HORMONAL CONTROL OF HEPATIC GLUCONEOGENESIS/GLYCOLYSIS

这项研究的目的是了解 调节肝脏中的糖酵解/促生途径通量 关注Fru 6-P/Fru 1，6-P2底物循环的酶， 包括6-磷酸果糖-1-激酶（6PFlK）、果糖-1，6- 二磷酸酶（Fru-1，6-P2酶）和6-磷酸果糖-2- 激酶/果糖-2，6-二磷酸酶（6PF 2K/Fru-2，6-P2 ase）。 的 建议的工作的主要重点将是6PF 2K/Fru 2，6-P2 ase。 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。 定点诱变 将被用来测试这一假设，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，保留其预测的 活动，假设将得到证实。 的性质 分离激酶和双磷酸酶结构域也将 与天然酶的那些进行比较，以阐明 酶功能的优势。 互惠的性质 通过cAMP依赖性磷酸化调节这两种活性 将研究突变形式的酶， 磷酸化位点发生了改变。 进化 双磷酸酶结构域与磷酸甘油酸之间的关系 特别是保护生物活性位点 双磷酸酶中的残基，为位点提供了基础- 在双磷酸酶活性位点的定向诱变。 推定 活性位点残基将被系统地改变， 确定关键的残基催化的激酶和 双磷酸酶反应。 定点诱变也将是 用于鉴定6PF 1 K和Fru-2，6-P2中的Fru 2，6-P2结合位点。 1，6-P2酶，并确定磷酸化在其 调控 6PF2K/Fru-2，6- P2酶、6PF 1 K和Fru-1，6-P2酶也将被表征， 这些影响的机制阐明，和这种关系， 从长期调控到全面控制 各种饮食和激素状态下的糖酵解/异生 评估。 如果确定控制这些数量， 酶是在转录水平，那么它们的基因组 将确定结构，包括位置和顺序 的假定的激素反应元件的知识， 上述研究将允许表达野生型和突变酶 在未来的肝细胞系中形成，并允许 这些变化将在整个功能和控制方面进行测试， 通路 阐明了激素 调节糖酵解/代谢通量应该导致新的 治疗疾病状态的方法， 控制这些通路的紊乱，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.