MOLECULAR GENETICS OF ISOCITRATE DEHYDROGENASES

异柠檬酸脱氢酶的分子遗传学

• 批准号: 7152889
• 负责人: Lee McAlister-Henn
• 金额: $ 28.83万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2008-02-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7152889
• 关键词: 5' Untranslated Regions; Address; Adenine Nucleotides; Affinity; Allosteric Regulation; Binding; Binding Sites; Buffers; Carboxy-Lyases; Catalysis; Catalytic Domain; Cell Respiration; Citrate; Citrates; Citric Acid Cycle; Collection; Communication; Complex; Condition; Crystallization; Defect; Enzymes; Evolution; Gene Expression; Genes; Goals; Growth; Holoenzymes; Imagery; In Vitro; Isocitrate Dehydrogenase; Isocitrates; Kinetics; Ligand Binding; Ligands; Measures; Membrane; Messenger RNA; Metabolic; Mitochondria; Mitochondrial Matrix; Modeling; Molecular Genetics; Monitor; Mutagenesis; Nicotinamide adenine dinucleotide; Numbers; Physiological; Production; Property; Proteins; Rate; Reporter; Research; Roentgen Rays; Site; Structure; Testing; Translations; Yeasts; base; design; in vivo; isocitrate; mitochondrial messenger RNA; mutant; novel; prevent; protein degradation; research study; respiratory; response; three dimensional structure

As a classic example for allosteric regulation by adenine nucleotides, yeast NAD(+) specific isocitrate dehydrogenase (IDH) is purported to be responsive to cellular energy levels and, as an enzyme in the tricarboxylic acid cycle, IDH in turn modulates these energy levels by controlling flux through oxidative metabolism. More recently, IDH has also been found to be an example of a metabolic enzyme with dual functions. The enzyme binds with significant affinity to specific sites in the 5'-untranslated regions of yeast mitochondrial mRNAs. This binding by IDH to mRNAs that encode subunits of inner-membrane respiratory complexes is hypothesized to transiently inhibit translation to prevent premature synthesis of these hydrophobic proteins in the mitochondrial matrix. We have shown that binding of mitochondrial mRNA also dramatically inhibits IDH activity, and that binding and inhibition by mRNA are alleviated by the allosteric activator of IDH, AMP. These observations suggest a novel mechanism for coordinate control of oxidative energy production and of mitochondrial gene expression through allosteric regulation of IDH. Proposed research will test hypotheses related to this mechanism. Yeast IDH is an octamer containing two types of homologous subunits. We have shown that the IDH2 subunit contains catalytic isocitrate/Mg(2+)-and NAD(+)-binding sites, whereas similar sites in the IDH1 subunit have evolved for regulatory binding of isocitrate and AMP. Proposed research seeks to clarify three-dimensional and quaternary structures of IDH to provide a novel model for co-evolution of homologous catalytic and regulatory subunits, and to elucidate the organization that structurally facilitates allosteric communication between these subunits. Concomitantly, mutant IDH enzymes with well-defined defects in specific catalytic and regulatory properties will be used in in vitro studies and to replace the wild-type enzyme in vivo to investigate the phenomena described above, i.e., allosteric control of oxidative metabolism and of mitochondrial gene expression at the level of translation.

作为腺嘌呤核苷酸变构调节的经典实例，酵母NAD（+）特异性异柠檬酸脱氢酶（IDH）据称响应于细胞能量水平，并且作为三羧酸循环中的酶，IDH进而通过氧化代谢控制通量来调节这些能量水平。最近，IDH也被发现是具有双重功能的代谢酶的一个例子。该酶以显著的亲和力结合酵母线粒体mRNA的5 '-非翻译区中的特定位点。IDH与编码内膜呼吸复合物亚基的mRNA的这种结合被假设为瞬时抑制翻译，以防止这些疏水蛋白在线粒体基质中的过早合成。我们已经表明，线粒体mRNA的结合也显着抑制IDH的活性，并通过mRNA的结合和抑制IDH，AMP的变构激活剂减轻。这些观察结果表明，通过IDH的变构调节协调控制氧化能的产生和线粒体基因表达的新机制。拟议的研究将测试与此机制有关的假设。 酵母IDH是含有两种类型的同源亚基的八聚体。我们已经表明，IDH 2亚基含有催化异柠檬酸/Mg（2+）-和NAD（+）-结合位点，而IDH 1亚基中的类似位点已经进化为异柠檬酸和AMP的调节结合。拟议的研究旨在阐明IDH的三维和四级结构，为同源催化和调节亚基的共同进化提供一种新的模型，并阐明在结构上促进这些亚基之间变构通信的组织。伴随地，在特定催化和调节特性中具有明确缺陷的突变IDH酶将用于体外研究并在体内替代野生型酶以研究上述现象，即，氧化代谢和线粒体基因表达在翻译水平的变构控制。