REGULATION OF THIAMINE DEPENDENT ENZYMES INVOLVED IN GLUCOSE METABOLISM

参与葡萄糖代谢的硫胺素依赖性酶的调节

• 批准号: 5200218
• 负责人: B J SONG
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5200218
• 关键词: animal tissue; arginine; computer assisted sequence analysis; enzyme activity; enzyme complex; enzyme induction /repression; enzyme inhibitors; enzyme structure; genetic polymorphism; glucose metabolism; mitochondria; molecular cloning; oxoglutarate dehydrogenase; phosphorylation; polymerase chain reaction; protein sequence; pyruvate dehydrogenase; single strand conformation polymorphism; site directed mutagenesis; transaldolase /transketolase

Because of their importance in the energy and intermediary metabolism, we studied the biochemical and molecular characteristics of thiamine- dependent enzymes [the mitochondrial pyruvate dehydrogenase (PDH) complex, the mitochondrial `-ketoglutarate dehydrogenase (`-KGDH) complex, and cytosolic transketolase (TK).] These enzymes play key roles in glucose metabolism which is critically important in cerebral energy supply. Reduced activities of these thiamine-dependent enzymes were reportedly observed in many neurodegenerative disease states including alcoholic Wernicke-Korsakoff patients. Catalytically active PDH E1 proteins of human and rat were over-produced in E. coli. Site-directed mutagenesis of the E1 subunit simulating naturally occurring mutations were performed to study the molecular mechanism of how the structural mutations affect the catalytic activity or the stability of the PDH E1 subunit. Methods to purify the over-produced proteins to near homogeneity were developed: cell homogenation, ammonium sulfate precipitation, and chromatography on phenyl-sepharose column connected to FPLC. We then examined the biochemical properties of the purified human PDH E1 proteins of both wild and mutant PDH E1 proteins over-produced in E. coli. Differential rate of protein phosphorylation was observed in the mutant PDH protein, which may correspond for the rapid loss of the activity in this mutant. In addition, DNA polymorphism in the human PDH gene is being studied by techniques of reverse transcription-polymerase chain reaction (RT-PCR) followed by analyses of single strand conformational polymorphism (SSCP). The critical role of arginine residue in the catalytic activity of transketolase was studied with the aid of site-directed mutagenesis technique. Among the four conservative arginine residues in transketolase (amino acids 62, 359, 455, and 528), only Arg359 appears to be essential for the TK activity. Computer- assisted homology modelling of the PDH E1 is being accomplished using the crystal structure of yeast transketolase obtained from the Swiss Protein Data Bank.

由于它们在能量和中间代谢中的重要性， 我们研究了硫胺素的生化和分子特征， 依赖酶[线粒体丙酮酸脱氢酶（PDH） 复合物，线粒体“-酮戊二酸脱氢酶（”-KGDH） 复合物和胞质转酮醇酶（TK）。 这些酶发挥着关键作用 葡萄糖代谢对大脑能量至关重要 供应 这些硫胺素依赖性酶的活性降低， 据报道，在许多神经退行性疾病状态中观察到， 酗酒的韦尼克-科萨科夫综合征患者 催化活性PDH E1 人和大鼠的蛋白质在E.杆菌 定点 模拟天然突变的E1亚基诱变 进行了研究的分子机制，如何结构 突变影响PDH E1的催化活性或稳定性 亚单位 将过量产生的蛋白质纯化至接近同质的方法是 开发：细胞匀浆，硫酸铵沉淀，和 在连接至FPLC的苯基-琼脂糖柱上进行色谱分离。 然后，我们检测了纯化的人PDH E1的生化特性 野生型和突变型PDH E1蛋白在E. 杆菌 蛋白质磷酸化的差异率观察到， 突变PDH蛋白，这可能对应于快速损失的 在这个突变体中。 此外，人类PDH中的DNA多态性 基因正在研究的逆转录聚合酶技术 链反应（RT-PCR），然后进行单链分析 构象多态性（SSCP）。 精氨酸的关键作用 研究了转酮醇酶催化活性中的残留物， 借助于定点诱变技术。 在四个保守的 转酮醇酶中的精氨酸残基（氨基酸62、359、455和528）， 只有Arg 359似乎是TK活性所必需的。 电脑- PDH E1的辅助同源性建模正在使用 从瑞士蛋白获得的酵母转酮醇酶的晶体结构 数据库。