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Dehydroascorbic acid irreversibly inhibits hexokinase activity
M Fiorani1, R De Sanctis, F Scarlatti
1Istituto di Chimica Biologica Giorgio Fornaini, Urbino (PU), Italy.
Insights
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.
- 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.
Abstract:
The oxidized form of vitamin C (dehydroascorbic acid, DHA) completely and irreversibly inactivates recombinant human hexokinase type I, in a pseudo-first order fashion. The inactivation reaction occurs without saturation, indicating that DHA does not form a reversible complex with hexokinase. Further characterization of this response revealed that the inactivation does not require oxygen and that dithiothreitol, while able to prevent the DHA-mediated loss of enzyme activity, failed to restore the activity of the DHA-inhibited enzyme. Inactivation was not associated with cleavage of the peptide chain or cross-linking. The decay in enzymatic activity was however both dependent on deprotonation of a residue with an alkaline pKa and associated with covalent binding of DHA to the protein. In addition, inactivation of hexokinase decreased or increased, respectively, in the presence of the substrates glucose or MgATP. Finally, amino acid analysis of the DHA-modified hexokinase revealed a decrease of cysteine residues. Taken together, the above results are consistent with the possibility that covalent binding of the reagent with a thiol group of cysteine is a critical event for the DHA-mediated loss of hexokinase activity.