HORMONAL CONTROL OF HEPATIC GLUCONEOGENESIS & GLYCOLYSIS

这项研究的目标是了解人类免疫缺陷的分子基础。 白三烯对肝脏糖酵解/糖异生途径流量的调节 聚焦于Fru6-P/Fru1，6-P2底物循环中的酶， 包括6-磷酸果糖-L-激酶(6PFlK)、果糖-1，6- 二磷酸酶(果糖-1，6-磷)和6-磷酸果糖-2- 激酶/果糖-2，6-二磷酸酶(6PF2K/果糖-2，6-P2ase)。这个 拟议工作的主要重点将是6PF2K/2，6-P2ase。 我们提出了这样的假设，即这种双功能酶 由两个独立的域组成。定点突变 将被用来检验这一假说并确定 酶的催化和调节的分子基础， 反应。如果每个域都可以在 异源表达系统及其预测的保留性 活动，假说将得到证实。的属性 单独的激酶和双磷酸酶结构域也将是 与天然酶进行比较，以阐明 酶功能的优势。互惠的本质 CAMP依赖的磷酸化对这两种活性的调节 将用这种酶的突变形式进行研究，其中 磷酸化位点发生了改变。进化论 二磷酸酶结构域与磷酸甘油酯的关系 变位酶，特别是变位酶活性部位的保守性 双磷酸酶中的残基，为该位点提供了基础- 针对双磷酸酶活性部位进行定向突变。推定 活性中心残基将被系统地改变，以便 确定在这两种蛋白中催化的关键残基 双磷酸酶反应。定点突变也将是 用来鉴定6PF1K和Fru2，6-P2结合位点 1，6-P2ase，并确定磷酸化在它们的 监管。6PF2K/Fru-2，6-基因表达的激素调控 还将对P2ase、6PF1K和Fru-1，6-P2ase进行鉴定， 阐明了这些作用的机理，以及它们之间的关系 长效调控，全面管控 不同饮食和激素状态下的糖异生/糖酵解 已评估。如果确定了对这些物质数量控制 酶是在转录水平上，然后是它们的基因组 将确定结构，包括位置和顺序 可能的激素反应元件的知识从 上述研究将允许表达野生型和突变型酶 在未来的肝细胞系中形成并允许作用于 这样的变化需要在功能和控制上进行测试 路径。激素参与的分子机制的阐明 调节糖酵解/糖异生通量应导致新的 治疗疾病状态的方法 这些通路的控制错乱，如糖尿病。

The goal of this research is to understand the molecular basis for regulation of glycolytic/gluconeogenic pathway flux in liver by focusing on the enzymes of the Fru 6-P/Fru 1,6-P2 substrate cycle, including 6-phosphofructo-l-kinase (6PFlK), fructose-1,6- bisphosphatase (Fru-1,6-P2ase), and 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-P2ase). The major emphasis of the proposed work will be on 6PF2K/Fru 2,6-P2ase. We have put forth the hypothesis that this bifunctional enzyme consists of two independent domains. Site-directed mutagenesis will be employed to test this hypothesis and to determine the molecular basis for catalysis and regulation of the enzymes, reactions. If each domain can be expressed separately in a heterologous expression system, with retention of its predicted activity, the hypothesis will be substantiated. The properties of the separate kinase and bisphosphatase domains will also be compared with those of the native enzyme to elucidate the advantages of enzyme functionality. The nature of the reciprocal regulation of the two activities by cAMP-dependent phosphorylation will be studied with mutant forms of the enzyme where the phosphorylation site has been altered. The evolutionary relationship between the bisphosphatase domain and phosphoglycerate mutase, in particular the conservation of the mutase active site residues in the bisphosphatase, provides a basis for the site- directed mutagenesis at the bisphosphatase active site. Putative active site residues will be systematically altered in order to identify the critical residues for catalysis in both the kinase and bisphosphatase reactions. Site-directed mutagenesis will also be employed to identify the Fru 2,6-P2 binding site in 6PF1K and Fru- 1,6-P2ase and to determine what role phosphorylation has in their regulation. Hormonal control of gene expression of 6PF2K/Fru-2,6- P2ase, 6PF1K, and Fru-1,6-P2ase will also be characterized, the mechanism of these effects elucidated, and the relationship of this long-term regulation to overall control of gluconeogenesis/glycolysis in various dietary and hormonal states evaluated. If it is determined that control of the amount of these enzymes is at the level of transcription then their genomic structures will be determined, including the location and sequence of putative hormone response element knowledge gained from the above studies will allow expression of wild-type and mutant enzyme forms in liver cell lines in the future and permit the effects of such changes to be tested on function and control of the entire pathway. Elucidation of the molecular mechanisms whereby hormones regulate glycolytic/gluconeogenic flux should lead to new approaches for treatment of disease states where there are derangements in control of these pathways, such as diabetes.