Mammalian Heart Isocitrate Dehydrogenases
哺乳动物心脏异柠檬酸脱氢酶
基本信息
- 批准号:6359790
- 负责人:
- 金额:$ 33.98万
- 依托单位:
- 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 2005-05-31
- 项目状态:已结题
- 来源:
- 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
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(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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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ROBERTA Fishman COLMAN其他文献
ROBERTA Fishman COLMAN的其他文献
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{{ truncateString('ROBERTA Fishman COLMAN', 18)}}的其他基金
Adenylosuccinate Lyase: Novel Intersubunit Active Sites
腺苷琥珀酸裂解酶:新型亚基间活性位点
- 批准号:
6414578 - 财政年份:2002
- 资助金额:
$ 33.98万 - 项目类别:
Adenylosuccinate Lyase: Novel Intersubunit Active Sites
腺苷琥珀酸裂解酶:新型亚基间活性位点
- 批准号:
6620282 - 财政年份:2002
- 资助金额:
$ 33.98万 - 项目类别:
Adenylosuccinate Lyase: Novel Intersubunit Active Sites
腺苷琥珀酸裂解酶:新型亚基间活性位点
- 批准号:
6696608 - 财政年份:2002
- 资助金额:
$ 33.98万 - 项目类别: