CRYSTAL STRUCTURE OF YEAST NAD-SPECIFIC ISOCITRATE DEHYDROGENASE

酵母 NAD 特异性异柠檬酸脱氢酶的晶体结构

• 批准号: 7726261
• 负责人: Peter JOHN HART
• 金额: $ 0.55万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-18 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7726261
• 关键词: Affinity; Anodes; Attenuated; Binding; Binding Sites; Citrate; Citrates; Citric Acid Cycle; Computer Retrieval of Information on Scientific Projects Database; Condition; Decarboxylation; Enzymatic Biochemistry; Enzymes; Funding; Future; Glycolysis; Glycolysis Pathway; Grant; Institution; Isocitrate Dehydrogenase; Isocitrates; Ligand Binding; Mitochondria; Multienzyme Complexes; Mutagenesis; NADH; Nicotinamide adenine dinucleotide; Pathway interactions; Production; Rate; Regulation; Relative (related person); Research; Research Personnel; Resources; Roentgen Rays; Site; Source; Structure; Tricarboxylic Acids; United States National Institutes of Health; Yeasts; alpha ketoglutarate; isocitrate; research study

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 由线粒体 NAD 特异性异柠檬酸脱氢酶催化的异柠檬酸氧化脱羧为 α-酮戊二酸是三羧酸 (TCA) 循环中的限速步骤。 酵母酶 (IDH) 对异柠檬酸的亲和力受变构调节，受 AMP 正向调节，受 ATP 和 NADH 负向调节。 已提出这种变构控制有助于通过氧化途径和糖酵解来反向调节能量产生速率。 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，并且三羧酸、柠檬酸和异柠檬酸将被转移到生物合成途径。酵母 IDH 是由四个 IDH1 和四个 IDH2 亚基组成的八聚体。 IDH1 (M.W. = 38,001) 和 IDH2 (M.W. = 37,755) 具有 42% 的序列同一性。 靶向诱变研究的结果表明，IDH2 亚基含有催化异柠檬酸/Mg2+ 和 NAD 结合位点，而 IDH1 亚基中的同源位点在异柠檬酸的协同结合和变构激活剂 AMP 的结合中发挥作用。 因此，对 IDH 寡聚结构的理解将阐明同源催化和调节配体结合位点之间的关系。 我们已经生产出在 Rigaku FR-D 旋转阳极 X 射线源上衍射至 3.2 英寸的 IDH 晶体。 我们预计晶体结构中 IDH 寡聚排列的测定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 酶学的实验。