Residues important for folding and dimerisation of recombinant Torpedo californica acetylcholinesterase.
The three-dimensional crystal structure of the glycosyl phosphatidylinositol (GPI)-modified form of Torpedo acetylcholinesterase reveals the participation of Arg-44 and Glu-92 in a salt bridge and a hydrogen bond between Asp-93 and Tyr-96. To investigate the biological significance of these interactions, we have made amino acid replacements in this form of AChE: R44E, R44K, E92Q, E92L, D93N, and D93V. None of the introduced mutations affected the production of the acetylcholinesterase polypeptide significantly. However, the mutations introduced at position 92, as well as the D93V and R44E mutations, resulted in a total loss of surface located, active acetylcholinesterase. Replacement of Asp-93 with Asn resulted in a reduced amount of active enzyme. This mutant enzyme was indistinguishable from the wild-type enzyme regarding catalytic constants, but was more sensitive to thermal inactivation. The results show that the salt bridge and hydrogen bond involving residues Arg-44, Glu-92, and Asp-93 have important structural roles and are needed for correct folding, required for transport to the cell surface of TcAChE. The GPI-modified form of acetylcholinesterase is a disulfide bonded dimer. Cys-537 is shown to be required for the formation of the intersubunit disulfide bond in the dimer. Replacement with Ser resulted in the production of an enzyme, that migrates as a monomer upon non-reducing SDS-PAGE and has a lower stability compared to the dimeric wild-type enzyme.
Bucht
G
G
Department of NBC Defence, National Defence Research Establishment, Umeå, Sweden.
Häggström
B
B
Radić
Z
Z
Osterman
A
A
Hjalmarsson
K
K
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D004220:Disulfides; D004795:Enzyme Stability; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D011994:Recombinant Proteins; D014101:Torpedo / Q000378:metabolism*
D004220:Disulfides; D011994:Recombinant Proteins; D000110:Acetylcholinesterase