ON THE PROTECTIVE MECHANISM OF THE THIOL-SPECIFIC ANTIOXIDANT ENZYME AGAINST THE OXIDATIVE DAMAGE OF BIOMACROMOLECULES

A thiol-specific antioxidant enzyme (TSA), which provides protection a gainst the inactivation of other enzymes by the thiol/Fe(III)/oxygen s ystem, was previously isolated and cloned. We investigated the mechani sm by which TSA protects biomolecules from oxidative damage caused by the thiol-containing oxidation system using the spin trapping method w ith 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Thiyl radicals from dithi othreitol (.DTT) were produced by horseradish peroxidase/H2O2 under ae robic and anaerobic conditions and by the Fe(III)/oxygen system. The f ormation of DMPO-.DTT radical adducts were inhibited by TSA regardless of the thiyl radical-generating conditions used. The active mutant C1 70S also quenched the signals of the radical adduct, whereas the inact ive mutant C47S did not exert any effect. It was also found that C170S has a higher rate at the initial stage of the reaction than that of t he native enzyme, although C170S failed to remove DMPO-.DTT radical ad ducts completely. These results indicate that only active TSA can cata lyze the removal of thiyl radicals, and cysteine 47 is required for th is activity. In addition, thiyl radicals react with oxygen to generate unidentified thiylperoxy species. Fe.EDTA reacts with this species to generate a reactive radical that can abstract hydrogen atom from etha nol to produce a hydroxyethyl radical. This reactive thiyl-oxygen radi cal is believed to be responsible for causing deleterious effects on b iomolecules. Together, our data indicate that TSA protects biomolecule s from oxidative damage by catalyzing the removal of thiyl radicals be fore they generate more reactive radicals. However, presently we canno t rule out the possibility that TSA can also use other thiol-containin g species as substrates.