MECHANISTIC & STRUCTURAL CHARACTERIZATION OF L-ASPARTASE

本研究项目的长期目标是了解 分子水平上的化学反应机理和调控 由L-天冬氨酸氨裂解酶（L-ASPA）催化。 许多天生的 代谢紊乱是由于低活性或缺乏 一种特殊的酶 在某些疾病中，一个点突变 是导致酶反应性急剧改变的原因 一 在分子水平上的结构-功能关系的知识是 在理解这种类型的缺陷之前， 可以尝试使用活性位点来修饰这些失活酶 定向试剂或定点诱变。 选择天冬氨酸酶进行详细研究有三个原因。 第一、 它是使用延胡索酸作为 衬底 相对较少的机械或结构信息， 了解这个家庭的成员。 第二，蛋白酶是一种 金属酶，使其非常适合结构功能研究 因为金属离子可以作为该结构的内置探针。 最后，在pH 7.5以上，酶显示非米氏动力学。 的 这种变构行为的结构原因还不清楚。 许多或不同的技术将被用来理解酶。 结构信息将来自X射线晶体学， 从天然酶和突变酶的光谱研究中。 机械论 信息将来自定点诱变结合 稳态动力学研究和化学改性研究。 成功完成这项研究计划将产生一套 化学反应过程中发生的所有中间体的结构 反应和更好地理解之间的转换 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.