On the protective mechanism of the thiol-specific antioxidant enzyme against the oxidative damage of biomacromolecules

• Published in: The Journal of biological chemistry Vol. 269; no. 3; pp. 1621 - 1626
• Main Authors: YIM, M. B, CHAE, H. Z, RHEE, S. G, CHOCK, P. B, STADTMAN, E. R
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 21.01.1994
• Subjects: Analytical, structural and metabolic biochemistry; Antioxidants - chemistry; Antioxidants - metabolism; Biological and medical sciences; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Fungal Proteins - biosynthesis; Fungal Proteins - metabolism; Horseradish Peroxidase - metabolism; Kinetics; Mathematics; Miscellaneous; Models, Theoretical; Oxidants - toxicity; Oxygen - pharmacology; Peroxidases; Peroxiredoxins; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Spin Labels; Thiol; Enzymatic activity; Enzyme; Escherichia coli; EPR spectrometry; Bacteria; Antioxidant; Mechanism of action; Enterobacteriaceae
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)42072-2

A thiol-specific antioxidant enzyme (TSA), which provides protection against the inactivation of other enzymes by the thiol/Fe(III)/oxygen system, was previously isolated and cloned. We investigated the mechanism by which TSA protects biomolecules from oxidative damage caused by the thiol-containing oxidation system using the spin trapping method with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Thiyl radicals from dithiothreitol (.DTT) were produced by horseradish peroxidase/H2O2 under aerobic and anaerobic conditions and by the Fe(III)/oxygen system. The formation of DMPO-.DTT radical adducts were inhibited by TSA regardless of the thiyl radical-generating conditions used. The active mutant C170S also quenched the signals of the radical adduct, whereas the inactive 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 the native enzyme, although C170S failed to remove DMPO-.DTT radical adducts completely. These results indicate that only active TSA can catalyze the removal of thiyl radicals, and cysteine 47 is required for this 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 ethanol to produce a hydroxyethyl radical. This reactive thiyl-oxygen radical is believed to be responsible for causing deleterious effects on biomolecules. Together, our data indicate that TSA protects biomolecules from oxidative damage by catalyzing the removal of thiyl radicals before they generate more reactive radicals. However, presently we cannot rule out the possibility that TSA can also use other thiol-containing species as substrates.