STRUCTURE AND MECHANISM OF L-ASPARTASE

L-天冬氨酸酶的结构和机制

• 批准号: 3285758
• 负责人: RONALD Edward VIOLA
• 金额: $ 7.74万
• 依托单位: UNIVERSITY OF AKRON
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-09-01 至 1991-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3285758
• 关键词: Escherichia coli; X ray crystallography; acidity /alkalinity; aspartate; chemical binding; dialysis; electron spin resonance spectroscopy; enzyme induction /repression; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; metalloenzyme; nuclear magnetic resonance spectroscopy; stereochemistry

This project involves a continuation of our studies on the enzyme L-aspartase, which catalyzes the reversible addition of ammonia to fumaric acid. The long term objectives of this project are to acquire a detailed understanding, at the molecular level, of the events involved in the mechanism and the regulation of the reaction catalyzed by L-aspartase, and by several other aspartate-utilizing metalloenzymes. The urea cycle enzyme argininosuccinase catalyzes a related reaction involving the addition of arginine to fumaric acid. Partial loss of argininosuccinase activity has been implicated in an inborn error of metabolism in humans associated with mental deficiencies. The bifunctional enzyme aspartokinase- homoserine dehydrogenase also occupies a key position in metabolism, the commitment step to the biosynthesis of four amino acids starting from L-aspartic acid. The specific aims proposed in this research plan are to: probe the detailed kinetic and chemical mechanism of this enzyme system; establish the role of metal ion activators and inhibitors; explore the dual role of L- aspartic acid as a substrate and an activator for the enzyme; and determine the structure and orientation of the binding sites on L- aspartase. The techniques that will be applied in order to address the specific aims of the research project will include equilibrium dialysis and kinetic binding studies of metal ions, substrate analogs, and activators. A range of kinetic studies, including initial velocity, product inhibition, pH, and isotope effect studies, will be utilized to elucidate dynamic information about the reaction catalyzed by L-aspartase. Magnetic resonance studies, including NMR paramagnetic relaxation studies to examine enzyme-- substrate and enzyme-activator interactions, NMR and EPR studies to examine metal ion interactions, low temperature NMR studies to characterize intermediates in the catalytic cycle, and x-ray crystallographic studies of enzyme-substrate and analog complexes, will be utilized to provide structural information about the interaction of metal ions, substrates and activators with L- aspartase. Taken together these techniques offer the opportunity to conduct a detailed examination of the events involved in the mechanism of L-aspartase and some related enzymes, and to examine the role played by metal ion and substrate effectors for these enzymes.

这个项目包括我们对酶的研究的继续 L-精氨酸酶，其催化氨的可逆加成， 富马酸 该项目的长期目标是 在分子水平上获得详细的了解， 参与反应机制和调控的事件 由L-淀粉酶和其他几种利用淀粉酶的酶催化， 金属酶 尿素循环酶黄嘌呤琥珀酸酶催化 一种涉及精氨酸与富马酸加成的相关反应 酸 已经发现， 与人类先天性代谢缺陷有关， 有精神缺陷 双功能酶葡激酶- 高丝氨酸脱氢酶也在 代谢，四个氨基的生物合成的承诺步骤 从L-天冬氨酸开始的酸。 提出的具体目标 在本研究计划是：探讨详细的动力学和 该酶系统的化学机制;确定 金属离子活化剂和抑制剂;探索L- 天冬氨酸作为酶的底物和激活剂;和 确定L-上结合位点的结构和方向 放轻松 将应用的技术，以解决 该研究项目的具体目标将包括平衡 金属离子、底物的透析和动力学结合研究 类似物和活化剂。 一系列动力学研究，包括 初始速度、产物抑制、pH和同位素效应 研究，将被用来阐明动态信息， 该反应由L-精氨酸酶催化。 磁共振研究， 包括核磁共振顺磁弛豫研究来检测酶-- 底物和酶-活化剂相互作用，NMR和EPR研究 为了检查金属离子的相互作用，低温NMR研究， 表征催化循环中的中间体， 酶-底物和类似物复合物的晶体学研究， 将被用来提供有关的结构信息 金属离子、底物和活化剂与L- 放轻松 综合这些技术， 详细审查了涉及的事件， L-精氨酸酶及相关酶的作用机制，并研究 金属离子和基底效应物对这些所起作用 内切酶