Purification, Characterization, and Localization of Aspartoacylase from Bovine Brain

Canavan disease, an autosomal recessive disorder, is characterized biochemically by N ‐acetylaspartic aciduria and aspartoacylase ( N ‐acyl‐L‐aspartate amidohydrolase; EC 3.5.1.15) deficiency. However, the role of aspartoacylase and N ‐acetylaspartic acid in brain metabolism is unknown. Aspartoacylase has been purified to apparent homogeneity with a specific activity of ∼ 19,000–20,000 nmol of aspartate released/mg of protein. The native enzyme is a 58‐kDa monomer. The purified aspartoacylase activity is enhanced by divalent cations, nonionic detergents, and dithiothreitol. Low levels of dithiothreitol or β‐mercaptoethanol are required for enzyme stability. Aspartoacylase has a K m of 8.5 × 10 −4 M and a V max of 43,000 nmol/min/mg of protein. Inhibition of aspartoacylase by glycyl‐L‐aspartate and amino derivatives of D‐aspartic acid suggests that the carbon backbone of the substrate is primarily involved in its interaction with the active site and that a blocked amino group is essential for the catalytic activity of aspartoacylase. Biochemical and immunocytochemical studies revealed that aspartoacylase is localized to white matter, whereas the N ‐acetylaspartic acid concentration is threefold higher in gray matter than in white matter. Our studies so far indicate that aspartoacylase is conserved across species during evolution and suggest a significant role for aspartoacylase and N ‐acetylaspartic acid in normal brain biology.