MOLECULAR GENETICS OF ISOCITRATE DEHYDROGENASES

异柠檬酸脱氢酶的分子遗传学

• 批准号: 6731860
• 负责人: Lee McAlister-Henn
• 金额: $ 30.17万
• 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 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6731860
• 关键词: Escherichia coli; Krebs' cycle; NAD(H) phosphate; aerobiosis; allosteric site; biosynthesis; carbon; enzyme deficiency; enzyme mechanism; enzyme structure; eukaryote; fungal genetics; gene expression; genetic strain; glucose 6 phosphate dehydrogenase; isocitrate dehydrogenase; isozymes; microorganism metabolism; mitochondria; molecular genetics; mutant; nucleic acid sequence; phenotype; polymerase chain reaction; structural biology; three dimensional imaging /topography; yeasts

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控的一个经典示例，据称酵母NAD（+）特异性异急塞脱氢酶（IDH）对细胞能级有反应，并且作为三羧酸周期中的酶，IDH中的酶是通过氧化剂通过氧化剂来控制这些能级的，从而调节这些能级。最近，还发现IDH是具有双重功能的代谢酶的一个例子。该酶与酵母线粒体mRNA的5'-非翻译区域中的特定位点具有显着亲和力结合。假设，IDH与mRNA结合编码内膜呼吸复合物的亚基以瞬时抑制翻译，以防止线粒体基质中这些疏水蛋白的过早合成。我们已经表明，线粒体mRNA的结合也极大地抑制了IDH活性，并且mRNA的结合和抑制作用被IDH的变构激活剂AMP减轻。这些观察结果表明了通过IDH的变构调节来协调对氧化能量产生和线粒体基因表达的新机制。拟议的研究将检验与该机制相关的假设。 酵母IDH是一个包含两种类型的同源亚基的八聚体。我们已经表明，IDH2亚基包含催化异位酸/mg（2+） - 和NAD（+） - 结合位点，而IDH1亚基中的相似位点已演变为用于调节异位酸和AMP的调节结合。拟议的研究旨在阐明IDH的三维和第四纪结构，为同源催化和调节亚基共同进化提供了一种新型模型，并阐明了该组织在结构上促进这些亚基之间变构通信的组织。在体外研究中，将使用具有特定催化和调节特性的明确缺陷的突变体IDH酶，并在体内替代野生型酶，以研究上述现象，即，在线粒体基因的氧化代谢性和线粒体基因的氧化作用中所描述的现象。