NMR STUDIES 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分子基础。 这些酶是解毒酶家族， 种类繁多，包括植物、昆虫和哺乳动物。 在 在人类中，谷胱甘肽转移酶在抵抗 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。 此外，本发明还提供了一种方法， 不同谷胱甘肽转移酶显示不同底物 特殊性 底物特异性的分子机制将 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来自于 存在几个功能性疏水结合位点， 在活性位点区域内。 2. 不同谷胱甘肽 转移酶可以利用底物结合的自由能来改变 沿反应坐标沿着不同位置的自由能。 不同酶中自由能的储存将通过以下方式评估： 测量配体结合对酰胺交换动力学的影响。 3. 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 松弛速率和二硫化物交联。 最大的 在这些蛋白质上进行NMR实验的困难在于它们 大小（55 KDa二聚体）。 在CMR上获得的高场TROSY谱 将是非常宝贵的，在实现配体和 非配体酶

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 that 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 chemo-therapeutic 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. In addition, 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. 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 N-15 nuclear relaxation rates and by disulfide cross-linking. The largest difficulty in performing NMR experiments on these proteins is their size (55 KDa dimer). The high field TROSY spectra obtained at CMR will be invaluable in attaining the assignment of liganded and unliganded enzymes.