Purification and characterization of a second type thioredoxin peroxidase (type II TPx) from Saccharomyces cerevisiae
Purification and characterization of a second type thioredoxin peroxidase (type II TPx) from Saccharomyces cerevisiae
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DOI：
10.1021/bi9817818
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发表时间：
1999-01-12
期刊：
影响因子：
2.9
通讯作者：
Kim, K
中科院分区：
文献类型：
--
作者：
Jeong, JS;Kwon, SJ;Kim, K
先前已鉴定出一种酵母过氧化物酶，其使用硫氧还蛋白提供的还原当量来还原H2 O2和烷基氢过氧化物，并命名为硫氧还蛋白过氧化物酶（TPX）[Chae，H. Z.，Chung，S，J.，和Rhee，S. G，（1994）J.Biol.Chem.269，27670-27678]。第二种类型的硫氧还蛋白依赖性过氧化物酶，命名为II型TPx，现已从酵母中纯化，并已获得几个肽序列。使用这些序列，已从GenBank数据库中鉴定出相应的cDNA。将II型TPx的176个氨基酸的预测序列与TPx的195个残基的预测序列进行比较，发现除了II型TPx的Cys（62）之前的一个短片段外，没有实质性的同源性。由I型和II型TPx催化的反应的动力学表征表明，I型优先还原H2 O2而不是烷基氢过氧化物，而II型则表现出相反的特异性。II型TPx在位置31、62和120处含有三个半胱氨酸残基。用突变蛋白进行的实验表明，这三个半胱氨酸残基分别被丝氨酸取代，Cys（62）-SH构成了过氧化物氧化的位点，氧化的Cys（62）与另一个II型TPx分子的Cys（120）-SH基团反应形成分子间二硫键。形成的二硫化物可以被硫氧还蛋白还原，但不能被谷胱甘肽还原。因此，缺乏Cys（62）或Cys（120）的II型TPx突变体显示没有可检测的TPx活性，而Cys（31）的突变对TPx活性没有影响。通过观察到过表达野生型蛋白的大肠杆菌细胞对烷基氢过氧化物抑制生长的敏感性低于对照细胞或过表达缺乏Cys的突变蛋白的细胞，证明了完整细胞中II型TPx的抗氧化功能（62）。
A yeast peroxidase that reduces H2O2 and alkyl hydroperoxides with the use of reducing equivalents provided by thioredoxin was identified previously and named thioredoxin peroxidase (TPx) [Chae, H. Z., Chung, S, J., and Rhee, S. G, (1994) J. Biol, Chem. 269, 27670-27678]. A second type thioredoxin-dependent peroxidase, named type II TPx, has now been purified from yeast, and several peptide sequences have been obtained. Using those sequences, the corresponding cDNA has been identified from the GenBank database. Comparison of the predicted sequence of 176 amino acids of type II TPx with that of the 195 residues of TPx, now renamed type I TPx, revealed no substantial homology except for a short segment preceding Cys(62) of type II TPx, Kinetic characterization of the reactions catalyzed by type I and II TPxs revealed that type I preferentially reduces H2O2 rather than alkyl hydroperoxides, whereas type II shows the reverse specificity. Type II TPx contains three cysteine residues at positions 31, 62, and 120. Experiments with mutant proteins in which these three cysteine residues were replaced individually with serine suggest that Cys(62)-SH constitutes the site of oxidation by peroxides and that the oxidized Cys(62) reacts with the Cys(120)-SH group of another type II TPx molecule to form an intermolecular disulfide linkage. The formed disulfide can then be reduced by thioredoxin, but not by glutathione. Thus, type II TPx mutants lacking Cys(62) or Cys(120) showed no detectable TPx activity, whereas mutation of Cys(31) had no effect on TPx activity. An antioxidant function of type II TPx in intact cells was demonstrated by the observation that Escherichia coli cells overexpressing wild-type protein were less sensitive to inhibition of growth by alkyl hydroperoxides than were control cells or cells overexpressing the mutant protein lacking Cys(62).