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（由申请方提供）：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，一种线粒体，另一种 细胞质，以及变构NAD依赖性异柠檬酸脱氢酶 位于线粒体中。异柠檬酸脱氢酶催化一种 柠檬酸循环中的限制步骤。证据表明 在心力衰竭中，NAD酶的减少与 减少氧化代谢和能量产生。而反观 NADP-特异性异柠檬酸脱氢酶在产生 NADPH用于还原性生物合成和抗氧化应激 心我们的目标是了解差异的结构基础， 两种哺乳动物线粒体的动力学、特异性和调节 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-异柠檬酸盐结合是保守的 异柠檬酸脱氢酶，但有更大的多样性在辅酶 和核苷酸调节位点。重组猪心线粒体 NADP特异性异柠檬酸脱氢酶是相同亚基的二聚体， 活性不是变构调节的。我们的目标是确定其关键氨基酸 通过定点诱变、亲和标记和X射线 结晶学突变的靶位点将根据以下因素选择： 异柠檬酸脱氢酶之间的序列比对、亲和标记 结果和晶体结构分析。突变酶将被纯化 并被广泛表征。我们有晶体（衍射到1.7 A）的 该重组野生型NADP酶的Mn-异柠檬酸复合物。我们现建议 解析其结构并确定其它野生型酶-配体的结构 复合物，以及那些选定的突变酶。哺乳动物 NAD特异性酶被ADP激活，并有3种类型的亚基存在于 2 α：1 β：1 γ的比例。人类酶的亚基最近 在E.杆菌为了阐明这些作用， 亚基，我们计划表达，纯化，表征和比较NAD酶 由野生型α、β和γ亚基组成，酶由2 野生型亚基+1个亚基，其中单个氨基酸（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 为了理解这些酶在人类心脏能量代谢中的作用， 以及细胞防御活性氧引起的损伤。 这些研究可能会导致合成活化剂的合理设计， 用于治疗人类疾病的异柠檬酸酯酶。