ALLOSTERIC PROPERTIES OF PHOSPHOFRUCTOKINASE

磷酸果糖激酶的变构特性

• 批准号: 2137396
• 负责人: ROBERT G KEMP
• 金额: $ 19.23万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-05-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2137396
• 关键词: 6 phosphofructokinase; Escherichia coli; Propionibacteriaceae; allosteric site; animal genetic material tag; chemical binding; chemical kinetics; conformation; enzyme mechanism; enzyme structure; fluorescence; isozymes; nucleic acid structure; protein sequence; proteolysis; site directed mutagenesis

This is a proposal to continue long-standing studies of structure/function relationships with respect to the catalytic and regulatory properties of phosphofructo-1-kinase (PFK). PFK is clearly established as the major rate controlling step in the metabolism of hexose phosphate to pyruvate in all mammalian tissues and almost all other organisms. A detailed study of the nature of the ligand binding sites and of the conformational changes associated with allosteric control is important to our understanding of the regulation of carbohydrate metabolism in normal and pathological states. The principal goal will be the detailed analysis of the regulatory properties of the recently cloned and expressed PFK (C isozyme) from rabbit brain. Critical residues involved in ligand binding and in the conformational change that transmits the allosteric signal will be identified using site-directed mutagenesis of residues that are thought to comprise each of the four binding sites for organic regulatory ligands. To distinguish between those mutations that prevent ligand binding and those that interfere with conformational changes, kinetic studies of the mutants will be supplemented by studies of equilibrium binding of regulatory ligands and by protein conformational analysis, examining the effects of ligands on the rate of thiol reactivity, the rate of limited proteolysis, and changes in intrinsic fluorescence. In addition, the genomic structure of rabbit PFK C isozyme will be examined to determine intron-exon junctions and the structural features of the 5' flanking region. Finally, site-directed mutagenesis studies of the role of specific amino acid residues in the interaction of phosphoryl donors of ATP-dependent PFK from Escherichia coli and the PPi-dependent PFK from Propionibacterium freudenriechii will be completed.

这是继续对结构/功能进行长期研究的建议 关于催化和调节特性的关系 磷酸果糖1-激酶（PFK）。显然将PFK确定为主要率 控制所有磷酸己糖代谢的步骤 哺乳动物组织和几乎所有其他生物。一项详细研究 配体结合位点和构象变化的性质 与变构控制相关 正常和病理中碳水化合物代谢的调节 国家。主要目标是对监管的详细分析 来自最近克隆和表达的PFK（C同工酶）的特性 兔子大脑。涉及配体结合的关键残基和 传输变构信号的构象变化将是 使用被认为是的残基的定点诱变确定 包括有机调节配体的四个结合位点中的每一个。到 区分那些防止配体结合的突变和 干扰了构象变化，突变体的动力学研究 调节的平衡结合的研究将补充 配体和蛋白质构象分析，检查 配体关于硫醇反应性速率，有限的蛋白水解速率， 和内在荧光的变化。另外，基因组结构 将检查兔PFK C同工酶以确定内含子外激素 连接和5'侧翼区域的结构特征。最后， 特定氨基酸作用的位置定向诱变研究 来自ATP依赖性PFK的磷酸供体相互作用的残基 大肠杆菌和PPI依赖性PFK来自丙肽的PFK Freudenriechii将完成。