Identification of the catalytic nucleophile of the family 31 alpha-glucosidase from aspergillus niger via trapping of a 5-fluoroglycosyl-enzyme intermediate.
Identification of the catalytic nucleophile of the family 31 alpha-glucosidase from aspergillus niger via trapping of a 5-fluoroglycosyl-enzyme intermediate.
The mechanism-based reagent 5-fluoro-alpha-d-glucopyranosyl fluoride (5F alpha GlcF) was used to trap a glycosyl-enzyme intermediate and identify the catalytic nucleophile at the active site of Aspergillus niger alpha-glucosidase (Family 31). Incubation of the enzyme with 5F alpha GlcF, followed by peptic proteolysis and comparative liquid chromatography/MS mapping allowed the isolation of a labelled peptide. Fragmentation analysis of this peptide by tandem MS yielded the sequence WYDMSE, with the label located on the aspartic acid residue (D). Comparison with the known protein sequence identified the labelled amino acid as Asp-224 of the P2 subunit.
381-386
Lee, Seung Seo
ee34fa26-5fb6-48c8-80c2-1f13ec4ccceb
He, Shouming
b6c067bc-20d1-4ae4-8114-8d7ce4ef793f
Withers, Stephen G.
2fa507a8-6772-41a0-97a0-a33f443bbcc1
15 October 2001
Lee, Seung Seo
ee34fa26-5fb6-48c8-80c2-1f13ec4ccceb
He, Shouming
b6c067bc-20d1-4ae4-8114-8d7ce4ef793f
Withers, Stephen G.
2fa507a8-6772-41a0-97a0-a33f443bbcc1
Lee, Seung Seo, He, Shouming and Withers, Stephen G.
(2001)
Identification of the catalytic nucleophile of the family 31 alpha-glucosidase from aspergillus niger via trapping of a 5-fluoroglycosyl-enzyme intermediate.
Biochemical Journal, 359 (Pt 2), .
(PMID:9620260)
Abstract
The mechanism-based reagent 5-fluoro-alpha-d-glucopyranosyl fluoride (5F alpha GlcF) was used to trap a glycosyl-enzyme intermediate and identify the catalytic nucleophile at the active site of Aspergillus niger alpha-glucosidase (Family 31). Incubation of the enzyme with 5F alpha GlcF, followed by peptic proteolysis and comparative liquid chromatography/MS mapping allowed the isolation of a labelled peptide. Fragmentation analysis of this peptide by tandem MS yielded the sequence WYDMSE, with the label located on the aspartic acid residue (D). Comparison with the known protein sequence identified the labelled amino acid as Asp-224 of the P2 subunit.
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Published date: 15 October 2001
Organisations:
Organic Chemistry: SCF
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Local EPrints ID: 345660
URI: http://eprints.soton.ac.uk/id/eprint/345660
ISSN: 1470-8728
PURE UUID: 92f67bd0-29f8-444e-a012-f20ed041bc1a
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Date deposited: 12 Feb 2013 16:54
Last modified: 09 Jan 2022 03:43
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Author:
Shouming He
Author:
Stephen G. Withers
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