MECHANISTIC & STRUCTURAL CHARACTERIZATION OF L-ASPARTASE

这项研究项目的长期目标是了解 分子水平上反应的化学机理和调控 由L-天冬氨酸氨解酶(天冬氨酸酶)催化。很多是天生的 代谢紊乱是由于低活动度或缺乏 一种特殊的酶。在其中一些疾病中，单点突变 是导致酶活性显著改变的原因。一个 在分子水平上的结构-功能关系知识是 在可以理解这种类型的缺陷之前和之前 可以尝试使用活性部位来修饰这些失活的酶 定向试剂或定点突变。 选择天冬氨酸酶进行详细研究有三个原因。第一, 它是一大类酶的成员，这些酶利用富马酸作为 底物。相对较少的机械性或结构性信息 了解这个家庭的成员。其次，天冬氨酸酶是一种 金属酶，使其非常适合结构功能研究 因为金属离子可以充当该结构的内置探头。 天冬氨酸氨基转移酶在pH 7.5以上表现为非米氏动力学。这个 这种变构行为的结构性原因还没有被很好地理解。 将使用许多或不同的技术来理解天冬氨酸酶。 结构信息将来自x射线结晶学和 来自对原生酶和突变酶的光谱研究。机械论 信息将来自两个点的定向突变结合 稳态动力学研究和化学修饰研究。 这项研究计划的成功完成将产生一套 化学过程中产生的所有中间体的结构 反应和对相互转化的更好的理解 这些中间体。

The long term goal of this research project is to understand at a molecular level the chemical mechanism and regulation of the reaction catalyzed by L-aspartic acid ammonia lyase (aspartase). Many inborn metabolic disorders are the result of the low activity or the absence of a particular enzyme. In some of these diseases, a single point mutation is responsible for the dramatically altered enzyme reactivity. A knowledge of structure-function relationships at the molecular level is needed before deficiencies of this type can be understood, and before attempts can be made to modify these inactive enzymes using active site directed reagents or site directed mutagenesis. Aspartase has been chosen for detailed study for three reasons. First, it is a member of a large family of enzymes which use fumarate as a substrate. Relatively little mechanistic or structural information is known about the members of this family. Second, aspartase is a metalloenzyme which makes it well suited for structure function studies since the metal ion can act as a built-in probe of the structure. Finally, aspartase shows non-Michaelis-Menten kinetics above pH 7.5. The structural causes of such allosteric behavior are not well understood. A number or different techniques will be used to understand aspartase. Structural information will be derived from x-ray crystallography and from spectroscopic studies of native and mutant enzymes. Mechanistic information will come from both site directed mutagenesis combined with steady state kinetic studies and from chemical modification studies. Successful completion of this research proposal will yield a set of structures of all of the intermediates which occur during the chemical reaction and an improved understanding of the transformation between these intermediates.