title: Epoxyalkyl glycosides of D-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10.
abstract: A series of omega-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xylotriose were tested as potential active-site-directed inhibitors of xylanases from glycoside hydrolase families10 and 11. Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resistant toelectrophilic attack of active-site carboxyl residues, glycosidehydrolases of family 11 (Thermomyces lanuginosus Xyn and Trichoderma reesei Xyn II) are irreversibly inhibited. Theapparent inactivation and association constants (k(i), 1/K(i)) are one order of magnitude higher for thexylobiose and xylotriose derivatives. The effects of the aglycone chainlength can clearly be described. Xylobiose and n-alkyl beta-D-xylopyranosides are competitive ligands and provide protectionagainst inactivation. MS measurements showed 1:1 stoichiometries inmost labelling experiments. Electrospray ionization MS/MS analysisrevealed the nucleophile Glu(86) as the modified residue inthe T. lanuginosus xylanase when 2,3-epoxypropyl beta-D-xylopyranoside was used, whereas the acid/base catalyst Glu(178) was modified by the 3,4-epoxybutyl derivative. The active-site residues Glu(86) and Glu(177) in T. reesei Xyn II are similarly modified, confirming earlier X-raycrystallographic data [Havukainen, Törrönen, Laitinen and Rouvinen (1996)Biochemistry 35, 9617-9624]. The inability of the omega-epoxyalkyl xylo(oligo)saccharide derivatives to inactivate family-10enzymes is discussed in terms of different ligand-subsiteinteractions.
authors.affiliation: Laboratory for Biochemistry, Department of Biochemistry, University of Ghent, K.L.Ledeganckstraat 35, B-9000 Ghent, Belgium.
authors.forename: P
authors.initials: P
authors.lastname: Ntarima
authors.forename: W
authors.initials: W
authors.lastname: Nerinckx
authors.forename: K
authors.initials: K
authors.lastname: Klarskov
authors.forename: B
authors.initials: B
authors.lastname: Devreese
authors.forename: M K
authors.initials: MK
authors.lastname: Bhat
authors.forename: J
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: M
authors.initials: M
authors.lastname: Claeyssens
mesh_terms: D000478:Alkylation; D001203:Ascomycota / Q000201:enzymology; D001665:Binding Sites; D001667:Binding, Competitive; D003013:Clostridium / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology; D043325:Xylan Endo-1,3-beta-Xylosidase; D014994:Xylose / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D014995:Xylosidases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000145:classification / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D004852:Epoxy Compounds; D006027:Glycosides; D008024:Ligands; D009844:Oligosaccharides; D010446:Peptide Fragments; D018698:Glutamic Acid; D014994:Xylose; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase