Crystallographic observation of a covalent catalytic intermediate in a beta-glycosidase.
The three-dimensional structure of a catalytically competent glycosyl-enzyme intermediate of a retaining beta-1,4-glycanase has been determined at a resolution of 1.8 A by X-ray diffraction. A fluorinated slow substrate forms an alpha-D-glycopyranosyl linkage to one of the two invariant carboxylates, Glu 233, as supported in solution by 19F-NMR studies. The resulting ester linkage is coplanar with the cyclic oxygen of the proximal saccharide and is inferred to form a strong hydrogen bond with the 2-hydroxyl of that saccharide unit in natural substrates. The active-site architecture of this covalent intermediate gives insights into both the classical double-displacement catalytic mechanism and the basis for the enzyme's specificity.
White
A
A
Protein Engineering Network of Centres of Excellence, Ontario Cancer Institute, Toronto, Canada.
Tull
D
D
Johns
K
K
Withers
S G
SG
Rose
D R
DR
D001665:Binding Sites; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D043364:Endo-1,4-beta Xylanases; D005960:Glucosides / Q000737:chemistry; D006093:Gram-Positive Asporogenous Rods / Q000201:enzymology; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D011487:Protein Conformation; D014995:Xylosidases / Q000737:chemistry*; D001617:beta-Glucosidase / Q000737:chemistry*
D005960:Glucosides; C081853:2',4'-dinitrophenyl-2-deoxy-2-fluorocellobioside; D014995:Xylosidases; D001617:beta-Glucosidase; D043364:Endo-1,4-beta Xylanases; C090001:XynB xylanase