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PMID:1517212

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Citation

Shafferman, A, Kronman, C, Flashner, Y, Leitner, M, Grosfeld, H, Ordentlich, A, Gozes, Y, Cohen, S, Ariel, N and Barak, D (1992) Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding. J. Biol. Chem. 267:17640-8

Abstract

Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.

Links

PubMed

Keywords

Acetylcholinesterase/chemistry; Acetylcholinesterase/genetics; Acetylcholinesterase/metabolism; Amino Acid Sequence; Antibodies, Monoclonal; Base Sequence; Binding Sites; Cell Line; Codon/genetics; Humans; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Protein Conformation; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Restriction Mapping; Transfection

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:ACES

located_in

GO:0005576: extracellular region

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:ACES

part_of

GO:0005576: extracellular region

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:ACES

enables

GO:0004104: cholinesterase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:ACES

enables

GO:0003990: acetylcholinesterase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:ACES

enables

GO:0042803: protein homodimerization activity

ECO:0000303: author statement without traceable support used in manual assertion

F

Seeded From UniProt

complete

HUMAN:ACES

enables

GO:0042166: acetylcholine binding

ECO:0000303: author statement without traceable support used in manual assertion

F

Seeded From UniProt

complete

HUMAN:ACES

enables

GO:0003990: acetylcholinesterase activity

ECO:0000315: mutant phenotype evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:ACES

involved_in

GO:0001507: acetylcholine catabolic process in synaptic cleft

ECO:0000303: author statement without traceable support used in manual assertion

P

Seeded From UniProt

complete

HUMAN:ACES

involved_in

GO:0006581: acetylcholine catabolic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:ACES

enables

GO:0016787: hydrolase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:ACES

involved_in

GO:0032223: negative regulation of synaptic transmission, cholinergic

ECO:0000305: curator inference used in manual assertion

GO:0006581

P

Seeded From UniProt

complete


See also

References

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