GENETICS OF PHOSPHOFRUCTOKINASE STRUCTURE AND FUNCTION

磷酸果糖激酶结构和功能的遗传学

• 批准号: 3230262
• 负责人: Simon H Chang
• 金额: $ 14.34万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3230262
• 关键词: 6 phosphofructokinase; Bacillus stearothermophilus; Escherichia coli; active sites; allosteric site; circular dichroism; complementary DNA; enzyme mechanism; enzyme structure; enzyme substrate; gene deletion mutation; gene expression; isozymes; laboratory rabbit; light scattering; molecular cloning; mutant; point mutation; polymerase chain reaction; protein structure function; site directed mutagenesis

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.

磷脂运动酶控制糖酵解的速率限制步骤 匹配正常和压力下的养分和能量的细胞需求 状况。该项目侧重于PFK酶学的两个方面： 兔肌肉PFK（RMPFK）和变构的变构控制 两个细菌PFK之间的差异。 （1）RMPFK的表达是DF1020大肠杆菌宿主和纯化 克隆的rmpfk：为了避免ECPFK-1活动，RMPFK cDNA将为 在PPL2（Lambda PL）载体中亚克隆并在大肠杆菌细胞中转化 ECPFK-1活性不足。克隆的rmpfk将被表示，并 使用新开发的协议进行纯化。作为一个长期目标， 克隆的RMPFK的纯化可能会导致对此的晶体学研究 酶。 （2）RMPFK的位置定向诱变和突变蛋白的性质： Kunkel的方法将用于创建点和删除突变 预定位点。将根据 RMPFK和细菌PFK与晶体之间的序列同源 后者的结构。突变的RMPFK将分析动力学， 底物和效应子的结合以及提交互动。一个 发现存在31个氨基酸（280-311）的截短的PFK亚基存在 自然在人类肌肉和其他组织中。该多肽的作用是 完全未知。缺乏这31个氨基酸的突变体将是 构造，表达和分析酶促活性，变构性 特性，与野生型肌肉PFK亚基的关联及其 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合作绑定F6P，但BSPFK提供双曲线轮廓VS {F6P}。关于PFK的T和R状态的详细信息是从他们的 晶体结构。定向诱变将用于分析 涉及变构过渡的残留物，并确定 负责两个PFK的变构差异。 （4） F6P和F2,6BP站点的立体特殊性：F6P的立体特定性 克隆的BSPFK及其某些突变体的位点将是研究 确定确切的残留物涉及DIN结合并洞悉 本网站发生的进化发生了变化。而且，残留物 涉及DIN F2,6bp变构现场将使用某些 RMPFK的突变体。在所有情况下，一系列结构锁定的类似物 F6P和F2,6bp将使用，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CD 光谱法。