Probing the Enzymatic Basis of Canavan Disease

探讨卡纳万病的酶学基础

• 批准号: 7100467
• 负责人: RONALD Edward VIOLA
• 金额: $ 1.8万
• 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/7100467
• 关键词: Probing; Enzymatic; Basis; Canavan; Disease

EXCEED THE SPACE PROVIDED. Canavan disease is a fatal neurodegenerative disorder caused by defects in the acy2 gene that encodes for the enzyme aspartoacylase. 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. Our goals for this project are to determine the specificity and the detailed mechanism of human aspartoacylase and also aspartate N- acetyltransferase. We will examine how the activities of these enzymes are regulated, and explore methods for the production of improved variants of aspartoacylase and selective inhibitors of aspartate N- acetyltransferase. Our 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 N-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. A directed evolution approach will screen for variants of aspartoacylase with improved catalytic and regulatory properties. The coordination application of these techniques in our laboratory will provide a detailed picture of the mechanism and inhibition of aspartate N-acetyltransferase. We will also learn how aspartoacylase functions, and why it malfunctions in Canavan disease. i PERFORMANCE SITE ========================================Section End===========================================

超出所提供的空间。Canavan病是一种致命的神经退行性疾病，由acy 2基因缺陷引起，acy 2基因编码的酶是乙酰化酰化酶。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，通常进展非常迅速，死亡通常发生在生命的最初几年。从患者身上提取的DNA已经鉴定出许多导致β-酰化酶缺陷的突变，然而，还没有系统的研究这些改变如何以及为什么影响催化活性，并且很少有β-酰化酶的详细表征。本课题的目的是研究人天冬酰化酶和天冬氨酸N-乙酰转移酶的特异性和详细的作用机制。我们将研究这些酶的活性是如何调节的，并探索生产改进的天冬酰化酶变体和天冬氨酸N-乙酰转移酶选择性抑制剂的方法。我们的长期目标是利用我们将了解的关于这些酶的基本知识来帮助克服人类中β-酰化酶的代谢缺陷。为了实现这些目标，我们将开发这些酶的改进的表达系统和优化的纯化方法。金属离子的作用，在β-酰化酶将被检查，和监管网站的功能将通过控制翻译后修饰和位点特异性共价修饰进行评估。将通过X-射线衍射方法确定人天冬酰化酶和天冬氨酸N-乙酰转移酶的高分辨率结构，沿着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。定向进化方法将筛选具有改进的催化和调节特性的β-酰化酶变体。这些技术在本实验室的协同应用将为天冬氨酸N-乙酰转移酶的作用机制和抑制提供详细的图像。我们还将了解乙酰化酶如何发挥作用，以及为什么它在卡纳万病中出现故障。i表演现场=