Mammalian Heart Isocitrate Dehydrogenases

哺乳动物心脏异柠檬酸脱氢酶

• 批准号: 6359790
• 负责人: ROBERTA Fishman COLMAN
• 金额: $ 33.98万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-15 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6359790
• 关键词: Krebs' cycle; NAD(H) phosphate; X ray crystallography; affinity labeling; allosteric site; aminoacid; animal genetic material tag; binding sites; catalyst; chemical kinetics; chemical stability; cofactor; enzyme mechanism; enzyme structure; gene expression; gene targeting; isocitrate dehydrogenase; mitochondria; nicotinamide adenine dinucleotide; protein binding; protein purification; site directed mutagenesis; swine

DESCRIPTION (provided by applicant): Mammalian heart tissues contain two types of NADP-specific isocitrate dehydrogenases, one mitochondrial and the other cytoplasmic, as well as an allosteric NAD-dependent isocitrate dehydrogenase located in the mitochondria. Isocitrate dehydrogenase catalyzes one of the rate limiting steps in the energy-producing Citric Acid Cycle. Evidence suggests that, in heart failure, decreases in the NAD enzyme are associated with decreased oxidative metabolism and energy production. In contrast, the NADP-specific isocitrate dehydrogenases have a major role in the generation of NADPH for reductive biosynthesis and protection against oxidative stress in the heart. Our goal is to understand the structural basis for differences in kinetics, specificity and regulation of the two mammalian mitochondrial isocitrate dehydrogenases. We hypothesize that certain critical amino acids involved in catalysis and metal-isocitrate binding are conserved among the isocitrate dehydrogenases, but that there is greater diversity at the coenzyme and nucleotide regulatory sites. The recombinant pig heart mitochondrial NADP-specific isocitrate dehydrogenase is a dimer of identical subunits and its activity is not allosterically regulated. We aim to identify its critical amino acids by site-directed mutagenesis, affinity labeling and x-ray crystallography. Target sites for mutation will be chosen on the basis of sequence alignments among the isocitrate dehydrogenases, affinity labeling results, and analysis of crystal structures. Mutant enzymes will be purified and extensively characterized. We have crystals (diffracting to 1.7 A) of the Mn-isocitrate complex of this recombinant wild type NADP enzyme. We now propose to solve its structure and to determine that of other wild type enzyme-ligand complexes, as well as those of selected mutant enzymes.The mammalian NAD-specific enzyme is activated by ADP and has 3 types of subunits present in the ratio 2alpha: 1beta: lgamma. The subunits of the human enzyme have recently been co-expressed in E. coli. With the aim of elucidating the roles of these subunits, we plan to express, purify, characterize and compare the NAD enzyme composed of wild type alpha, beta and gamma subunits, with enzyme composed of 2 wild type subunits + 1 subunit in which a single amino acid (proposed to participate in catalysis, substrate or ADP binding) is replaced by mutagenesis. Knowledge of the isocitrate dehydrogenases at the molecular level is important for understanding the role of these enzymes in human cardiac energy metabolism, and in the cellular defense against damage caused by reactive oxygen species. These studies may lead to the rational design of synthetic activators of isocitratedehydrogenases useful for treating human disease.

描述(申请人提供)：哺乳动物心脏组织包含两种类型 NADP特异性异柠檬酸脱氢酶，一个线粒体，另一个 细胞质，以及依赖NAD的变构异柠檬酸脱氢酶 位于线粒体中。异柠檬酸脱氢酶催化其中一种速率 限制产生能量的柠檬酸循环的步骤。有证据表明 在心力衰竭中，NAD酶的减少与 氧化新陈代谢和能量产生减少。相比之下， NADP特异的异柠檬酸脱氢酶在产生 NADPH在生物还原合成中的作用及对氧化应激的保护作用 心。我们的目标是了解 两种哺乳动物线粒体的动力学、特异性及其调控 异柠檬酸脱氢酶。我们假设某些关键氨基酸 参与催化和金属-异柠檬酸结合是保守的 异柠檬酸脱氢酶，但辅酶有更大的多样性 和核苷酸调控位点。重组猪心线粒体 NADP特异性异柠檬酸脱氢酶是一种相同亚基的二聚体，其 活动不是变构调控的。我们的目标是确定它的关键氨基酸 定点诱变、亲和标记和X-射线分析 结晶学。突变的目标位置的选择将基于 异柠檬酸脱氢酶的序列比对、亲和标记 结果，并进行了晶体结构分析。突变的酶将被提纯 和广泛的特色化。我们有晶体(衍射率为1.7埃) 该重组野生型NADP酶的锰-异柠檬酸复合体。我们现在提议 解决其结构和确定其他野生型酶-配体的结构 复合体以及所选突变酶的复合体。哺乳动物 NAD特异性酶由ADP激活，有3种亚基存在于 2阿尔法：1贝塔：1伽马。人类酵素的亚基最近 在大肠杆菌中共表达。目的是阐明这些因素的作用 亚基，我们计划表达、纯化、鉴定和比较NAD酶 由野生型α、β和伽马亚基组成，酶由2 野生型亚基+1亚基，其中一个氨基酸(建议 参与催化、底物或ADP结合)被诱变取代。 在分子水平上了解异柠檬酸脱氢酶是很重要的 为了了解这些酶在人体心脏能量代谢中的作用， 以及在细胞防御活性氧物种造成的损害方面。 这些研究可能导致合成激活剂的合理设计。 用于治疗人类疾病的异柠檬酸脱氢酶。