Avian sulfhydryl oxidase is not a metalloenzyme: Adventitious binding of divalent metal ions to the enzyme
Avian sulfhydryl oxidase is not a metalloenzyme: Adventitious binding of divalent metal ions to the enzyme
复制标题
DOI：
10.1021/bi0301385
复制
发表时间：
2003-09-23
期刊：
影响因子：
2.9
通讯作者：
Thorpe, C
中科院分区：
文献类型：
--
作者：
Brohawn, SG;Miksa, IR;Thorpe, C
金属和黄素依赖性巯基氧化酶催化二硫键的产生，并将氧还原为过氧化氢。哺乳动物皮肤酶已被报道是铜依赖性的，但最近的蛋白质序列显示它属于Quiescin/巯基氧化酶（QSOX）黄素蛋白家族。这项工作表明，禽QSOX是不是一种金属酶，铜和锌离子抑制氧化还原胰腺核糖核酸酶的酶。与锌2+，作为铜的氧化还原非活性替代品的研究表明，一个锌2+绑定到四个电子还原QSOX转移电子远离黄素，并进入两个的三个氧化还原活性二硫桥的酶。由此产生的锌络合物是适度的空气稳定的，恢复到光谱的天然蛋白质的t（1/2）为40分钟，而四电子还原的天然QSOX在不到一秒的时间内在可比的条件下被再氧化。使用三（2-羧乙基）膦盐酸盐（TCEP），QSOX的替代底物，结合Zn 2+相对较弱（不像二硫苏糖醇），允许在低微摩尔金属水平下快速抑制氧化酶活性。锌结合后，快速扫描分光光度法。铜还结合四电子还原形式的QSOX，其可见光谱暗示活性位点占据。除了与还原酶的相互作用，透析实验表明，多个铜和锌离子可以结合到氧化酶，而没有前面看到的黄素光谱的扰动。这些数据表明，皮肤巯基氧化酶的金属含量的重新调查是必要的。锌的氧化还原调节结合QSOX被认为是在光的证据锌硫醇盐的相互作用中的氧化还原信号和锌动员的作用。
Metal- and flavin-dependent sulfhydryl oxidases catalyze the generation of disulfide bonds with reduction of oxygen to hydrogen peroxide. The mammalian skin enzyme has been reported to be copper-dependent, but a recent protein sequence shows it belongs to the Quiescin/sulfhydryl oxidase (QSOX) flavoprotein family. This work demonstrates that avian QSOX is not a metalloenzyme, and that copper and zinc ions inhibit the oxidation of reduced pancreatic ribonuclease by the enzyme. Studies with Zn2+, as a redox inactive surrogate for copper, show that one Zn2+ binds to four-electron-reduced QSOX by diverting electrons away from the flavin and into two of the three redox active disulfide bridges in the enzyme. The resulting zinc complex is modestly air-stable, reverting to a spectrum of the native protein with a t(1/2) of 40 min, whereas the four-electron-reduced native QSOX is reoxidized in less than a second under comparable conditions. Using tris(2-carboxyethyl)phosphine hydrochloride (TCEP), an alternate substrate of QSOX that binds Zn2+ relatively weakly (unlike dithiothreitol), allows rapid inhibition of oxidase activity to be demonstrated at low micromolar metal levels. Zinc binding was followed by rapid-scanning spectrophotometry. Copper also binds the four-electron-reduced form of QSOX with a visible spectrum suggestive of active site occupancy. In addition to interactions with the reduced enzyme, dialysis experiments show that multiple copper and zinc ions can bind to the oxidized enzyme without the perturbation of the flavin spectrum seen earlier. These data suggest that a reinvestigation of the metal content of skin sulfhydryl oxidases is warranted. The redox-modulated binding of zinc to QSOX is considered in light of evidence for a role of zinc-thiolate interactions in redox signaling and zinc mobilization.