Identification of the zinc binding ligands and the catalytic residue in human aspartoacylase, an enzyme involved in Canavan disease.
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.
Herga
S
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Université Paul Cézanne, Biosciences FRE-3005 (Université Aix Marseille III/CNRS), Laboratoire de Biochimie et Biologie de la Nutrition, Av Escardrille Nomandie Niemen, 13397 Marseille Cedex 20, France.
Berrin
J-G
JG
Perrier
J
J
Puigserver
A
A
Giardina
T
T
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D017825:Canavan Disease / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000378:metabolism
D018076:DNA, Complementary; D008024:Ligands; D011994:Recombinant Proteins; D018698:Glutamic Acid; D006639:Histidine; D000581:Amidohydrolases; C019751:aspartoacylase; D015032:Zinc