Hoober K L, Thorpe C
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
Biochemistry. 1999 Mar 9;38(10):3211-7. doi: 10.1021/bi9820816.
The flavin-dependent sulfhydryl oxidase from chicken egg white catalyzes the oxidation of sulfhydryl groups to disulfides with reduction of oxygen to hydrogen peroxide. The oxidase contains FAD and a redox-active cystine bridge and accepts a total of 4 electrons per active site. Dithiothreitol (DTT; the best low molecular weight substrate known) reduces the enzyme disulfide bridge with a limiting rate of 502/s at 4 degrees C, pH 7.5, yielding a thiolate-to-flavin charge-transfer complex. Further reduction to EH4 is limited by the slow internal transfer of reducing equivalents from enzyme dithiol to oxidized flavin (3.3/s). In the oxidative half of catalysis, oxygen rapidly converts EH4 to EH2, but Eox appearance is limited by the slow internal redox equilibration. During overall turnover with DTT, the thiolate-to-flavin charge-transfer complex accumulates with an apparent extinction coefficient of 4.9 mM-1 cm-1 at 560 nm. In contrast, glutathione (GSH) is a much slower reductant of the oxidase to the EH2 level and shows a kcat/Km 100-fold smaller than DTT. Full reduction of EH2 by GSH shows a limiting rate of 3.6/s at 4 degrees C comparable to that seen with DTT. Reduced RNase is an excellent substrate of the enzyme, with kcat/Km per thiol some 1000- and 10-fold better than GSH and DTT, respectively. Enzyme-monitored steady-state turnover shows that RNase is a facile reductant of the oxidase to the EH2 state. This work demonstrates the basic similarity in the mechanism of turnover between all of these three substrates. A physiological role for sulfhydryl oxidase in the formation of disulfide bonds in secreted proteins is discussed.
来自鸡蛋白的黄素依赖性巯基氧化酶催化巯基氧化为二硫键，同时将氧气还原为过氧化氢。该氧化酶含有黄素腺嘌呤二核苷酸（FAD）和一个氧化还原活性的胱氨酸桥，每个活性位点总共接受4个电子。二硫苏糖醇（DTT；已知最佳的低分子量底物）在4℃、pH 7.5条件下以502/s的极限速率还原酶的二硫键，生成硫醇盐-黄素电荷转移复合物。进一步还原为EH4受到还原当量从酶二硫醇缓慢向内转移至氧化型黄素（3.3/s）的限制。在催化的氧化阶段，氧气迅速将EH4转化为EH2，但Eox的出现受到缓慢的内部氧化还原平衡的限制。在用DTT进行的整体周转过程中，硫醇盐-黄素电荷转移复合物在560nm处积累，表观消光系数为4.9 mM-1 cm-1。相比之下，谷胱甘肽（GSH）将氧化酶还原至EH2水平的速度要慢得多，其kcat/Km比DTT小100倍。GSH将EH2完全还原在4℃下的极限速率为3.6/s，与DTT的情况相当。还原型核糖核酸酶是该酶极好的底物，每个巯基的kcat/Km分别比GSH和DTT约高100和10倍。酶监测的稳态周转表明，核糖核酸酶是将氧化酶还原至EH2状态的便捷还原剂。这项工作证明了这三种底物在周转机制上的基本相似性。文中还讨论了巯基氧化酶在分泌蛋白中二硫键形成过程中的生理作用。
