ACTIVE AND ALLOSTERIC SITES ISOCITRATE DEHYDROGENASES

异柠檬酸脱氢酶活性和变构位点

• 批准号: 6140742
• 负责人: ROBERTA Fishman COLMAN
• 金额: $ 3.75万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6140742
• 关键词: NAD(P)H dehydrogenase; active sites; affinity labeling; allosteric site; animal tissue; cofactor; crystallization; enzyme activity; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate complex; high performance liquid chromatography; isocitrate dehydrogenase; isozymes; nuclear magnetic resonance spectroscopy; protein sequence; protein structure function; site directed mutagenesis

Mammalian tissues contain an NADP- and an NAD-specific isocitrate dehydrogenase which both catalyze a metal-dependent oxidative decarboxylation of isocitrate, but differ in their affinities for substrate, their mode of regulation and their structures. The pig heart NADP-dependent enzyme is a dimer of identical subunits and is not subject to control by modifiers; whereas the NAD-specific enzyme from the same species and tissue is activated by ADP and is composed of 3 types of subunits present in the ratio of 2alpha:1beta:1gamma. This study aims at identifying and ascertaining the role of those amino acids critical for function in both enzymes and in elucidating the structural basis for kinetic differences between the two enzymes. The sequence of the pig heart NADP enzyme is now known; and good progress has been made in determining its tertiary structure by X-ray crystallography. For this NADP-specific enzyme in solution, we aim to identify amino acids contributing to catalysis and/or binding of metal-isocitrate and coenzyme. The major tool to be used is site-directed mutagenesis, with the target sites for mutation selected by analysis of affinity modification results, crystal structures and sequence alignments with functionally comparable enzymes. Mutant enzymes will be expressed in E. coli, purified and characterized. Affinity labeling by reactive nucleotide analogues synthesized in this laboratory will also be used to locate sub-regions of the coenzyme site. For the NAD enzyme, we will focus on the roles of the dissimilar subunits, which have recently been sequenced. The enzyme has 2 binding sites/enzyme tetramer. Subunit types of the NAD-enzyme may have specialized functions; alternatively, all subunits may have the potential to bind every type of ligand but only half may actually bind at a time. These possibilities will be evaluated by identification of the subunit types (and peptides) affected by site-specific labels and affinity cleavage, and by studying the reassembly of enzyme from unmodified or affinity labeled separated subunits. Striking similarities exist between the NADP-dependent isocitrate dehydrogenase of pig and human hearts, suggesting that these studies will be relevant for understanding human cardiac energy metabolism.

哺乳动物组织中含有NADP和NAD特异性等异位酸盐 脱氢酶都催化金属依赖性氧化 等二羧酸脱羧，但其亲和力的不同 底物，调节方式及其结构。 猪心 NADP依赖性酶是相同亚基的二聚体，不是受试者 通过修饰符控制；而NAD特异性酶来自同一酶 物种和组织被ADP激活，由3种类型的 2 alpha的比例存在亚基：1beta：1gamma。 这项研究的目的是 识别和确定这些氨基酸的作用至关重要 在酶以及阐明结构基础的功能 两种酶之间的动力学差异。 猪的序列 心脏NADP酶现在已知。并取得了良好的进步 通过X射线晶体学确定其三级结构。 为了这 溶液中的NADP特异性酶，我们旨在鉴定氨基酸 有助于金属 - 异构酸和辅酶的催化和/或结合。 要使用的主要工具是定向诱变，目标是 通过分析亲和力修饰结果选择突变的位点， 晶体结构和序列比对与功能可比性 酶。 突变酶将在大肠杆菌中表达，纯化和 特征。 反应性核苷酸类似物的亲和力标记 该实验室合成的合成也将用于定位 辅酶部位。 对于NAD酶，我们将专注于 最近已经测序的不同亚基。 酶具有 2个结合位点/酶四聚体。 NAD-酶的亚基类型可能具有 专业功能；或者，所有亚基都有潜力 绑定每种类型的配体，但只有一半可以一次绑定。 这些可能性将通过识别亚基来评估 受特定地点标签和亲和力影响的类型（和肽） 通过研究未修饰或 亲和力标记为分离的亚基。 存在惊人的相似之处 猪和人类心脏的NADP依赖性异位酸脱氢酶， 暗示这些研究将与了解人类有关 心脏能量代谢。