Article

Molecular and Cellular Biochemistry

, Volume 209, Issue 1, pp 145-153

First online:

Dehydroascorbic acid irreversibly inhibits hexokinase activity

  • Mara FioraniAffiliated withIstituto di Chimica Biologica 'Giorgio Fornaini'
  • , Roberta De SanctisAffiliated withIstituto di Chimica Biologica 'Giorgio Fornaini'
  • , Francesca ScarlattiAffiliated withIstituto di Chimica Biologica 'Giorgio Fornaini'
  • , Luciana ValloraniAffiliated withIstituto di Chimica Biologica 'Giorgio Fornaini'
  • , Roberta De BellisAffiliated withIstituto di Chimica Biologica 'Giorgio Fornaini'
  • , Giordano SerafiniAffiliated withIstituto di Chimica Biologica 'Giorgio Fornaini'
  • , Marzia BianchiAffiliated withIstituto di Chimica Biologica 'Giorgio Fornaini'
  • , Vilberto StocchiAffiliated withIstituto di Chimica Biologica 'Giorgio Fornaini'

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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.

hexokinase dehydroascorbic acid thiol groups protein modification