title: Insights into transition state stabilization of the beta-1,4-glycosidase Cex by covalent intermediate accumulation in active site mutants.
abstract: The catalytic mechanism of 'retaining' beta-glycosidases has been the subject of considerable interest and debate for many years. The visualization of a covalent glycosyl enzyme intermediate by X-ray crystallography was first accomplished with a saccharide substrate substituted with fluorine at its 2-position. The structure implicated major roles for residue His 205 and for the 2-hydroxyl position of the proximal saccharide in binding and catalysis. Here we have studied the kinetic behavior of various His 205 mutants. One of these mutants, a double mutant H205N/E127A, has been used to stabilize a covalent glycosyl-enzyme intermediate involving an unsubstituted sugar, permitting crystallographic analysis of the interactions between its 2-hydroxyl group and the enzyme.
authors.affiliation: Protein Engineering Network of Centres of Excellence, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Canada.
authors.forename: V
authors.initials: V
authors.lastname: Notenboom
authors.forename: C
authors.initials: C
authors.lastname: Birsan
authors.forename: M
authors.initials: M
authors.lastname: Nitz
authors.forename: D R
authors.initials: DR
authors.lastname: Rose
authors.forename: R A
authors.initials: RA
authors.lastname: Warren
authors.forename: S G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D043364:Endo-1,4-beta Xylanases; D004795:Enzyme Stability / Q000235:genetics; D016988:Gram-Positive Asporogenous Rods, Irregular / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D055672:Static Electricity; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D006639:Histidine; D014995:Xylosidases; D001617:beta-Glucosidase; D043364:Endo-1,4-beta Xylanases; C090001:XynB xylanase