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Related Experiment Videos

Dehydroascorbic acid irreversibly inhibits hexokinase activity.

M Fiorani1, R De Sanctis, F Scarlatti

  • 1Istituto di Chimica Biologica Giorgio Fornaini, Urbino (PU), Italy.

Molecular and Cellular Biochemistry
|August 15, 2000
PubMed
Summary
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The oxidized form of vitamin C, dehydroascorbic acid (DHA), irreversibly inactivates hexokinase type I. This inactivation involves DHA covalently binding to cysteine residues, leading to a loss of enzyme activity.

Area of Science:

  • Biochemistry
  • Enzymology

Background:

  • Hexokinase type I is a crucial enzyme in glucose metabolism.
  • Vitamin C (ascorbic acid) and its oxidized form (dehydroascorbic acid, DHA) play vital roles in biological systems.
  • Understanding enzyme regulation and inhibition is essential for metabolic research.

Purpose of the Study:

  • To investigate the mechanism by which dehydroascorbic acid (DHA) inactivates recombinant human hexokinase type I.
  • To characterize the kinetics and molecular basis of DHA-induced hexokinase inactivation.

Main Methods:

  • Enzyme activity assays were performed to monitor hexokinase inactivation.
  • Kinetic analysis was used to determine the reaction order and saturation behavior.
  • Experiments involving oxygen, dithiothreitol, and substrates were conducted.

Related Experiment Videos

  • Amino acid analysis was employed to identify modifications on the enzyme.

    • Main Results:

    • Dehydroascorbic acid (DHA) caused complete and irreversible inactivation of hexokinase type I in a pseudo-first order manner.
    • The inactivation reaction did not show saturation, suggesting no reversible complex formation.
    • Dithiothreitol prevented inactivation but could not restore activity, and inactivation was independent of oxygen.
    • Inactivation was dependent on deprotonation of an alkaline pKa residue and involved covalent binding of DHA, with decreased cysteine residues observed.
    • Substrates like glucose and MgATP modulated the inactivation rate.

    Conclusions:

    • Dehydroascorbic acid (DHA) irreversibly inactivates hexokinase type I through a non-saturable mechanism.
    • The primary mechanism involves the covalent modification of cysteine residues by DHA, leading to loss of enzymatic function.
    • These findings highlight a novel regulatory pathway for hexokinase activity involving oxidized vitamin C.