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Crystallographic observation of a covalent catalytic intermediate in a β-glycosidase

Nature Structural Biology volume 3, pages 149–154 (1996)Cite this article

Abstract

The three-dimensional structure of a catalytically competent glycosyl-enzyme intermediate of a retaining β-1,4-glycanase has been determined at a resolution of 1.8 Å by X-ray diffraction. A f luorinated slow substrate forms an α-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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Authors and Affiliations

  1. Protein Engineering Network of Centres of Excellence, Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, M5G 2M9, Canada

    André White, Kathy Johns & David R. Rose

  2. Protein Engineering Network of Centres of Excellence, Department of Chemistry, University of British Columbia, Vancouver, V6T 1Z1, Canada

    Dedreia Tull & Stephen G. Withers

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White, A., Tull, D., Johns, K. et al. Crystallographic observation of a covalent catalytic intermediate in a β-glycosidase. Nat Struct Mol Biol 3, 149–154 (1996). https://doi.org/10.1038/nsb0296-149

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