β-Glucocerebrosidase: Affinity Purification and Characterization of its Active Site with N-Alkyl Derivatives of 1-Deoxynojirimycin

  • Günter Legler
  • Harald Liedtke
Part of the NATO ASI Series book series (NSSA, volume 116)


An essential feature of the catalytic site of practically all β-glucosidases is a carboxylate group in close proximity to the glucosyl C-1 of the bound substrate. It participates in the bond breaking step by stabilizing a positive charge developing at this carbon atom and/or by forming an α-glucosyl enzyme intermediate which subsequently hydrolyses to β-glucose. The presence and orientation with respect to the bound substrate of this carboxylate is revealed by the covalent inhibition of these enzymes by epoxides related to glucose and by their strong non-covalent inhibition by glucose derivatives with a basic group at C-1 which is up to 103-times better than by the corresponding non-basic derivativesl. We have used the latter property to develop an affinity purification for β-glucocerebrosidase from calf spleen and to explore the aglycon part of its active site with N-alkyl derivatives of 1-deoxynojirimycin (dNM, 1,5-dideoxy-1,5-imino-D-glucitol), a basic analogue of D-glucose where the pyranose oxygen has been replaced by an NH-group.


Affinity Purification Sebacic Acid Basic Analogue Enzyme Intermediate Reductive Alkylation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    P. Lalégerie, G. Legler, and J.M. Yon, The Use of Inhibitors in the Study of Glycosidases, Biochimie 64: 977 (1982)PubMedCrossRefGoogle Scholar
  2. 2.
    H. Hettkamp, G. Legler, and E. Bause, Purification by Affinity Chromatography of Glucosidase I, an Endoplasmatic Reticulum Hydrolase involved in the Processing of Asparagine-linked Oligosaccharides, Europ. J. Biochem. 142: 85 (1984)PubMedCrossRefGoogle Scholar
  3. 3.
    S. F. Furbish, H. E. Blair, J. Shiloach, P. G. Pentchev, and R. O. Brady, Glucocerebrosidase from Human Placenta, in ‘Meth. Enzymol.’, S. P. Colowick and N. O. Kaplan, ed., Vol 50:529 (1979)Google Scholar
  4. 4.
    S. L. Berent and N. S. Radin, β-Glucosidase Activator Protein from Bovine Spleen (’Coglucosidase’) Arch. Biochem. Biophy. 208: 248 (1981)CrossRefGoogle Scholar
  5. 5.
    T. Dinur, G. A. Grabowski, R. J. Desnick, and S. Gatt, Synthesis of a Fluorescent Derivative of Glucosyl Ceramide for the Sensitive Determination of Glucocerebrosidase Activity, Analyt. Biochem. 231: 144 (1984)Google Scholar
  6. 6.
    G. A. Grabowski, S. Gatt, J. Kruse, and R. J. Desnick, Human Lysosomal β-Glucosidase: Kinetic Characterization of the Catalytic, Aglycon and Hydrophobic Binding Sites, Arch. Biochem. Biophys. 231: 144 (1984)PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1986

Authors and Affiliations

  • Günter Legler
    • 1
  • Harald Liedtke
    • 1
  1. 1.Institut für BiochemieUniversität zu KölnGermany

Personalised recommendations