Purification and characterization of a second type thioredoxin peroxidase (type II TPx) from Saccharomyces cerevisiae

被引:104
作者
Jeong, JS
Kwon, SJ
Kang, SW
Rhee, SG
Kim, K [1 ]
机构
[1] Chonnam Natl Univ, Coll Home Econ, Dept Food & Nutr, Kwangju 500757, South Korea
[2] NHLBI, Lab Cell Signaling, NIH, Bethesda, MD 20892 USA
关键词
D O I
10.1021/bi9817818
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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).
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页码:776 / 783
页数:8
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