STRUCTURE AND MECHANISM OF L-ASPARTASE

这个项目涉及到我们对这种酶的继续研究。 L-天冬氨酸酶，催化氨可逆加成 富马酸。这项计划的长期目标是 在分子水平上获得对 参与反应机制和调节的事件 由L-天冬氨酸酶和其他几种天冬氨酸利用酶催化 金属酶。尿素循环酶精氨酸琥珀酸酶催化 与富马酸的精氨酸加成有关的反应 酸。精氨酸琥珀酸酶活性的部分丧失 与人类先天新陈代谢错误有关 有精神缺陷。双功能酶天冬氨酸激酶- 高丝氨酸脱氢酶也在 新陈代谢，四种氨基酸生物合成的承诺步骤 从L开始的酸-天冬氨酸。建议的具体目标 在本研究计划中是：探索详细的动力学和 这一酶系统的化学机制；确立了 金属离子活化剂和抑制剂；探索L的双重作用-- 天冬氨酸作为酶的底物和激活剂；以及 确定L结合部位的结构和取向-- 天门冬氨酸酶。将应用的技术，以解决 研究项目的具体目标将包括均衡 金属离子底物的渗析和动力学结合研究 类似物和激活剂。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，将被用来阐明关于 该反应由L-天冬氨酸酶催化。磁共振研究， 包括核磁共振顺磁松弛研究，以检查酶-- 底物和酶-激活剂相互作用的核磁共振和电子顺磁共振研究 为了研究金属离子的相互作用，低温核磁共振研究 表征催化循环中的中间体，以及x射线 酶-底物及其类似物的结晶学研究， 将用于提供有关 金属离子、底物和活化剂与L的相互作用 天门冬氨酸酶。总而言之，这些技术提供了一个机会 对涉及的事件进行详细审查 L-天冬氨酸酶的作用机理及相关酶的研究 金属离子和底物效应器对此所起的作用 酵素。

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.