STRUCTURAL ANALYSIS OF 6-PF-2-K/FRU-2,6-P2ASE ISOZYMES

描述(改编自申请人的摘要)： 2，6-二磷酸果糖(Fru-2，6-P2)通过 它能有效激活磷酸果糖激酶(PFK)。细胞内 Fru2，6-P2的浓度由拮抗剂的平衡决定 一种独特的双功能酶的激酶和磷酸酶活性 6-phosphofructo-2-kinase/fructose-2，6-bisphosphatase(6-PF-2-K/Fru2，6-P2 对糖酵解活性的需求随着时间和组织的不同而不同 到组织。因此，组织特异性的6-PF-2-K/Fru-2，6-P2酶亚型有 被鉴定为几种细胞类型(肝脏、骨骼肌、心脏 肌肉、睾丸、胎盘和大脑)。这些亚型中的每一个都有一个 激酶与磷酸酶活性的特征相对比率，以及 由各种不同的抑制剂和激活剂独一无二地调节 浓度与细胞的代谢需要(即PEP，PI NTPs， 柠檬酸盐、磷酸甘油酸酯)。肝脏和心脏同工酶的活性 进一步受荷尔蒙诱导的NH2-和蛋白质磷酸化的调节 分别为COOH-末端调节域。该蛋白的磷酸化 肝脏同工酶受胰高血糖素的刺激，导致抑制 6-PF-2-K活性，伴随着Fru-2，6-P2酶的激活。这个 肾上腺素刺激心脏同工酶的磷酸化，并且 结果激活6-PF-2-K活性，但不影响 FRU-2，6-P2酶。因此，不仅这两个蛋白的磷酸化位点 多肽链两端的同工酶，但它们有相反的 对6-PF-2-K活性的影响。 这个系统提供了一个独特的机会来观察单个酶是如何 进化以平衡组织中的两种拮抗催化活性 特定的时尚，包括配基介导的和 依赖于磷酸化的催化调节机制。它是 拟议研究的目标是确定这些物质的分子基础 现象。他们已经确定了2.0年的结构。 未调控的大鼠睾丸同工酶。他们现在将确定三个 不同配基结合的精巢同工酶的空间结构 构象。此外，它们还将结晶并解决结构问题 肝脏、心脏和胎盘同工酶，在它们的磷酸化和 非磷酸化状态。

DESCRIPTION (Adapted from the applicant's abstract): Fructose-2,6-bisphosphate (Fru-2,6-P2) modulates the rate of glycolysis via its potent activation of phosphofructokinase (PFK). The intracellular concentration of Fru-2,6-P2 is determined by the balance of the antagonistic kinase and phosphatase activitie of the unique bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6-PF-2-K/Fru-2,6-P2 ase) The need for glycolytic activity varies both with time and from tissue to tissue. Thus tissue specific isoforms of 6-PF-2-K/Fru-2,6-P2 ase have been identified for several cell types (liver, skeletal muscle, heart muscle, testis, placenta, and brain). Each of these isoforms has a characteristic relative ratio of kinase to phosphatase activity, and are uniquely regulated b various inhibitors and activators which vary in concentration with the metabolic needs of the cell (i.e., PEP, Pi NTPs, citrate, phosphoglycerate). The activities of the liver and heart isozymes are further regulated by hormon induced protein phosphorylation of NH2- and COOH-terminal regulatory domains respectively. The phosphorylation of the liver isozyme is stimulated by glucagon, and results in inhibition of the 6-PF-2-K activity, with a concomitant activation of the Fru-2,6-P2 ase. The phosphorylation of the heart isozyme is stimulated by epinephrine, and results in an activation of the 6-PF-2-K activity, with no effect on the Fru-2,6-P2 ase. So not only are the sites of phosphorylation for these two isozymes at opposite ends of the polypeptide chain, but they have opposite effects on the 6-PF-2-K activity as well. This system offers a unique opportunity to observe how a single enzyme has evolved to balance two antagonistic catalytic activities in a tissue specific fashion, involving both ligand-mediated and phosphorylation-dependent mechanisms for the regulation of catalysis. It is the goal of the proposed research to determine the molecular basis of these phenomena. They have alread determined the 2.0Aring; structure of the unregulated rat testis isozyme. They will now determine the three dimensional structure of the testis isozyme in various ligand-bound conformations. In addition, they will crystallize and solve the structure of the liver, heart, and placenta isozymes, in both their phosphorylated and unphosphorylated states.