GENETICS OF PHOSPHOFRUCTOKINASE STRUCTURE AND FUNCTION

磷酸果糖激酶控制糖酵解的限速步骤， 匹配细胞在正常和压力下对营养和能量的需求 条件本项目主要研究PFK酶学的两个方面： 兔肌肉PFK（RMPFK）的变构控制， 两种细菌PFK之间的差异。 (1)RMPFK的表达载体为DF 1020 E.大肠杆菌宿主和纯化 克隆的RMPFK：为了避免EcPFK-1活性，将RMPFK cDNA 亚克隆到pPL 2（λ pL）载体中并转化到大肠杆菌细胞中 缺乏EcPFK-1活性。克隆的RMPFK将被表达， 使用新开发的协议进行纯化。作为长期目标， 克隆的RMPFK的纯化可以导致对此的晶体学研究。 酵素 (2)RMPFK的定点诱变和突变蛋白的性质： Kunkel的方法将被应用于创建点突变和缺失突变， 预定的地点。诱变位点的选择将基于 RMPFK和细菌PFKs之间的序列同源性以及晶体 后者的结构。将分析突变的RMPFK的动力学， 底物和效应物的结合以及提交相互作用。一 PFK亚基缺失31个氨基酸（280-311） 天然存在于人体肌肉和其他组织中。这种多肽的作用是 完全未知缺乏这31种氨基酸的突变体将被 构建、表达和分析酶活性、变构 性质，其与野生型肌肉PFK亚基的相关性及其 对一般肌肉PFK酶学的影响。 (3)EcPFK-1和BsPFK的变构差异的结构基础： EcPFK-1协同结合F6 P，但BsPFK给出双曲线曲线， {F6p}。关于PFK的T和R状态的细节从它们的 晶体结构将使用定点诱变来分析 参与变构转换的残基，并鉴定那些 负责两种PFK的变构差异。（四） F6 P和F2，6 BP位点的立体特异性：F6 p的立体特异性 将研究克隆的BsPFK及其某些突变体的位点， 确定涉及din结合的确切残基， 这个地方所经历的进化变化此外，残留物 涉及F2，6 BP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F6 P和F2，6 BP，并将动力学数据与 生物物理学研究，即，直接结合、光散射、UV和CD 光谱法

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.