Identification of Glu-519 as the catalytic nucleophile in beta-mannosidase2A from Cellulomonas fimi

Incubation of the beta -mannosidase Man2A from Cellulomonas fimi with 2-deo xy-2-fluoro-beta -D-mannosyl fluoride (2-FMan betaF) resulted in time-depen dent 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 -mannos idase 2A (2FMan-Man2A) enzyme intermediate, as observed by electrospray ion ization mass spectrometry. The stoichiometry of inactivation was 1:1. Remov al of excess inactivator and regeneration of active enzyme by transglycosyl ation of the covalently attached inhibitor to gentiobiose [Glc beta (1-b)Gl c] 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-Man 2A, and one of m/z 1036.5 that was unique to a Man2A peptide, Their sequenc es, determined by collision-induced fragmentation, were CSEFGFQGPPTW and FG FQGPPTW, corresponding to residues 517-528 and 520-528 of Man2A respectivel y. 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 co ncluded that in 2FMan-Man2A, the 2-fluoromannose esterified to Glu-519 bloc ks 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 memb ers of family 2 of the glycosyl hydrolases. This represents the first ever labelling and identification of an active-site nncleophile in a beta -manno sidase.