Yim M B, Chae H Z, Rhee S G, Chock P B, Stadtman E R
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1994 Jan 21;269(3):1621-6.
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.
一种硫醇特异性抗氧化酶（TSA），它能保护其他酶不被硫醇/铁（III）/氧系统灭活，此前已被分离和克隆。我们使用5,5-二甲基-1-吡咯啉N-氧化物（DMPO）的自旋捕获方法，研究了TSA保护生物分子免受含硫醇氧化系统引起的氧化损伤的机制。在有氧和无氧条件下，辣根过氧化物酶/H2O2以及铁（III）/氧系统可产生二硫苏糖醇（.DTT）的硫自由基。无论使用何种产生硫自由基的条件，TSA均能抑制DMPO-.DTT自由基加合物的形成。活性突变体C170S也能淬灭自由基加合物的信号，而无活性突变体C47S则没有任何作用。还发现，尽管C170S不能完全去除DMPO-.DTT自由基加合物，但在反应初始阶段其速率比天然酶更高。这些结果表明，只有活性TSA能催化硫自由基的去除，而半胱氨酸47是该活性所必需的。此外，硫自由基与氧反应生成未鉴定的硫过氧物种。Fe.EDTA与该物种反应生成一种活性自由基，该自由基可从乙醇中提取氢原子以产生羟乙基自由基。这种活性硫-氧自由基被认为是对生物分子造成有害影响的原因。总之，我们的数据表明，TSA通过在硫自由基产生更多活性自由基之前催化其去除，从而保护生物分子免受氧化损伤。然而，目前我们不能排除TSA也可以使用其他含硫醇物种作为底物的可能性。
