ALLOSTERIC PROPERTIES OF PHOSPHOFRUCTOKINASE

磷酸果糖激酶的变构特性

• 批准号: 3226582
• 负责人: ROBERT G KEMP
• 金额: $ 15.46万
• 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 1988-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3226582
• 关键词: 6 phosphofructokinase; adenosine triphosphate; affinity chromatography; allosteric site; aminoacid analyzer; brain metabolism; carbohydrate metabolism; conformation; cyclic AMP; enzyme structure; fluorescent dye /probe; immunochemistry; isozymes; liver metabolism; molecular rearrangement; monoclonal antibody; muscle metabolism; phosphorylation; radiotracer; striated muscles

Phosphofructokinase (PFK) is clearly established as the enzyme catalyzing one of the principal rate controlling steps of glycolysis in virtually all tissues. The mechanism of regulation of this key pacemaker is a subject of continued interest, as mechanistic knowledge acquired in the study of this enzyme has general applicability to other allosteric enzymes. This continuing project will: (1) Study the allosteric regulatory properties and tissue distribution of phosphofructokinase-C, which is a major, unique isozyme of the brain. Among the properties to be examined are the kinetic consequence of protein phosphorylation, the regulatory role of fructose-2,6-P2, and the possible role of other regulatory ligands that may be important to the control of carbohydrate metabolism in the brain. As a part of these studies, monoclonal antibodies will be prepared against the A, B, and C isozymes of PFK. The antibodies will be used in isozyme isolation and in tissue distribution studies, and may provide tools for the isolation of homologous sequences among PFK isozymes. (2) Continue ongoing studies of the association of muscle PFK with F-actin, and to examine the role of PFK phosphorylation and the role of regulatory ligands on the binding phenomenon. (3) Extend studies of the tertiary structure and the multiple conformational forms of PFK by employing the technique of limited proteolysis coupled with primry structure analysis. (4) Study the kinetic consequences of the chemical modification of allosteric and catalytic sites, including modification of the AMP site with 2-azido-AMP, and modification of the ATP catalytic site with dialdehyde ATP, dialdehyde ITP, and 5[p-(fluorosulfonyl)benzoyl]adenosine. In each of these last instances the modified enzyme will be used in a parallel project to determine the complete covalent structure of PFK and to place these sites in the primary structure of the enzyme.

磷酸果糖激酶（PFK）清楚地确定为酶催化 几乎所有的糖酵解步骤控制糖酵解步骤的主要速率之一 组织。 该关键起搏器调节的机制是 持续的兴趣，因为在研究中获得的机械知识 酶对其他变构酶具有一般适用性。 这 持续项目将：（1）研究变构调节特性 和磷酸果糖激酶-C的组织分布，这是主要的，独特的 大脑的同工酶。 在要检查的属性中有动力学 蛋白质磷酸化的结果，调节作用 果糖2,6-P2，以及其他调节配体的可能作用 对于控制大脑中的碳水化合物代谢很重要。 作为 这些研究的一部分，将准备单克隆抗体 PFK的A，B和C同工酶。 抗体将用于同工酶 隔离和组织分布研究，并可能为 PFK同工酶之间同源序列的分离。 （2）继续进行 研究肌肉PFK与F-肌动蛋白的关联，并检查 PFK磷酸化的作用和调节配体在 结合现象。 （3）扩展了三级结构的研究和 PFK的多种构象形式通过采用有限的技术 蛋白水解结合启动结构分析。 （4）研究动力学 变构和催化的化学修饰的后果 站点，包括用2-齐多 - AMP修改AMP站点，以及 用尿素ATP，尿醛ITP修饰ATP催化位点， 和5 [p-（氟磺酰基）苯甲烷]腺苷。 在最后一个实例中 修饰的酶将在平行项目中使用，以确定 PFK的完整共价结构，并将这些位点放置在初级 酶的结构。