REGULATION OF THIAMINE DEPENDENT ENZYMES INVOLVED IN GLUCOSE METABOLISM

因为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物化学和分子特性。 依赖酶[线粒体丙酮酸脱氢酶 线粒体`-酮戊二酸脱氢酶(`-KGDH) 复合体和胞质转酮醇酶(TK)。这些酶起着关键作用。 在对大脑能量至关重要的葡萄糖代谢中 供给。这些硫胺素依赖的酶的活性降低是 据报道，在许多神经退行性疾病中观察到了这种情况，包括 酗酒的韦尼克-科萨科夫患者。催化活性PDH E1 人和大鼠的蛋白在大肠杆菌中得到了高效表达。站点定向 模拟自然发生突变的E1亚单位的诱变 都是为了研究结构如何变化的分子机制 突变会影响PDH-E1的催化活性或稳定性 亚单位。 将过量生产的蛋白质提纯到接近均一的方法是 发展：细胞匀浆，硫酸铵沉淀，以及 与FPLC连接的苯基琼脂糖柱层析。 然后我们检测了纯化的人PDH E1的生化性质 野生型和突变型PDH-E1蛋白在E. Coli.观察到不同的蛋白质磷酸化速率。 突变的PDH蛋白，这可能对应于 这个突变体的活性。此外，人类PDH的DNA多态 基因正在用逆转录聚合酶技术进行研究 链式反应(RT-PCR)和单链分析 构象多态(SSCP)。精氨酸的关键作用 对转酮醇酶催化活性中的残基进行了研究。 借助定点突变技术。在四位保守派中 转酮醇酶中的精氨酸残基(氨基酸62、359、455和528)， 似乎只有Arg359对TK活动是必需的。计算机- PDH E1的辅助同源建模是使用 瑞士蛋白酵母转酮醇酶的晶体结构 数据库。

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.