Mutational analysis of aspartoacylase: implications for Canavan disease.
Mutations that result in near undetectable activity of aspartoacylase, which catalyzes the deacetylation of N-acetyl-l-aspartate, correlate with Canavan Disease, a neurodegenerative disorder usually fatal during childhood. The underlying biochemical mechanisms of how these mutations ablate activity are poorly understood. Therefore, we developed and tested a three-dimensional homology model of aspartoacylase based on zinc dependent carboxypeptidase A. Mutations of the putative zinc-binding residues (H21G, E24D/G, and H116G), the general proton donor (E178A), and mutants designed to switch the order of the zinc-binding residues (H21E/E24H and E24H/H116E) yielded wild-type aspartoacylase protein levels and undetectable ASPA activity. Mutations that affect substrate carboxyl binding (R71N) and transition state stabilization (R63N) also yielded wild-type aspartoacylase protein levels and undetectable aspartoacylase activity. Alanine substitutions of Cys124 and Cys152, residues indicated by homology modeling to be in close proximity and in the proper orientation for disulfide bonding, yielded reduced ASPA protein and activity levels. Finally, expression of several previously tested (E24G, D68A, C152W, E214X, D249V, E285A, and A305E) and untested (H21P, A57T, I143T, P183H, M195R, K213E/G274R, G274R, and F295S) Canavan Disease mutations resulted in undetectable enzyme activity, and only E285A and P183H showed wild-type aspartoacylase protein levels. These results show that aspartoacylase is a member of the caboxypeptidase A family and offer novel explanations for most loss-of-function aspartoacylase mutations associated with Canavan Disease.
Hershfield
Jeremy R
JR
Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814, USA.
Pattabiraman
Nagarajan
N
Madhavarao
Chikkathur N
CN
Namboodiri
M A Aryan
MA
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001923:Brain Chemistry / Q000235:genetics*; D017825:Canavan Disease / Q000201:enzymology* / Q000235:genetics*; D043422:Carboxypeptidases A / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D004789:Enzyme Activation / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D020022:Genetic Predisposition to Disease / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D010802:Phylogeny; D017434:Protein Structure, Tertiary / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D043422:Carboxypeptidases A; D000581:Amidohydrolases; C019751:aspartoacylase