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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS045664-02
Application #
7014049
Study Section
Special Emphasis Panel (ZRG1-MDCN-L (02))
Program Officer
Tagle, Danilo A
Project Start
2005-02-15
Project End
2010-01-31
Budget Start
2006-02-01
Budget End
2007-01-31
Support Year
2
Fiscal Year
2006
Total Cost
$227,622
Indirect Cost
Name
University of Toledo
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
051623734
City
Toledo
State
OH
Country
United States
Zip Code
43606
Zano, Stephen; Malik, Radhika; Szucs, Sylvia et al. (2011) Modification of aspartoacylase for potential use in enzyme replacement therapy for the treatment of Canavan disease. Mol Genet Metab 102:176-80
Camper, DeMarco V; Viola, Ronald E (2009) Fully automated protein purification. Anal Biochem 393:176-81
Le Coq, Johanne; Pavlovsky, Alexander; Malik, Radhika et al. (2008) Examination of the mechanism of human brain aspartoacylase through the binding of an intermediate analogue. Biochemistry 47:3484-92
Le Coq, Johanne; An, Hyun-Joo; Lebrilla, Carlito et al. (2006) Characterization of human aspartoacylase: the brain enzyme responsible for Canavan disease. Biochemistry 45:5878-84