Mechanism and Molecular Recognition in Human Pyruvate Dehydrogenase Complex

人丙酮酸脱氢酶复合体在丙酮酸氧化中的守门人S作用 来源于碳水化合物和几种氨基酸的碳骨架，每天约50% 卡路里摄入量(根据碳通量)是由这种酶调节的。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)、二氢硫胺 乙酰转移酶(E2)和二氢硫胺脱氢酶(E3)；E3结合蛋白(BP)，以及两个 调节酶：PDH激酶(PDK，4个同工酶)和磷酸酶(PDP，2个同工酶)。遗传 PDC成分的缺陷说明了PDC在能量动态平衡中的重要性及其原因 儿童大脑发育受损，智力发育迟缓，常常过早死亡。PDC的减少 在慢性疾病中观察到组成蛋白和活性水平，如2型糖尿病、肥胖症 和心血管疾病以及神经退行性疾病，如阿尔茨海默病和S病， 帕金森病、S病和韦尼克-科萨科夫综合征。HPDC已演变为独特的结构 原核PDCs和α-酮酸脱氢酶复合体家族其他成员的组织 通过将BP作为附加成分和高度复杂的调节机制从所有物种中分离出来 通过PDKs和PDP的多种同工酶。最近，我们已经解决了人体PDH的三维结构 以及与BP的E3结合域结合的人E3。基于这些发展，我们的三个 具体目的是(I)研究人PDH催化过程中中间体的形成，(Ii) 研究hPDH中的活性部位通讯，以及(Iii)确定HE3之间的相互作用位置 和HBP之间，也介于hE2和HBP之间。将采用定点突变技术来引入所需的 突变和人类蛋白将通过亲和层析进行过度表达和纯化。我们会 采用最先进的仪器(如圆二色谱、核磁共振、质谱学、快速 停流圆二色谱和等温滴定量热法)和化学合成的类似物 识别和监控PDH反应的中间产物。石油化工中间体的时程分析 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，并 描述两个活性部位之间的通信机制(触发器机制)。角色 HBP和HE3以及HBP和hE2在蛋白质-蛋白质相互作用中的特定氨基酸残基将 采用动力学分析、凝胶过滤、表面等离子体共振、恒温滴定等方法进行测定 量热法和超速离心法。拟议的研究结合了两个 实验室大大提高了我们对hPDH的催化机理和结构功能的了解 HPDC组分之间的相互关系，将为某些基因的研究提供生化基础 PDC中的缺陷。

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.