Nordberg J, Zhong L, Holmgren A, Arnér E S
Medical Nobel Institute for Biochemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
J Biol Chem. 1998 May 1;273(18):10835-42. doi: 10.1074/jbc.273.18.10835.
The immunostimulatory dinitrohalobenzene compound 1-chloro-2, 4-dinitrobenzene (DNCB) irreversibly inhibits mammalian thioredoxin reductase (TrxR) in the presence of NADPH, inducing an NADPH oxidase activity in the modified enzyme (Arnér, E. S. J., Björnstedt, M., and Holmgren, A. (1995) J. Biol. Chem. 270, 3479-3482). Here we have further analyzed the reactivity with the enzyme of DNCB and analogues with varying immunomodulatory properties. We have also identified the reactive residues in bovine thioredoxin reductase, recently discovered to be a selenoprotein. We found that 4-vinylpyridine competed with DNCB for inactivation of TrxR, with DNCB being about 10 times more efficient, and only alkylation with DNCB but not with 4-vinylpyridine induced an NADPH oxidase activity. A number of nonsensitizing DNCB analogues neither inactivated the enzyme nor induced any NADPH oxidase activity. The NADPH oxidase activity of TrxR induced by dinitrohalobenzenes generated superoxide, as detected by reaction with epinephrine (the adrenochrome method). Addition of superoxide dismutase quenched this reaction and also stimulated the NADPH oxidase activity. By peptide analysis using mass spectrometry and Edman degradation, both the cysteine and the selenocysteine in the conserved carboxyl-terminal sequence Gly-Cys-Sec-Gly (where Sec indicates selenocysteine) were determined to be dinitrophenyl-alkylated upon incubation of native TrxR with NADPH and DNCB. A model for the interaction between TrxR and dinitrohalobenzenes is proposed, involving a functional FAD in the alkylated TrxR generating an anion nitroradical in a dinitrophenyl group, which in turn reacts with oxygen to generate superoxide. Production of reactive oxygen species and inhibited reduction of thioredoxin by the modified thioredoxin reductase after reaction with dinitrohalobenzenes may play a major role in the inflammatory reactions provoked by these compounds.
免疫刺激剂二硝基卤代苯化合物1-氯-2,4-二硝基苯（DNCB）在NADPH存在下不可逆地抑制哺乳动物硫氧还蛋白还原酶（TrxR），并在修饰后的酶中诱导NADPH氧化酶活性（阿诺尔，E.S.J.，比约恩斯泰德，M.，和霍尔姆格伦，A.（1995年）《生物化学杂志》270，3479 - 3482）。在此，我们进一步分析了DNCB及其具有不同免疫调节特性的类似物与该酶的反应活性。我们还确定了牛硫氧还蛋白还原酶（最近发现它是一种硒蛋白）中的反应性残基。我们发现4-乙烯基吡啶与DNCB竞争使TrxR失活，DNCB的效率约高10倍，并且只有用DNCB而非4-乙烯基吡啶进行烷基化会诱导NADPH氧化酶活性。许多无致敏性的DNCB类似物既不会使该酶失活，也不会诱导任何NADPH氧化酶活性。如通过与肾上腺素反应（肾上腺色素法）所检测到的，二硝基卤代苯诱导的TrxR的NADPH氧化酶活性会产生超氧化物。添加超氧化物歧化酶可终止此反应，并且还会刺激NADPH氧化酶活性。通过使用质谱和埃德曼降解进行肽分析，在天然TrxR与NADPH和DNCB一起孵育后，保守的羧基末端序列Gly-Cys-Sec-Gly（其中Sec表示硒代半胱氨酸）中的半胱氨酸和硒代半胱氨酸都被确定为发生了二硝基苯基烷基化。提出了一个TrxR与二硝基卤代苯之间相互作用的模型，其中涉及烷基化的TrxR中起作用的FAD在二硝基苯基基团中产生一个阴离子硝基自由基，该自由基进而与氧反应生成超氧化物。与二硝基卤代苯反应后，修饰后的硫氧还蛋白还原酶产生活性氧物种并抑制硫氧还蛋白的还原，这可能在这些化合物引发的炎症反应中起主要作用。
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