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Related Experiment Videos

Solvent isotope effects on alpha-glucosidase.

Anne H O'donnell1, Xiaojie Yao, Larry D Byers

  • 1Department of Chemistry, Tulane University, New Orleans, LA 70118, USA.

Biochimica Et Biophysica Acta
|December 14, 2004
PubMed
Summary

Yeast alpha-glucosidase hydrolysis kinetics were studied using solvent kinetic isotope effects. An unusually large solvent isotope effect was observed for p-nitrophenyl-D-glucopyranoside, indicating significant proton transfer during catalysis.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Chemical Kinetics

Background:

  • Yeast alpha-glucosidase is a key enzyme in carbohydrate metabolism.
  • Understanding its catalytic mechanism is crucial for various applications.
  • Solvent kinetic isotope effects (SKIE) provide insights into transition states and proton transfer events.

Purpose of the Study:

  • To investigate the catalytic mechanism of yeast alpha-glucosidase.
  • To measure SKIE on the hydrolysis of p-nitrophenyl-D-glucopyranoside (pNPG) and methyl-D-glucopyranoside.
  • To elucidate the role of proton transfer in the enzyme's active site.

Main Methods:

  • Enzyme kinetics assays were performed at 25°C in H2O and D2O.
  • Solvent kinetic isotope effects (SKIE) were measured for rate constants (kcat, Km) and inhibition constants (Kis).

Related Experiment Videos

  • pH/pD profiles were analyzed to determine the effects on ionization states.

    • Main Results:

    • An unusually large SKIE of 1.9 was observed for the second-order rate constant of pNPG hydrolysis.
    • The pKa values of catalytic residues shifted in D2O, with a small shift for pKa1 and a typical shift for pKa2.
    • Substrate inhibition was observed for pNPG, with a small SKIE on the inhibition constant.
    • Methyl glucoside showed significantly lower reactivity and a smaller SKIE compared to pNPG.

    Conclusions:

    • The large SKIE for pNPG suggests significant proton transfer in the transition state of the deglycosylation step.
    • The differential SKIEs between substrates indicate distinct catalytic mechanisms or transition state involvements.
    • Yeast alpha-glucosidase catalysis involves complex proton transfer events influenced by the solvent deuterium composition.