Peskin Alexander V, Low Felicia M, Paton Louise N, Maghzal Ghassan J, Hampton Mark B, Winterbourn Christine C
Free Radical Research Group, Department of Pathology, University of Otago, P. O. Box 4345, Christchurch, New Zealand.
J Biol Chem. 2007 Apr 20;282(16):11885-92. doi: 10.1074/jbc.M700339200. Epub 2007 Feb 27.
Peroxiredoxin 2 is a member of the mammalian peroxiredoxin family of thiol proteins that is important in antioxidant defense and redox signaling. We have examined its reactivity with various biological oxidants, in order to assess its ability to act as a direct physiological target for these species. Human erythrocyte peroxiredoxin 2 was oxidized stoichiometrically to its disulfide-bonded homodimer by hydrogen peroxide, as monitored electrophoretically under nonreducing conditions. The protein was highly susceptible to oxidation by adventitious peroxide, which could be prevented by treating buffers with low concentrations of catalase. However, this did not protect peroxiredoxin 2 against oxidation by added H(2)O(2). Experiments measuring inhibition of dimerization indicated that at pH 7.4 catalase and peroxiredoxin 2 react with hydrogen peroxide at comparable rates. A rate constant of 1.3 x 10(7) M(-1) s(-1) for the peroxiredoxin reaction was obtained from competition kinetic studies with horseradish peroxidase. This is 100-fold faster than is generally assumed. It is sufficiently high for peroxiredoxin to be a favored cellular target for hydrogen peroxide, even in competition with catalase or glutathione peroxidase. Reactions of t-butyl and cumene hydroperoxides with peroxiredoxin were also fast, but amino acid chloramines reacted much more slowly. This contrasts with other thiol compounds that react many times faster with chloramines than with hydrogen peroxide. The alkylating agent iodoacetamide also reacted extremely slowly with peroxiredoxin 2. These results demonstrate that peroxiredoxin 2 has a tertiary structure that facilitates reaction of the active site thiol with hydrogen peroxide while restricting its reactivity with other thiol reagents.
过氧化物酶2是哺乳动物硫醇蛋白过氧化物酶家族的成员，在抗氧化防御和氧化还原信号传导中起重要作用。我们研究了它与各种生物氧化剂的反应性，以评估其作为这些物质直接生理靶点的能力。在非还原条件下通过电泳监测，人红细胞过氧化物酶2被过氧化氢化学计量地氧化为其二硫键连接的同型二聚体。该蛋白极易被偶然产生的过氧化物氧化，用低浓度过氧化氢酶处理缓冲液可防止这种情况发生。然而，这并不能保护过氧化物酶2免受添加的H₂O₂的氧化。测量二聚化抑制的实验表明，在pH 7.4时，过氧化氢酶和过氧化物酶2与过氧化氢的反应速率相当。通过与辣根过氧化物酶的竞争动力学研究，得到过氧化物酶反应的速率常数为1.3×10⁷ M⁻¹ s⁻¹。这比通常假设的快100倍。即使与过氧化氢酶或谷胱甘肽过氧化物酶竞争，这个速率也足够高，使过氧化物酶成为过氧化氢青睐的细胞靶点。叔丁基过氧化氢和异丙苯过氧化氢与过氧化物酶的反应也很快，但氨基酸氯胺的反应要慢得多。这与其他硫醇化合物形成对比，其他硫醇化合物与氯胺的反应速度比与过氧化氢的反应速度快很多倍。烷基化剂碘乙酰胺与过氧化物酶2的反应也极其缓慢。这些结果表明，过氧化物酶2具有一种三级结构，这种结构有利于活性位点硫醇与过氧化氢反应，同时限制其与其他硫醇试剂的反应性。
