CRYSTAL STRUCTURE OF YEAST NAD-SPECIFIC ISOCITRATE DEHYDROGENASE
CRYSTAL STRUCTURE OF YEAST NAD-SPECIFIC ISOCITRATE DEHYDROGENASE
批准号:
7602328
负责人:
Peter JOHN HART
金额:
$0.43万
依托单位国家:
美国
项目类别:
财政年份:
2007
资助国家:
美国
项目状态:
已结题
起止时间:
2007-07-01 至 2008-06-30
关键词:
AffinityAnodesAttenuatedBindingBinding SitesCitrateCitratesCitric Acid CycleComputer Retrieval of Information on Scientific Projects DatabaseConditionDecarboxylationEnzymatic BiochemistryEnzymesFundingFutureGlycolysisGlycolysis PathwayGrantInstitutionIsocitrate DehydrogenaseIsocitratesLigand BindingMitochondriaMultienzyme ComplexesMutagenesisNADHNicotinamide adenine dinucleotidePathway interactionsProductionRateRegulationRelative (related person)ResearchResearch PersonnelResourcesRoentgen RaysSiteSourceStructureTricarboxylic AcidsUnited States National Institutes of HealthYeastsalpha ketoglutarateisocitrateresearch study
中文摘要
这个子项目是许多研究子项目中利用
资源由NIH/NCRR资助的中心拨款提供。子项目和
调查员(PI)可能从NIH的另一个来源获得了主要资金,
并因此可以在其他清晰的条目中表示。列出的机构是
该中心不一定是调查人员的机构。
线粒体NAD特异性异柠檬酸脱氢酶催化异柠檬酸氧化脱酸为α-酮戊二酸是三羧酸(TCA)循环的限速步骤。酵母酶(IDH)对异柠檬酸的亲和力是变构调节的,AMP是正的,而ATP和NADH是负的。这种变构控制被认为有助于通过氧化途径和糖酵解对能量产生速率进行反向调节。因此,在能量充足的条件下,即当[ATP]/[AMP]和[NADH]/[NAD]的相对细胞比率较高时,通过TCA循环的通量将在IDH水平上减弱,糖酵解速率将增加,三元酸、柠檬酸和异柠檬酸将被转移到生物合成途径。酵母IDH是由4个IDH1和4个IDH2亚基组成的八聚体。IDH1(M.W.=38,001)和IDH2(M.W.=37,755)有42%的序列同源性。定向突变研究结果表明,IDH2亚基中含有催化异柠檬酸/镁离子和NAD结合位点,而IDH1亚基中的同源位点在异柠檬酸协同结合和变构激活剂AMP的结合中起作用。因此,了解IDH的寡聚体结构将有助于阐明同源催化和调节配体结合位点之间的关系。
我们在Rigaku FR-D旋转阳极X射线源上生产了衍射率为3.2°&的IDH晶体。我们预计,测定晶体结构中IDH的低聚排列将揭示这种复杂酶的变构控制模式。此外,该结构提供的信息将指导未来研究IDH酶的实验。
英文摘要
This subproject is one of many research subprojects utilizing the
resources provided by a Center grant funded by NIH/NCRR. The subproject and
investigator (PI) may have received primary funding from another NIH source,
and thus could be represented in other CRISP entries. The institution listed is
for the Center, which is not necessarily the institution for the investigator.
The oxidative decarboxylation of isocitrate to alpha-ketoglutarate catalyzed by mitochondrial NAD-specific isocitrate dehydrogenases is a rate limiting step in the tricarboxylic acid (TCA) cycle. The affinity of the yeast enzyme (IDH) for isocitrate is allosterically regulated, positively by AMP and negatively by ATP and NADH. This allosteric control has been proposed to contribute to inverse regulation of rates of energy production by oxidative pathways and by glycolysis. Thus, under conditions of energy sufficiency, i.e. when relative cellular ratios of [ATP]/[AMP] and of [NADH]/[NAD] are high, flux through the TCA cycle would be attenuated at the level of IDH, rates of glycolysis would increase, and the tricarboxylic acids, citrate and isocitrate, would be diverted into biosynthetic pathways. Yeast IDH is an octamer composed of four IDH1 and four IDH2 subunits. IDH1 (M.W. = 38,001) and IDH2 (M.W. = 37,755) share 42% sequence identity. Results of targeted mutagenesis studies suggest that the IDH2 subunit contains catalytic isocitrate/Mg2+ and NAD binding sites, whereas homologous sites in the IDH1 subunit function in cooperative binding of isocitrate and in binding of the allosteric activator AMP. An understanding of the oligomeric structure of IDH would thus illuminate relationships between homologous catalytic and regulatory ligand binding sites.
We have produced crystals of IDH that diffract to 3.2¿¿¿& on a Rigaku FR-D rotating anode X-ray source. We anticipate that the determination of the oligomeric arrangement of IDH in the crystal structure will reveal the mode of allosteric control of this complex enzyme. Additionally, the information provided by the structure will direct future experiments investigating IDH enzymology.
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