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

描述（改编自申请人摘要）： 果糖-2，6-二磷酸（Fru-2，6-P2）通过以下途径调节糖酵解速率： 其对磷酸果糖激酶（PFK）的有效激活。 细胞内 Fru-2，6-P2的浓度取决于拮抗剂的平衡。 独特的双功能酶的激酶和磷酸酶活性 6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶（6-PF-2-K/Fru-2，6-P2 糖酵解活性的需要随时间和组织而变化 到组织。 因此，6-PF-2-K/Fru-2，6-P2酶的组织特异性同种型具有 已经鉴定了几种细胞类型（肝脏、骨骼肌、心脏 肌肉、睾丸、胎盘和脑）。 这些同种型中的每一种都具有 激酶与磷酸酶活性的特征性相对比率， 独特调节的B各种抑制剂和激活剂， 浓度与细胞的代谢需要（即，PEP，Pi NTPs， 柠檬酸盐、磷酸甘油酸盐）。 肝脏和心脏同工酶的活性 进一步受激素诱导的蛋白质磷酸化NH 2-和 COOH-末端调节结构域。 的磷酸化 肝同工酶被胰高血糖素刺激，并导致抑制 6-PF-2-K活性，同时激活Fru-2，6-P2酶。 的 心脏同工酶的磷酸化受到肾上腺素的刺激， 导致6-PF-2-K活性的活化，而对 Fru-2，6-P2酶。 所以这两个磷酸化位点 同工酶在多肽链的两端，但它们具有相反的 对6-PF-2-K活性也有影响。 该系统提供了一个独特的机会，观察如何一个单一的酶， 进化来平衡组织中两种对立的催化活性 特异性方式，包括配体介导和 磷酸化依赖的催化调节机制。 是 拟议研究的目标是确定这些分子基础， 现象。 他们已经确定了2.0Aring的结构， 未调节的大鼠睾丸同工酶。 他们现在将决定 不同配体结合的睾丸同工酶的三维结构 构象 此外，它们会结晶并溶解结构 肝脏、心脏和胎盘同工酶的磷酸化和 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.