Mammalian Heart Isocitrate Dehydrogenases

哺乳动物心脏异柠檬酸脱氢酶

基本信息

批准号：
6737476
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
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-07-15 至 2006-05-31
项目状态：
已结题

项目摘要

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，一种是线粒体，另一种是细胞质以及变构 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其中一种速率限制产生能量的柠檬酸循环中的步骤。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。相比之下， NADP 特异性异柠檬酸脱氢酶在生成 NADPH 用于还原生物合成并防止氧化应激心。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-异柠檬酸盐结合的基因在异柠檬酸脱氢酶，但辅酶具有更大的多样性和核苷酸调节位点。重组猪心脏线粒体 NADP 特异性异柠檬酸脱氢酶是相同亚基的二聚体，其活性不受变构调节。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 射线检测酸晶体学。突变的目标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。突变酶将被纯化并进行了广泛的表征。我们有晶体（衍射至 1.7 A）该重组野生型 NADP 酶的锰-异柠檬酸复合物。我们现在提议解析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 特异性酶由 ADP 激活，具有 3 种类型的亚基 2alpha: 1beta: lgamma 的比率。最近，人类酶的亚基在大肠杆菌中共同表达。为了阐明这些角色的作用亚基，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很重要为了了解这些酶在人体心脏能量代谢中的作用，以及细胞防御活性氧造成的损伤。这些研究可能会导致合成激活剂的合理设计可用于治疗人类疾病的异柠檬酸脱氢酶。