Purification, Characterization, and Localization of Aspartoacylase from Bovine Brain

Abstract

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 approximately 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 beta-mercaptoethanol are required for enzyme stability. Aspartoacylase has a Km of 8.5 x 10(-4) M and a Vmax 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.

Mutational analysis of aspartoacylase: Implications for Canavan Disease

“…The carbon backbone of NAA has been suggested to interact with ASPA's active site because substrates with more electronegative carbonyl groups are better substrates for ASPA-mediated hydrolysis ,Kaul et al 1991. A nucleophilic attack is then believed to occur at the carbonyl group, irrespective of the α and β-carboxyl groups (Moore et al 2003).…”

“…Consistent with this possibility, divalent cation chelators drastically reduced ASPA activity in bovine brain homogenates and in dialyzed recombinant human ASPA from E. coli (Le Coq et al 2006) (Kaul et al 1991,Moore et al 2003. Zinc chloride slightly increased purified bovine brain ASPA activity at low concentrations and inhibited activity at higher concentrations (Kaul et al 1991). Roughly two atoms of zinc were detected per enzyme subunit following dialysis and denaturation of recombinant human ASPA expressed in E. coli, though zinc-treated ASPA had the same activity as the native enzyme refolded without additional metal ions (Moore et al 2003).…”

“…Consistent with this possibility, divalent cation chelators drastically reduced ASPA activity in bovine brain homogenates and in dialyzed recombinant human ASPA from E. coli (Le Coq et al 2006) (Kaul et al 1991,Moore et al 2003. Zinc chloride slightly increased purified bovine brain ASPA activity at low concentrations and inhibited activity at higher concentrations (Kaul et al 1991).…”

“…Mutations at Cys152 have been associated with CD and yield undetectable ASPA activity upon in vitro expression in COS cells (Kaul et al 1996,Zeng et al 2002. Also, low concentrations of DTT are required to maintain ASPA activity, whereas high concentrations ablate activity (Kaul et al 1991,Madhavarao et al 2002. Therefore, it has been suggested that there is an intramolecular disulfide bond in ASPA involving Cys152.…”

N-Acetylaspartate in the CNS: From neurodiagnostics to neurobiology

“…et al, 1977). ASPA was purified from bovine brain to apparent homogeneity by Kaul and colleagues, who also showed that it was expressed at higher levels in white matter than in gray matter (Kaul et al, 1991). They predicted that the active enzyme existed as a 58 kD monomer.…”