ACTIVE AND ALLOSTERIC SITES OF ISOCITRATE DEHYDROGENASES

异柠檬酸脱氢酶的活性位点和变构位点

• 批准号: 2140769
• 负责人: ROBERTA Fishman COLMAN
• 金额: $ 25.12万
• 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 1996-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2140769
• 关键词: NAD(H) phosphate; X ray crystallography; active sites; affinity labeling; allosteric site; animal tissue; chemical binding; cofactor; enzyme mechanism; enzyme structure; enzyme substrate complex; isocitrate dehydrogenase; isozymes; nicotinamide adenine dinucleotide; nuclear magnetic resonance spectroscopy; protein sequence; protein structure function; site directed mutagenesis

Mammalian tissues contain both an NADP-specific and an NAD-dependent isocitrate dehydrogenase which exhibit similarities in their catalytic reactions, but differ in their physical characteristics as well as in their mode of regulation. 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. The primary structure of the pig heart NADP enzyme has recently been completed in our collaborative studies involving nucleotide and protein sequencing; and studies are in progress to determine its tertiary structure by X-ray crystallography. For this NADP-specific enzyme in solution, we now aim to locate those regions in which metal-isocitrate and coenzyme bind and to evaluate the residues which contribute directly to binding and/or catalysis. 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, purified and characterized. In addition, affinity labeling by reactive nucleotide analogues synthesized in this laboratory will be used to locate sub-regions of the coenzyme site; and affinity cleavage by metal- isocitrate will help to localize the substrate binding site. The modified or cleaved peptides will be isolated and their amino acid sequences determined. For the NAD enzyme, which has 2 binding sites/enzyme tetramer for all ligands, a major focus will be on assessing the roles of the dissimilar subunits. Substantial segments of these subunits have already been sequenced and this work will continue. 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 affected by site-specific labels and affinity cleavage, and of the sequences of modified peptides derived from the distinct subunits. Kinetic and ligand binding studies on modified enzymes will also be conducted. NMR studies will use 113Cd and 13C-enriched substrates to explore the metal-substrate sites of the NAD enzyme for comparison with the NADP-enzyme. Relationships between the NAD and NADP enzymes may be found in the active site despite differences in their physicochemical properties.

哺乳动物组织同时含有 NADP 特异性和 NAD 依赖性 异柠檬酸脱氢酶在催化方面表现出相似性 反应，但其物理特性和 他们的监管模式。 猪心NADP依赖性酶是二聚体 具有相同的亚基并且不受修饰符的控制；然而 来自同一物种和组织的 NAD 特异性酶被激活 ADP 由 3 种亚基组成，其比例为 2α：1β：1γ。 本研究旨在识别和确定 对两种酶的功能至关重要的氨基酸的作用。 这 猪心 NADP 酶的一级结构最近被 我们在涉及核苷酸和蛋白质的合作研究中完成了 测序；正在进行研究以确定其第三级 通过X射线晶体学确定结构。 对于这种 NADP 特异性酶 解决方案，我们现在的目标是找到金属异柠檬酸盐存在的区域 和辅酶结合并评估直接贡献的残基 结合和/或催化。 要使用的主要工具是站点定向 诱变，通过分析选择突变的目标位点 亲和力修饰结果、晶体结构和序列比对 与功能相当的酶。 会表达突变酶， 纯化并表征。 此外，通过反应性亲和标记 该实验室合成的核苷酸类似物将用于 定位辅酶位点的子区域；和金属亲和力裂解 异柠檬酸盐将有助于定位底物结合位点。 这 修饰或切割的肽将被分离，并且它们的氨基酸 序列确定。 对于NAD酶，它有2个结合 所有配体的位点/酶四聚体，主要重点是评估 不同亚基的作用。 其中很大一部分 亚基已经测序，这项工作将继续进行。 NAD 酶的亚基类型可能具有特殊的功能； 或者，所有亚基都有可能结合每种类型的 配体，但一次实际上只有一半可以结合。 这些可能性 将通过识别受影响的亚基类型进行评估 位点特异性标记和亲和力切割，以及序列 来自不同亚基的修饰肽。 动力学和配体 还将进行修饰酶的结合研究。 核磁共振研究 将使用富含 113Cd 和 13C 的基底来探索金属基底 NAD 酶的位点，用于与 NADP 酶进行比较。 NAD 和 NADP 酶之间的关系可能存在于活性 尽管它们的物理化学性质不同，但它们的位置不同。