NMR STUDIES OF GLUTATHIONE TRANSFERASES

谷胱甘肽转移酶的核磁共振研究

• 批准号: 6355174
• 负责人: GORDON S RULE
• 金额: $ 0.21万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6355174
• 关键词: biological products; biomedical equipment development; biomedical resource; technology /technique

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.

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。 这些酶是解毒酶家族，可在 种类繁多，包括植物、昆虫和哺乳动物。在……里面 人类，谷胱甘肽转移酶在抵抗 致癌物与肿瘤抗药性的发展 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 在几种不同的底物上具有催化活性。此外, 不同的谷胱甘肽转移酶显示不同的底物 具体细节。底物专一性的分子机制将 通过测试三个不一定是排他性的工作来进行调查 假设：1.广泛的底物专一性可能源于 含有几个功能性疏水结合部位的存在 在活动站点区域内。2.不同的谷胱甘肽 转铁酶可以利用底物结合的自由能来改变 反应坐标上不同位置的自由能。 不同酶中自由能的储存将通过以下方式进行评估 测定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 通过计算机模拟进行研究，测量N-15核 松弛速率和二硫键交联。最大的 对这些蛋白质进行核磁共振实验的困难在于它们 大小(55 KDa二聚体)。CMR获得的高场TROSY谱 将在获得配基和配基的分配方面是无价的 未连接的酶。