REGULATION OF GLYCEROL KINASE CATALYTIC ACTIVITY

甘油激酶催化活性的调节

• 批准号: 3309130
• 负责人: DONALD W PETTIGREW
• 金额: $ 9.18万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3309130
• 关键词: Escherichia coli; X ray crystallography; active sites; adenosine triphosphate; bacterial proteins; chemical binding; enzyme activity; enzyme complex; enzyme inhibitors; enzyme mechanism; enzyme structure; fructose phosphate; genetic regulation; glycerol kinase; immunoelectron microscopy; laboratory rabbit; mutant; phosphorylation; spectrometry

The long-term objective is understanding mechanisms of control of the catalytic activity of the key enzyme in a signal transduction pathway which modulates gene expression in response to the availability of carbon sources. The enzyme is Escherichia coli glycerol kinase (GK), which catalyzes the MgATP-dependent phosphorylation of glycerol to sn-glycerol- 3-phosphate, which is the inducer for the elements of the glp regulon. Its catalytic activity is inhibited by the binding of fructose 1,6- bisphosphate (FBP) or dephospho-enzyme lllglc of the phosphoenolpyruvate:sugar phosphotransferase system, and the kinetics with respect to ATP show apparent negative cooperativity. The inhibition by lllglc is relieved by reversible phosphorylation of lllglc. the regulatory properties are physiologically-relevant and result in efficient inducer synthesis if glucose becomes unavailable while glycerol is available. Conversely, if glucose again becomes available, inducer synthesis is shutdown by V-system controls which operate even in the presence of high glycerol concentrations. The regulatory behavior will be rigorously characterized by using kinetics, ligand binding, and spectroscopic methods. The specific aims are to characterize the mechanisms and energetics of the regulation and the mechanism by which phosphorylation of lllglc relieves its inhibition. both wild type GK and lllglc will be characterized, as well as mutants of each enzyme which have been shown to affect FBP or lllglc regulation. The availability of the crystal structures of GK and the GK-lllglc complex greatly facilitates interpretation of the results, and additional mutants may be constructed to test hypotheses that develop from further refinement of the structures. Possible effects of regulation of GK activity on its subcellular localization will be studied by using electron microscopy and immunolocalization. Regulatory properties of other members of a widely- distributed family of bacterial sugar kinases that have primary structures that are similar to GK will be compared to those of GK to investigate relations between enzyme structure and regulation and control of gene expression. The results will provide insights into mechanisms of the control of gene expression in living systems.

长期目标是了解控制机制 信号转导途径中关键酶的催化活性 响应碳的可用性调节基因表达 来源。 酶是大肠杆菌甘油激酶（GK），它 催化甘油依赖MGATP的磷酸化 3-磷酸​​盐，这是GLP调节元素元素的诱导剂。 果糖1,6-的结合抑制其催化活性 双磷酸（FBP）或dephospho-enzyme lllGlc 磷酸烯醇丙酮酸：糖磷酸转移酶系统和动力学 关于ATP，显示出明显的负合作性。 抑制作用 通过LLLGLC的可逆磷酸化可以缓解LLLGLC。 这 监管特性在生理上与众不足，并导致 如果葡萄糖不可用，而甘油则有效诱导剂合成 可用。 相反，如果再次可用葡萄糖，诱导剂 V-System控件即使在 高甘油浓度的存在。 监管行为将 通过动力学，配体结合和 光谱法。 具体目的是表征 调节的机制和能量学和机制 LLLGLC的磷酸化可缓解其抑制作用。 野生型GK和 将表征LLLGLC，以及每个酶的突变体 已显示影响FBP或LLLGLC调节。 可用性 GK和GK-LLLGLC复合物的晶体结构极大 促进对结果的解释，其他突变体可能是 构建是为了测试从进一步改进而发展的假设 结构。 GK活性调节对其的可能影响 通过使用电子显微镜和 免疫定位。 广泛的其他成员的监管特性 具有主要的细菌糖激酶的分布式家族 将与GK相似的结构与GK的结构进行比较 研究酶结构与调节与控制之间的关系 基因表达。 结果将提供有关机制的见解 在生活系统中控制基因表达的控制。