Inactivation of Ascorbate Peroxidase by Thiols Requires Hydrogen Peroxide

The hydrogen peroxide-dependent oxidation of ascorbate by ascorbate peroxidase from tea leaves was inhibited by thiols, such as dithiothreitol, glutathione, mercaptoethanol and cysteine. These thiols themselves did not inactivate the enzyme. However, they inactivated the enzyme when hydrogen peroxide was produced by the metal-catalyzed oxidation of thiols or when exogenous hydrogen peroxide was added. Thiols were oxidized by ascorbate peroxidase and hydrogen peroxide to thiyl radicals, as detected by the ESR spectra of the thiyl radical-5,5′-dimethyll- pyrroline-N-oxidie adducts. Inactivation of ascorbate peroxidase by thiols and hydrogen peroxide is caused by the interaction of the enzyme with the thiyl radicals produced at its reaction center.