Mechanism and Molecular Recognition in Human Pyruvate Dehydrogenase Complex
人丙酮酸脱氢酶复合物的机制和分子识别
基本信息
- 批准号:7624775
- 负责人:
- 金额:$ 19.81万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2008
- 资助国家:美国
- 起止时间:2008-09-01 至 2010-05-31
- 项目状态:已结题
- 来源:
- 关键词:AbbreviationsAcetyl Coenzyme AActive SitesAffinity ChromatographyAmino AcidsArtsBacillus stearothermophilusBindingBinding ProteinsBiochemicalBiological AssayBrainCalorimetryCarbohydratesCarbonCardiovascular DiseasesCatalysisCessation of lifeChildChronic DiseaseCircular DichroismCommunicationComplexDailyDataDepthDevelopmentDiseaseEnzymesEscherichia coliEukaryotaEukaryotic CellFamilyGel ChromatographyGene MutationGoalsHomeostasisHumanIndividualIntakeInvestigationIsoenzymesKeto AcidsKineticsLaboratoriesLiteratureMass Spectrum AnalysisMental RetardationMethodsMonitorMutationNeuraxisNeurodegenerative DisordersNon-Insulin-Dependent Diabetes MellitusObesityOrganismOxidoreductasePDH kinasePathway interactionsPhosphoric Monoester HydrolasesPhosphorylationPlayProcessProductionProtein DephosphorylationProtein IsoformsProteinsPyruvatePyruvate DecarboxylasePyruvate Dehydrogenase (Lipoamide)-PhosphatasePyruvate Dehydrogenase ComplexPyruvatesPyruvic AcidRateReactionRegulationReportingRoleSite-Directed MutagenesisSkeletonSpecificityStructureSurface Plasmon ResonanceTechniquesTextThiamine PyrophosphateTimeTitrationsUltracentrifugationWernicke-Korsakoff SyndromeYeastsanalogbasedesiredihydrolipoamide dehydrogenasedihydrolipoyllysine-residue acetyltransferaseinstrumentationionizationmembermolecular recognitionoxidationprotein protein interactionpyruvate dehydrogenasepyruvate dehydrogenase complex E2pyruvate dehydrogenase kinase 4three dimensional structuretool
项目摘要
Human pyruvate dehydrogenase complex (hPDC) plays a gatekeeper¿s role in the oxidation of pyruvate
derived from carbohydrates and the carbon-skeletons of several amino acids, and about 50 percent of daily
calorie intake (in term of carbon-flux) is regulated by this enzyme. hPDC is composed of multiple copies of
three catalytic components: heterotetrameric (a2¿2) pyruvate dehydrogenase (PDH), dihydrolipoamide
acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3); E3-binding protein (BP), and two
regulatory enzymes: PDH kinase (PDK, 4 isoenzymes) and phosphatase, (PDP, 2 isoenzymes). Genetic
defects in PDC components have illustrated the importance of PDC in energy homeostasis and cause
impaired brain development, mental retardation and often early death in children. Reductions in PDC
component proteins and activity levels are observed in chronic diseases such as type 2 diabetes, obesity
and cardiovascular diseases and also in neurodegenerative diseases such as Alzheimer¿s disease,
Parkinson¿s disease and Wernicke-Korsakoff syndrome. hPDC has evolved to unique structural
organization over prokaryotic PDCs and other members of the a-keto acid dehydrogenase complex family
from all species by having BP as additional component and a highly sophisticated mechanism of regulation
by multiple isoenzymes of PDKs and PDPs. Recently, we have solved the 3-D structures of human PDH
and also of human E3 bound to the E3-binding domain of BP. Based on these developments our three
specific aims are to (i) investigate the formation of intermediates in the catalysis of human PDH, (ii)
investigate the active site communication in hPDH, and (iii) determine the loci of interactions between hE3
and hBP and also between hE2 and hBP. Site-directed mutagenesis will be employed to introduce desired
mutations and human proteins will be over-expressed and purified by affinity chromatography. We will
employ the state-of-art instrumentation (such as circular dichroism, 1H NMR, mass spectrometry, rapid
stopped-flow circular dichroism, and isothermal titration calorimetry) and chemically synthesized analogs to
identify and monitor the intermediates of the PDH reaction. The time-course analysis of the intermediates of
PDH reaction will allow us to determine function of individual amino acid residues in catalysis and to
characterize the mechanism of communication (flip-flop mechanism) between the two active sites. The roles
of specific amino acid residues of hBP and hE3 as well as of hBP and hE2 in protein-protein interactions will
be determined by kinetic analysis, gel filtration assay, surface plasmon resonance, isothermal titration
calorimetry and ultracentrifugation. The proposed studies combine the extensive expertise of two
laboratories to greatly enhance our understanding of the catalytic mechanism of hPDH and the structurefunction
relationships of hPDC components and would provide the biochemical basis for some genetic
defects in PDC.
