Probing the Enzymatic Basis of Canavan Disease

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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===========================================