Epoxyalkyl glycosides of D-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10

A series of omega-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xy lotriose were tested as potential active-site-directed inhibitors of xylana ses from glycoside hydrolase families 10 and 11. Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resist ant to electrophilic attack of active-site carboxyl residues, glycoside hyd rolases of family Il (Thermomyces lanuginosus Xyn and Trichoderma reesei Xy n II) are irreversibly inhibited. The apparent inactivation and association constants (k(i), 1/K-i) are one order of magnitude higher for the xylobios e and xylotriose derivatives. The effects of the aglycone chain length can clearly be described, Xylobiose and n-alkyl beta-D-xylopyranosides are comp etitive ligands and provide protection against inactivation. MS measurement s showed 1 : 1 stoichiometries in most labelling experiments. Electrospray ionization MS/MS analysis revealed the nucleophile Glu(86) as the modified residue in the T. lanuginosus xylanase when 2,3-epoxypropyl beta-D-xylopyra noside was used, whereas the acid/base catalyst Glu(178) was modified by th e 3,4-epoxybutyl derivative. The active-site residues Glu(86) and Glu(177) in T. reesei Xyn II are similarly modified, confirming earlier X-ray crysta llographic data [Havukainen, Torronen, Laitinen and Rouvinen (1996) Biochem istry 35, 9617-9624]. The inability of the omega-epoxyalkyl xylo(oligo)sacc haride derivatives to inactivate family-10 enzymes is discussed in terms of different ligand-subsite interactions.