人丙酮酸脱氢酶复合物(hPDC)在丙酮酸氧化中起着看门人的作用
来自碳水化合物和几种氨基酸的碳骨架,每天大约有50%的
卡路里摄入(就碳通量而言)由这种酶调节。hPDC由多个副本组成,
三种催化组分:异四聚体(a2 <$2)丙酮酸脱氢酶(PDH),二氢硫辛酰胺
乙酰转移酶(E2)和二氢硫辛酰胺脱氢酶(E3); E3结合蛋白(BP),以及两种
调节酶:PDH激酶(PDK,4种同工酶)和磷酸酶(PDP,2种同工酶)。遗传
PDC部件中的缺陷已经说明了PDC在能量稳态中的重要性,
儿童大脑发育受损、智力迟钝和往往早逝。PDC减少
在慢性疾病如2型糖尿病、肥胖症、糖尿病性糖尿
和心血管疾病,以及神经退行性疾病,如阿尔茨海默病,
帕金森病和韦尼克-科萨科夫综合征。hPDC已经发展成独特的结构
组织超过原核PDC和其他成员的α-酮酸脱氢酶复合物家族
通过将BP作为额外成分和高度复杂的调节机制,来自所有物种
PDK和PDP的多种同工酶。最近,我们已经解决了人PDH的三维结构,
以及与BP的E3结合结构域结合的人E3。根据这些发展,我们的三个
具体目标是(i)研究人PDH催化中中间体的形成,(ii)
研究hPDH中的活性位点通讯,以及(iii)确定hE 3之间相互作用的位点
hBP和hE 2之间的关系。将采用定点诱变来引入期望的突变。
突变和人蛋白将被过表达并通过亲和层析纯化。我们将
使用最先进的仪器(如圆二色谱、1H NMR、质谱、快速质谱、质谱仪
停流圆二色性和等温滴定量热法)和化学合成的类似物,
识别和监测PDH反应的中间体。中间体的时程分析
PDH反应将使我们能够确定单个氨基酸残基在催化中的功能,
表征两个活性位点之间的通信机制(触发机制)。的角色
hBP和hE 3以及hBP和hE 2的特定氨基酸残基在蛋白质-蛋白质相互作用中的作用将
通过动力学分析、凝胶过滤分析、表面等离子体共振、等温滴定
量热法和超离心法。拟议的研究联合收割机结合了两个
实验室大大提高了我们对hPDH的催化机制和结构功能的理解
hPDC组分之间的关系,并将提供一些遗传生化基础
PDC中的缺陷。
项目成果
期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Characterization of interactions of dihydrolipoamide dehydrogenase with its binding protein in the human pyruvate dehydrogenase complex.
人丙酮酸脱氢酶复合物中二氢硫辛酰胺脱氢酶与其结合蛋白相互作用的表征。
- DOI:10.1016/j.bbrc.2010.04.038
- 发表时间:2010
- 期刊:
- 影响因子:3.1
- 作者:Park,Yun-Hee;Patel,MulchandS
- 通讯作者:Patel,MulchandS
Furoates and thenoates inhibit pyruvate dehydrogenase kinase 2 allosterically by binding to its pyruvate regulatory site.
- DOI:10.1080/14756366.2016.1201812
- 发表时间:2016
- 期刊:
- 影响因子:5.6
- 作者:Masini T;Birkaya B;van Dijk S;Mondal M;Hekelaar J;Jäger M;Terwisscha van Scheltinga AC;Patel MS;Hirsch AK;Moman E
- 通讯作者:Moman E
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MULCHAND S PATEL其他文献
MULCHAND S PATEL的其他文献
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{{ truncateString('MULCHAND S PATEL', 18)}}的其他基金
Alpha Lipoic Acid as a Maternal Supplement in Obese Pregnancies
α-硫辛酸作为肥胖孕妇的母体补充剂
- 批准号:
10573241 - 财政年份:2022
- 资助金额:
$ 19.81万 - 项目类别:
Alpha Lipoic Acid as a Maternal Supplement in Obese Pregnancies
α-硫辛酸作为肥胖孕妇的母体补充剂
- 批准号:
10373662 - 财政年份:2022
- 资助金额:
$ 19.81万 - 项目类别:
Novel drug treatments for pyruvate dehydrogenase complex deficiency
丙酮酸脱氢酶复合物缺乏症的新药治疗
- 批准号:
8951447 - 财政年份:2015
- 资助金额:
$ 19.81万 - 项目类别:
Maternal Hyperinsulinemia and Fetal Programming
母亲高胰岛素血症和胎儿编程
- 批准号:
6369332 - 财政年份:2001
- 资助金额:
$ 19.81万 - 项目类别:
Maternal Hyperinsulinemia and Fetal Programming
母亲高胰岛素血症和胎儿编程
- 批准号:
6928500 - 财政年份:2001
- 资助金额:
$ 19.81万 - 项目类别:
Maternal Hyperinsulinemia and Fetal Programming
母亲高胰岛素血症和胎儿编程
- 批准号:
6525248 - 财政年份:2001
- 资助金额:
$ 19.81万 - 项目类别:
Maternal Hyperinsulinemia and Fetal Programming
母亲高胰岛素血症和胎儿编程
- 批准号:
6607540 - 财政年份:2001
- 资助金额:
$ 19.81万 - 项目类别:
Maternal Hyperinsulinemia and Fetal Programming
母亲高胰岛素血症和胎儿编程
- 批准号:
6785277 - 财政年份:2001
- 资助金额:
$ 19.81万 - 项目类别:
Maternal Hyperinsulinemia and Fetal Programming
母亲高胰岛素血症和胎儿编程
- 批准号:
7581309 - 财政年份:2001
- 资助金额:
$ 19.81万 - 项目类别:
Maternal Hyperinsulinemia and Fetal Programming
母亲高胰岛素血症和胎儿编程
- 批准号:
8134882 - 财政年份:2001
- 资助金额:
$ 19.81万 - 项目类别:
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