|Title||Crystallographic observation of a covalent catalytic intermediate in a beta-glycosidase|
|Publication Type||Journal Article|
|Year of Publication||1996|
|Authors||White, A, TULL, D, Johns, K, Withers, SG, ROSE, DR|
|Journal||NATURE STRUCTURAL BIOLOGY|
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 Angstrom 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 F-19-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.