Human placenta thioredoxin reductase - Isolation of the selenoenzyme, steady state kinetics, and inhibition by therapeutic gold compounds
Human placenta thioredoxin reductase - Isolation of the selenoenzyme, steady state kinetics, and inhibition by therapeutic gold compounds
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DOI：
10.1074/jbc.273.32.20096
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发表时间：
1998-08-07
影响因子：
4.8
通讯作者：
Becker, K
中科院分区：
文献类型：
--
作者：
Gromer, S;Arscott, LD;Becker, K
人硫氧还蛋白还原酶是一种与谷胱甘肽还原酶密切相关的吡啶核苷酸二硫氧化还原酶，但与后者的不同之处在于，它有一个Cys-SeCys（硒代半胱氨酸）序列作为额外的氧化还原中心。由于硒蛋白还不能在异源系统中表达，我们从胎盘中优化了硒蛋白的纯化，包括最终产量（1- 2mg）、比活性（42单位/mg）和硒含量（0.94 +/- 0.03 mol/mol亚单位）。稳态动力学表明，该酶以乒乓机制起作用；k（cat）值为3330 +/- 882 min(-1)， NADPH和大肠杆菌硫氧还蛋白的k - M值分别为18 μ M和25 μ M。该反应的活化能为53.2 kJ/mol，可以将稳态数据与稳态前的测量结果进行比较。在生理上，nadph降低的形式，酶被广泛用于治疗类风湿性关节炎的有机金化合物强烈抑制；在50 μ M硫氧还蛋白存在下，金糠蛋白的K-i为4 nM。在1000倍的浓度下，即在微摩尔水平下，这些药物还能抑制人谷胱甘肽还原酶和谷胱甘肽过氧化物酶的硒酶。
Human thioredoxin reductase is a pyridine nucleotide-disulfide oxidoreductase closely related to glutathione reductase but differing from the latter in having a Cys-SeCys (selenocysteine) sequence as an additional redox center. Because selenoproteins cannot be expressed yet in heterologous systems, we optimized the purification of the protein from placenta with respect to final yield (1-2 mg from one placenta), specific activity (42 units/mg), and selenium content (0.94 +/- 0.03 mol/mol subunit). The steady state kinetics showed that the enzyme operates by a ping-pong mechanism; the value of k(cat) was 3330 +/- 882 min(-1), and the K-m values were 18 mu M for NADPH and 25 mu M for Escherichia coli thioredoxin. The activation energy of the reaction was found to be 53.2 kJ/mol, which allows comparisons of the steady state data with previous pre-steady state measurements. In its physiological, NADPH-rednced form, the enzyme is strongly inhibited by organic gold compounds that are widely used in the treatment of rheumatoid arthritis; for auranofin, the K-i was 4 nM when measured in the presence of 50 mu M thioredoxin. At 1000-fold higher concentrations, that is at micromolar levels, the drugs also inhibited human glutathione reductase and the selenoenzyme glutathione peroxidase.