Probing the Enzymatic Basis of Canavan Disease

探讨卡纳万病的酶学基础

• 批准号: 6918914
• 负责人: RONALD Edward VIOLA
• 金额: $ 23.31万
• 依托单位: UNIVERSITY OF TOLEDO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-15 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6918914
• 关键词: X ray crystallography; amidohydrolases; aminoacid metabolism; chemical synthesis; drug design /synthesis /production; drug screening /evaluation; enzyme activity; enzyme mechanism; enzyme structure; mass spectrometry; matrix assisted laser desorption ionization; myelinopathy; neural degeneration; pathologic process; posttranslational modifications

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.

描述（由申请人提供）：卡纳万病是一种致命的神经退行性疾病，由编码天冬氨酸酰基酶的 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，出现在婴儿早期，并且通常进展非常迅速，死亡通常发生在生命的最初几年内。从患者身上提取的 DNA 已鉴定出许多导致天冬氨酸酰化酶缺陷的突变，然而，尚未对这些改变如何以及为何影响催化活性进行系统研究，也很少对天冬氨酸酰化酶进行详细表征。该项目的目标是确定人类天冬氨酸酰化酶以及负责在大脑中合成 NAA 的酶天冬氨酸 A/-乙酰转移酶的特异性和详细机制。我们将研究如何调节这些酶的活性，识别和定义关键活性位点氨基酸的作用，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学到的有关这些酶的基础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，我们将为这些酶开发改进的表达系统和优化的纯化方法。将检查金属离子在天冬氨酸酰化酶中的作用，并通过受控的翻译后修饰和位点特异性共价修饰来评估调节位点的功能。人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以及选定的活性位点和调节位点突变体以及酶抑制剂复合物的结构将通过X射线衍射方法确定。这些技术在我们实验室的协调应用将提供天冬氨酸A/-乙酰转移酶的机制和抑制的详细图片。我们还将了解天冬氨酸酰化酶如何发挥作用，以及它在卡纳万病中出现故障的原因。