The oxidation of selenocysteine is involved in the inactivation of glutathione peroxidase by nitric oxide donor
The oxidation of selenocysteine is involved in the inactivation of glutathione peroxidase by nitric oxide donor
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DOI：
10.1074/jbc.272.31.19152
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发表时间：
1997-08-01
影响因子：
4.8
通讯作者：
Taniguchi, N
中科院分区：
文献类型：
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作者：
Asahi, M;Fujii, J;Taniguchi, N
谷胱甘肽过氧化物酶(GPx)被一氧化氮供体S-亚硝基-N-乙酰-D，L-青霉胺(SNAP)灭活。化学。21035-21039)。对失活的结构基础进行了研究。我们还发现，过氧亚硝酸前体3-吗啉-西诺酮亚胺-N-乙基尿素以及合成的过氧亚硝酸根也能使牛GPX失活。质谱学结果表明，SNAP预处理后，N-辛基二硫代硝基苯甲酸在GPX中的掺入程度降低。为了确定SNAP对该酶的修饰部位，将SNAP处理过的GPx和未处理过的GPx分别与N-辛基二硫代硝基苯甲酸反应，用赖氨酰内肽酶消化，得到的多肽进行了质谱分析。这项技术发现了两个肽之间的桥梁，其中一个在催化中心含有SEC(45)和Cys(74)，另一个含有Cys(91)。虽然有两种可能的组合，硒半胱氨酸45(SEC(45))和半胱氨酸(91)或半胱氨酸(74)和半胱氨酸(91)，但GPX的三级结构表明SEC(45)和半胱氨酸(91)之间的交联更可行。这与SNAP特异性失活GPX的实验证据是一致的，在GPX中，SEC(45)形成催化中心。因此，我们得出结论，SNAP主要氧化SEC(45)与游离的硫醇形成硒基硫化物(Se-S)，导致该酶失活。这些数据表明，一氧化氮及其衍生物通过产生一种硒基硫化物，以一种特定的方式直接使GPX失活，导致导致细胞损伤的细胞内过氧化物质增加。
Glutathione peroxidase (GPx) was inactivated by S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitric oxide donor (Asahi, M., Fujii, J., Suzuki, K., See, H. G., Kuzuya, T., Hori, M., Tada, M., Fujii, S., and Taniguchi, N. (1995) J. Biol. Chem. 270, 21035-21039). The structural basis of the inactivation was studied. We also show that 3-morpholinosydnonimine N-ethylcarbamide, a peroxynitrite precursor, as well as synthetic peroxynitrite also inactivated bovine GPx. The degree of incorporation of a sulfhydryl reagent, n-octyldithionitrobenzoic acid, into GPx decreased after pretreatment with SNAP as evidenced by mass spectrometry. To identify the modification site of this enzyme by SNAP, both SNAP-pretreated and untreated GPxs were reacted with n-octyldithionitrobenzoic acid and digested with lysylendopeptidase, and the resulting peptides were subjected to mass spectrometry. This technique identified a bridge between two peptides, one of which contains Sec(45) at the catalytic center and Cys(74), and the other contains Cys(91). Although there are two possible combinations, selenocysteine 45 (Sec(45)) and Cys(91) or Cys(74) and Cys(91), the tertiary structure of GPx indicates that a cross-link between Sec(45) and Cys(91) is more feasible. This is consistent with the experimental evidence that SNAP specifically inactivates GPx, in which Sec(45) forms the catalytic center. Thus, we conclude that SNAP mainly oxidized Sec(45) to form a selenenyl sulfide (Se-S) with a free thiol, leading to the inactivation of the enzyme. These data suggest that nitric oxide and its derivatives directly inactivate GPx in a specific manner via the production of a selenenyl sulfide, resulting in an increase in intracellular peroxides that are responsible for cellular damage.