在化疗药物的设计中至关重要， 被这些酶灭活。 上述研究的一个关键组成部分是 使用NMR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.