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Identification of Glu-519 as the catalytic nucleophile in beta-mannosidase 2A from Cellulomonas fimi

TitleIdentification of Glu-519 as the catalytic nucleophile in beta-mannosidase 2A from Cellulomonas fimi
Publication TypeJournal Article
Year of Publication2000
AuthorsStoll, D, He, SM, Withers, SG, WARREN, RAJ
Date PublishedNOV 1

Incubation of the beta -mannosidase Man2A from Cellulomonas fimi with 2-deoxy-2-fluoro-beta -D-mannosyl fluoride (2-FMan betaF) resulted in time-dependent inactivation of the enzyme (inactivation rate constant k(1) = 0.57 min(-1), dissociation constant for the inactivator k(1) = 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-b)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 nncleophile in a beta -mannosidase.