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: 2140770
• 负责人: ROBERTA Fishman COLMAN
• 金额: $ 26.82万
• 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/2140770
• 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.

哺乳动物组织既包含NADP特异性和NAD依赖性的 异位酸脱氢酶，在其催化中表现出相似性 反应，但其身体特征以及 他们的监管方式。 猪心NADP依赖性酶是二聚体 相同的亚基，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种类型的亚基组成 2alpha：1beta：1gamma。 这项研究旨在确定和确定 那些氨基酸的作用对于两种酶的功能至关重要。 这 猪心NADP酶的主要结构最近已经 在我们涉及核苷酸和蛋白质的协作研究中完成 测序；并且正在进行研究以确定其三级 X射线晶体学结构。 对于这种NADP特异性酶 解决方案，我们现在旨在定位金属 - 异位酸盐的那些区域 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的 诱变，通过分析选择突变的目标位点 亲和力修改结果，晶体结构和序列比对 具有功能可比的酶。 突变酶将被表达， 纯化和特征。 另外，通过反应性标记 该实验室中合成的核苷酸类似物将用于 定位辅酶位点的子区域；和金属的亲和力分裂 等齿将有助于定位底物结合位点。 这 修饰或裂解的肽将分离出来，其氨基酸 确定的序列。 对于具有2个结合的NAD酶 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。 这些的大量部分 亚基已经进行了测序，这项工作将继续。 NAD-酶的亚基类型可能具有专门的功能； 或者，所有亚基都有可能绑定每种类型的 配体但只有一半可以一次绑定。 这些可能性 将通过识别受影响的亚基类型来评估 位点特异性标签和亲和力裂解，以及 从不同亚基得出的修饰肽。 动力学和配体 还将对改良酶进行结合研究。 NMR研究 将使用113CD和13C富含的底物探索金属覆盖物 NAD酶的位点与NADP-酶进行比较。 在活动中可以找到NAD和NADP酶之间的关系 尽管其物理化学特性差异差异。