ArticleThe Biochemical journal2000
Identification of Glu-519 as the catalytic nucleophile in beta-mannosidase 2A from Cellulomonas fimi.
Article in The Biochemical journal, 2000. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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Who cites it
4 citing papers in PubMed.
- Structure and function ofThe Journal of biological chemistry · 2020Article
- Two exo-beta-D-glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases.The Biochemical journal · 2006Article
- Cloning and heterologous expression of a beta-D-mannosidase (EC 3.2.1.25)-encoding gene from Thermobifida fusca TM51.Applied and environmental microbiology · 2003Article
- Identification of the catalytic nucleophile of the Family 31 alpha-glucosidase from Aspergillus niger via trapping of a 5-fluoroglycosyl-enzyme intermediate.The Biochemical journal · 2001Article
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4 authors.
Funding
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Abstract
Incubation of the beta-mannosidase Man2A from Cellulomonas fimi with 2-deoxy-2-fluoro-beta-D-mannosyl fluoride (2FMan beta F) resulted in time-dependent inactivation of the enzyme (inactivation rate constant k(i)=0.57 min(-1), dissociation constant for the inactivator K(i)=0.41 mM) through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-mannosyl-beta-mannosidase 2A (2FMan-Man2A) enzyme intermediate, as observed by electrospray ionization mass spectrometry. The stoichiometry of inactivation was 1:1. Removal of excess inactivator and regeneration of active enzyme by transglycosylation of the covalently attached inhibitor to gentiobiose [Glc beta(1-6)Glc] demonstrated that the covalent intermediate was catalytically competent. Comparison by MS of the peptic digests of 2FMan-Man2A with peptic digests of native Man2A revealed a peptide of m/z 1520 that was unique to 2FMan-Man2A, and one of m/z 1036.5 that was unique to a Man2A peptide. Their sequences, determined by collision-induced fragmentation, were CSEFGFQGPPTW and FGFQGPPTW, corresponding to residues 517-528 and 520-528 of Man2A respectively. The difference in mass of 483.5 between the two peptides equals the sum of the masses of the tripeptide CSE plus that of 2-fluoromannose. It was concluded that in 2FMan-Man2A, the 2-fluoromannose esterified to Glu-519 blocks hydrolysis of the Glu-519-Phe-520 peptide bond, and that Glu-519 is the catalytic nucleophile in this enzyme. This residue is conserved in all members of family 2 of the glycosyl hydrolases. This represents the first ever labelling and identification of an active-site nucleophile in a beta-mannosidase.
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11042141PMC1221426What OpenQuestion holds
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