GENETICS OF PHOSPHOFRUCTOKINASE STRUCTURE AND FUNCTION

磷酸果糖激酶控制糖酵解的限速步骤， 在正常和压力下匹配细胞对营养和能量的需求 条件。本项目的重点是两个方面的PFK酶学： 兔肌肉PFK(RMPFK)的变构调控及其变构 两种细菌PFK的差异。 (1)RMPFK在DF1020大肠杆菌中的表达及纯化 RMPFK的克隆：为了避免EcPFK-1的活性，RMPFK的cDNA将被 PPL2(Lambda Pl)载体的亚克隆及其在大肠杆菌中的转化 EcPFK-1活性缺乏。克隆的RMPFK将被表达并 使用一种新开发的方案进行纯化。作为一个长期目标， 克隆的RMPFK的纯化可能导致对此的结晶学研究 酵素。 (2)RMPFK的定点突变及其突变蛋白的性质： 昆克尔的方法将被应用于创建点和缺失突变 预先确定的地点。突变地点的选择将基于以下因素 RMPFK与细菌PFKs的序列同源性及晶体 后者的结构。突变的RMPFK将被分析动力学， 底物和效应器的结合以及提交相互作用。一个 发现存在缺失31个氨基酸(280-311)的截短的PFK亚基 天然存在于人体肌肉和其他组织中。这种多肽的作用是 完全未知。缺少这31个氨基酸的突变体将是 构建、表达和分析酶活性、变构 特性及其与野生型肌肉PFK亚基的关系及其意义 对全身肌肉PFK酶学的影响。 (3)EcPFK-1和BsPFK变构差异的结构基础： EcPFK-1协同结合F6P，但BsPFK呈双曲线型 {F6p}。有关PFK的T和R状态的详细信息可从它们的 晶体结构。定点突变将被用来分析 参与变构转变的残基及其鉴定 解释了两种全氟辛烷基酮的变构差异。(4) F6P和F2，6BP位点的立体特异性：F6p的立体特异性 克隆的BsPFK及其某些突变体的位置将被研究 确定与DIN结合有关的确切残留物并深入了解 这个遗址所经历的进化变化。此外，残留物 涉及DIN的F2，6BP变构位点将使用某些 RMPFK的突变体。在所有情况下，一系列结构锁定的类似物 将使用F6P和F2，6BP，动力学数据将与 生物物理研究，即直接结合、光散射、紫外线和镉 光谱分析。

Phosphofructokinase controls the rate-limiting step of glycolysis which matches cellular demand for nutrients and energy under normal and stressful conditions. This project focuses on two aspects on PFK enzymology: the allosteric control of rabbit muscle PFK (RMPFK), and the allosteric differences between two bacterial PFKs. (1) expression of RMPFK is DF1020 E. coli hosts and purification of the cloned RMPFK: In order to avoid EcPFK-1 activity, RMPFK cDNA will be subcloned in a pPL2 (lambda pL) vector and transformed in E.coli cells deficient in EcPFK-1 activity. The cloned RMPFK will be expressed and purified using a newly-developed protocol. As a long term goal, purification of cloned RMPFK could lead to crystallographic studies on this enzyme. (2) Site-directed mutagenesis of RMPFK and properties of mutated proteins: Kunkel's method will be applied to create point and deletion mutations at predetermined sites. Sites for mutagenesis will be selected on the basis of the sequence homologies between RMPFK and bacterial PFKs and the crystal structures of the latter. Mutated RMPFK will be analyzed for kinetics, binding of substrates and effectors as well as submit interaction. A truncated PFK subunit lacking 31 amino acids (280-311) was found to exist naturally in human muscle and other tissue. The role of this polypeptide is entirely unknown. A mutant lacking these 31 amino acids will be constructed, expressed and analyzed for enzymatic activity, allosteric properties, its association with wild-type muscle PFK subunits and its effects on general muscle PFK enzymology. (3) Structural basis for the allosteric differences of EcPFK-1 and BsPFK: EcPFK-1 binds F6P cooperatively but BsPFK gives a hyperbolic profile vs {F6p}. Details about the T and R states of the PFKs are known from their crystal structures. Site-directed mutagenesis will be used to analyzed the residues involved in the allosteric transition and to identify those responsible for the allosteric differences of the two PFKs. (4) Stereospecificity at F6P and F2,6BP sites: The stereospecificity of F6p site of cloned BsPFK and certain mutants thereof will be studies to ascertain the exact residues involve din binding and to gain insight into the evolutionary changes that this site underwent. Moreover, the residues involve din the F2,6BP allosteric site will be investigated using certain mutant of RMPFK. In all cases, a series of structurally locked analogues of F6P and F2,6BP will be used and the kinetic data will be correlated with biophysical studies, i.e., direct binding, light scattering, UV and CD spectrometry.