Identification of Glu-519 as the catalytic nucleophile in beta-mannosidase 2A from Cellulomonas fimi.
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.
Stoll
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Protein Engineering Network of Centres of Excellence of Canada, Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver, BC, Canada V6T 1Z3.
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Withers
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Warren
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D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002384:Catalysis; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000378:metabolism*; D006868:Hydrolysis; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D021241:Spectrometry, Mass, Electrospray Ionization; D044902:beta-Mannosidase
D018698:Glutamic Acid; D008361:Mannosidases; D044902:beta-Mannosidase