CLONING, EXPRESSION & CHARACTERIZATION OF GDH MUTANTS

克隆、表达

• 批准号: 3439270
• 负责人: JOHN E BELL
• 金额: $ 10.16万
• 依托单位: GUSTAVUS ADOLPHUS COLLEGE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-06-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3439270
• 关键词: acidity /alkalinity; active sites; affinity chromatography; enzyme mechanism; enzyme structure; glutamate dehydrogenase; molecular cloning; mutant; protein purification; protein structure function; site directed mutagenesis; transfection /expression vector

Mammalian Glutamate Dehydrogenases play an essential role in nitrogen metabolism in the liver and appear to play a vital role in glutamate metabolism in other tissues, especially the brain. Glutamate Dehydrogenase is a complex, allosterically regulated enzyme that in mammalian systems appears to be associated with ammonia metabolism. The proposed work will define structure function relationships involved in this enzyme. The work, in conjunction with the determination of the three-dimensional structure by a collaborating group, will define amino acid side chains involved in substrate binding, in cofactor and allosteric regulator binding, and in the regions of subunit interaction in this hexameric enzyme. The gene for Bovine Glutamate Dehydrogenase will be cloned, and an expression system developed to allow site-directed mutants to be constructed.This will involve using a yeast expression system in order to obtain appropriate processing of this mammalian intramitochondrial protein. Expressed protein will be purified using a novel affinity chromatography system specific for mammalian Glutamate Dehydrogenase. pH dependence and chemical reactivity studies of defined residues will help to elucidate the functional characteristics of residues that are not apparent simply from the structure. This work will form the basis of site-directed mutagenesis approaches and will be conducted in conjunction with the necessary enzyme characterization by kinetic methods as well as structure determination of mutants. Site-directed mutants will be used to examine the role of potential functional residues in the active site, in the ADP regulatory site, and in subunit interactions in this enzyme. The successful completion of this project will give new insights into this complex and important regulatory enzyme, and should provide, for the first time, a clear understanding of both structure-function relationships in the active site of this enzyme, and the structural basis of, and potential role for, negative homotropic interactions in a mammalian Glutamate Dehydrogenase.

哺乳动物谷氨酸脱氢酶在氮中起重要作用 在肝脏中的代谢，并似乎发挥重要作用，谷氨酸 其他组织的新陈代谢，尤其是大脑。 谷氨酸 脱氢酶是一种复杂的变构调节酶， 哺乳动物系统似乎与氨代谢有关。 的 拟议的工作将确定结构功能关系， 这种酶。 这项工作，连同确定 三维结构，将定义氨基 参与底物结合的酸性侧链，参与辅因子和 变构调节剂结合，并在亚基相互作用的区域 在这个六聚体酶中。 牛谷氨酸脱氢酶基因将被克隆， 表达系统的开发，以允许定点突变体， 这将涉及使用酵母表达系统，以便 获得这种哺乳动物线粒体内的适当处理 蛋白 表达的蛋白质将使用新的亲和性纯化， 哺乳动物谷氨酸脱氢酶的特异性层析系统。 确定残留物的pH依赖性和化学反应性研究将 有助于阐明残基的功能特性， 从结构上看很明显。 这项工作将成为 定点诱变方法，并将结合 通过动力学方法进行必要的酶表征， 突变体的结构测定。 将使用定点突变体 为了检查活性位点中潜在功能残基的作用， 在ADP调节位点，以及这种酶的亚基相互作用中。 该项目的成功完成将使我们对以下方面有新的认识： 这种复杂而重要的调节酶，应该提供， 第一次，清楚地了解了两者的结构-功能 这种酶的活性位点的关系，和结构基础 负同向性相互作用的潜在作用， 哺乳动物谷氨酸脱氢酶。