Probing the Enzymatic Basis of Canavan Disease

描述(申请人提供)：Canavan病是一种致命的神经退行性疾病，由编码天冬氨酸酶的acy2基因缺陷引起，从而导致A/-乙酰天冬氨酸(NAA)水平异常高。这种疾病的症状，包括运动技能和肌肉控制的丧失，在婴儿期早期就出现，通常进展非常快，死亡通常发生在生命的第一年。从患者身上提取的DNA已经发现了许多导致天冬氨酸氨基转移酶缺陷的突变，然而，对于这些改变如何以及为什么影响催化活性还没有系统的研究，也很少有天冬氨酸酯酶的详细表征。这个项目的目标是确定人类天冬氨酸酰基酶的特异性和详细的机制，以及大脑中负责合成NAA的酶-天冬氨酸A/-乙酰转移酶。我们将研究这些酶的活性是如何调节的，识别和定义关键活性部位氨基酸的作用，并合成和筛选天冬氨酸A/-乙酰转移酶的选择性抑制剂。长期目标是利用我们将了解的这些酶的基本知识来帮助克服人类天冬氨酸酯酶的代谢缺陷。为了实现这些目标，我们将为这些酶开发改进的表达系统和优化的纯化方法。我们将研究金属离子在天冬氨酸酰基酶中的作用，并通过受控的翻译后修饰和特定部位的共价修饰来评估调节位点的功能。人天冬氨酸氨基转移酶和天冬氨酸/V-乙酰转移酶的高分辨结构将用X射线衍射法测定，以及选定的活性部位和调节部位突变体和酶抑制物复合体的结构。这些技术在我们实验室的协同应用将为天冬氨酸A/-乙酰转移酶的机制和抑制提供详细的图景。我们还将学习天冬氨酸酯酶是如何发挥作用的，以及为什么它会在加拿大人病中失灵。

DESCRIPTION (provided by applicant): Canavan disease is a fatal neurodegenerative disorder caused by defects in the acy2 gene that encodes for the enzyme aspartoacylase, thus resulting in abnormally high levels of A/-acetylaspartate (NAA). Symptoms of this disorder, including loss of motor skills and muscle control, appear in early infancy and typically progress very rapidly, with death usually occurring within the first years of life. DNA taken from patients has identified numerous mutations that result in defective aspartoacylase, however, there have been no systematic studies of how and why these alterations affect catalytic activity, and little detailed characterization of aspartoacylase. The goals for this project are to determine the specificity and the detailed mechanism of human aspartoacylase and also the enzyme responsible for the synthesis of NAA in the brain, aspartate A/-acetyltransferase. We will examine how the activities of these enzymes are regulated, identify and define the roles of critical active site amino acids, and synthesize and screen selective inhibitors of aspartate A/-acetyltransferase. The long-range objectives are to use the basic knowledge that we will learn about these enzymes to help overcome the metabolic defects of aspartoacylase in humans. To accomplish these goals we will develop improved expression systems and optimized purification methods for these enzymes. The role of metal ions in aspartoacylase will be examined, and the function of regulatory sites will be assessed by controlled posttranslational modifications and by site-specific covalent modifications. The high-resolution structures of human aspartoacylase and aspartate /V-acetyltransferase will be determined by X-ray diffraction methods, along with the structures of selected active site and regulatory site mutants and enzyme-inhibitor complexes. The coordination application of these techniques in our laboratory will provide a detailed picture of the mechanism and inhibition of aspartate A/-acetyltransferase. We will also learn how aspartoacylase functions, and why it malfunctions in Canavan disease.