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Nat Struct Biol. 1996 Feb;3(2):149-54.

Crystallographic observation of a covalent catalytic intermediate in a beta-glycosidase.

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  • 1Protein Engineering Network of Centres of Excellence, Ontario Cancer Institute, Toronto, Canada.


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.

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