Probing the Enzymatic Basis of Canavan Disease

描述（由申请人提供）：Canavan病是一种致命的神经退行性疾病，由编码天冬氨酸酰化酶的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，出现在婴儿期早期，通常进展非常迅速，通常在生命的头几年内死亡。从患者身上提取的DNA已经确定了许多导致天冬氨酸酰化酶缺陷的突变，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，也很少有详细的天冬氨酸酰化酶的表征。该项目的目的是确定人类天冬氨酸酰化酶的特异性和详细机制，以及脑内负责合成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被调节的，识别和定义关键活性位点氨基酸的作用，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学习的这些酶的基本知识来帮助克服人类天冬氨酸酰化酶的代谢缺陷。为了实现这些目标，我们将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化酶中的作用，并通过控制翻译后修饰和位点特异性共价修饰来评估调控位点的功能。人类天冬氨酸酰化酶和天冬氨酸/ v -乙酰转移酶的高分辨率结构将通过x射线衍射方法确定，以及选定的活性位点和调节位点突变体和酶抑制剂复合物的结构。这些技术在本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一个详细的图景。我们还将了解天冬氨酸酰化酶的功能，以及它在Canavan病中出现故障的原因。

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.