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Related Experiment Videos

Trapping covalent intermediates on beta-glycosidases.

Jacqueline Wicki1, David R Rose, Stephen G Withers

  • 1Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.

Methods in Enzymology
|November 7, 2002
PubMed
Summary

Mechanism-based inactivation identified the active-site nucleophile in beta-glycosidases. X-ray crystallography of trapped complexes, like with Cex, revealed catalytic insights and residue roles.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Beta-glycosidases are crucial enzymes involved in carbohydrate metabolism.
  • Identifying the active-site nucleophile is key to understanding enzyme mechanisms.
  • Cex (C. fimi exoglycanase) serves as a model system for studying beta-glycosidase activity.

Purpose of the Study:

  • To elucidate the catalytic mechanism of beta-glycosidases.
  • To identify the active-site nucleophilic residue in Cex.
  • To gain insights into enzyme-substrate interactions during catalysis.

Main Methods:

  • Mechanism-based inactivation of Cex followed by mass spectrometry to identify the nucleophilic residue.
  • X-ray crystallography of trapped covalent glycosyl-enzyme intermediates, including a fluorocellobiosyl-enzyme complex.

Related Experiment Videos

  • Analysis of a mutant form of Cex to study natural sugar conformation and active site interactions.

    • Main Results:

    • The nucleophilic residue in Cex was identified using mass spectrometry.
    • The crystal structure of a trapped fluorocellobiosyl-enzyme complex provided insights into Cex catalysis.
    • Structural data revealed the roles of specific active-site residues and the conformation of the bound sugar.

    Conclusions:

    • Mechanism-based inactivation and mass spectrometry are effective for identifying active-site nucleophiles in beta-glycosidases.
    • X-ray crystallography of trapped intermediates offers valuable information on enzyme catalysis and active-site architecture.
    • Cex structure and mutant analysis illuminate the conformational states and interactions of natural substrates during catalysis.