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Identification of the zinc binding ligands and the catalytic residue in human aspartoacylase, an enzyme involved in Canavan disease
Author(s) -
Herga S.,
Berrin J.-G.,
Perrier J.,
Puigserver A.,
Giardina T.
Publication year - 2006
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2006.09.056
Subject(s) - enzyme , active site , chemistry , biochemistry , site directed mutagenesis , stereochemistry , escherichia coli , binding site , recombinant dna , mutant , gene
Canavan disease is an autosomal‐recessive neurodegenerative disorder caused by a lack of aspartoacylase, the enzyme that degrades N ‐acetylaspartate (NAA) into acetate and aspartate. With a view to studying the mechanisms underlying the action of human aspartoacylase (hASP), this enzyme was expressed in a heterologous Escherichia coli system and characterized. The recombinant protein was found to have a molecular weight of 36 kDa and kinetic constants K m and k cat of 0.20 ± 0.03 mM and 14.22 ± 0.48 s −1 , respectively. Sequence alignment showed that this enzyme belongs to the carboxypeptidase metalloprotein family having the conserved motif H 21 xxE 24 (91aa)H 116 . We further investigated the active site of hASP by performing modelling studies and site‐directed mutagenesis. His21, Glu24 and His116 were identified here for the first time as the residues involved in the zinc‐binding process. In addition, mutations involving the Glu178Gln and Glu178Asp residues resulted in the loss of enzyme activity. The finding that wild‐type and Glu178Asp have the same K m but different k cat values confirms the idea that the carboxylate group contributes importantly to the enzymatic activity of aspartoacylase.