Mammalian Heart Isocitrate Dehydrogenases

哺乳动物心脏异柠檬酸脱氢酶

• 批准号: 6638799
• 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/6638799
• 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特异性异位酸盐脱氢酶，一种线粒体，另一个线粒体 细胞质以及变构的NAD依赖性异位酸脱氢酶 位于线粒体。异氯酸酯脱氢酶催化速率之一 限制产生能源的柠檬酸周期的步骤。有证据表明 在心力衰竭中，NAD酶的减少与 氧化代谢和能量产生减少。相反， NADP特异性异位酸脱氢酶在产生中具有重要作用 NADPH用于还原性生物合成和防止氧化应激的保护 心。我们的目标是了解差异的结构基础 两个哺乳动物线粒体的动力学，特异性和调节 异位酸盐脱氢酶。我们假设某些临界氨基酸 参与催化和金属 - 异构酸盐结合在 异位酸盐脱氢酶，但辅酶的多样性更大 和核苷酸调节位点。重组猪心线粒体 NADP特异性异位酸脱氢酶是相同亚基及其的二聚体 活动不受变构调节。我们旨在确定其关键氨基 通过位置定向诱变，亲和力标记和X射线酸的酸 晶体学。将根据 异氯酸酯脱氢酶之间的序列比对，亲和力标记 结果和晶体结构的分析。突变酶将被纯化 并广泛特征。我们有晶体（衍射为1.7 a） 这种重组野生型NADP酶的Mn-异氯酸盐复合物。我们现在建议 解决其结构并确定其他野生型酶 - 配体的结构 复合物以及选定的突变酶的复合物。哺乳动物 NAD特异性酶被ADP激活，并具有3种类型的亚基 比率2alpha：1beta：lgamma。人类酶的亚基最近有 在大肠杆菌中共表达。为了阐明这些角色 亚基，我们计划表达，净化，表征和比较NAD酶 由野生型α，β和伽马亚基组成，酶由2个组成 野生型亚基 + 1个亚基，其中一个氨基酸（提出 参与催化，底物或ADP结合）被诱变所取代。 在分子水平上了解异氯酸盐脱氢酶很重要 为了理解这些酶在人类心能代谢中的作用， 并在细胞防御中抵抗由活性氧引起的损害。 这些研究可能导致合成激活因子的合理设计 异源性氢化酶用于治疗人类疾病。