Site-directed mutagenesis of active-site-related residues in Torpedo acetylcholinesterase. Presence of a glutamic acid in the catalytic triad.
Site-directed mutagenesis was used to investigate the role of acidic amino acid residues close to the active site of Torpedo acetylcholinesterase. The recently determined atomic structure of this enzyme shows the conserved Glu-327, together with His-440 and Ser-200 as forming a catalytic triad, while the adjacent conserved Asp-326 points away from the active site. Transfection of appropriately mutated DNA into COS cells showed that the mutation of Asp-326----Asn had little effect on catalytic activity or the molecular forms expressed, suggesting no crucial structural or functional role for this residue. Mutation of Glu-327 to Gln or to Asp led to an inactive product. These results support the conclusions of the structural analysis for the two acidic residues.
Laboratoire de Neurobiologie, CNRS UA 295, Ecole Normale Supérieure, Paris, France.
N
N
Duval
S
S
Bon
I
I
Silman
J
J
Sussman
J
J
Massoulié
D000110:Acetylcholinesterase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D016297:Mutagenesis, Site-Directed; D014101:Torpedo / Q000235:genetics*
D005971:Glutamates; D018698:Glutamic Acid; D000110:Acetylcholinesterase