Roos Goedele, Garcia-Pino Abel, Van Belle Karolien, Brosens Elke, Wahni Khadija, Vandenbussche Guy, Wyns Lode, Loris Remy, Messens Joris
Department of Molecular and Cellular Interactions, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
J Mol Biol. 2007 May 4;368(3):800-11. doi: 10.1016/j.jmb.2007.02.045. Epub 2007 Feb 22.
Nature uses thioredoxin-like folds in several disulfide bond oxidoreductases. Each of them has a typical active site Cys-X-X-Cys sequence motif, the hallmark of thioredoxin being Trp-Cys-Gly-Pro-Cys. The intriguing role of the highly conserved proline in the ubiquitous reducing agent thioredoxin was studied by site-specific mutagenesis of Staphylococcus aureus thioredoxin (Sa_Trx). We present X-ray structures, redox potential, pK(a), steady-state kinetic parameters, and thermodynamic stabilities. By replacing the central proline to a threonine/serine, no extra hydrogen bonds with the sulphur of the nucleophilic cysteine are introduced. The only structural difference is that the immediate chemical surrounding of the nucleophilic cysteine becomes more hydrophilic. The pK(a) value of the nucleophilic cysteine decreases with approximately one pH unit and its redox potential increases with 30 mV. Thioredoxin becomes more oxidizing and the efficiency to catalyse substrate reduction (k(cat)/K(M)) decreases sevenfold relative to wild-type Sa_Trx. The oxidized form of wild-type Sa_Trx is far more stable than the reduced form over the whole temperature range. The driving force to reduce substrate proteins is the relative stability of the oxidized versus the reduced form Delta(T(1/2))(ox/red). This driving force is decreased in the Sa_Trx P31T mutant. Delta(T(1/2))(ox/red) drops from 15.5 degrees C (wild-type) to 5.8 degrees C (P31T mutant). In conclusion, the active site proline in thioredoxin determines the driving potential for substrate reduction.
自然界在几种二硫键氧化还原酶中使用硫氧还蛋白样折叠结构。它们每一个都有一个典型的活性位点Cys-X-X-Cys序列基序，硫氧还蛋白的标志是Trp-Cys-Gly-Pro-Cys。通过对金黄色葡萄球菌硫氧还蛋白（Sa_Trx）进行位点特异性诱变，研究了普遍存在的还原剂硫氧还蛋白中高度保守的脯氨酸的有趣作用。我们给出了X射线结构、氧化还原电位、pK(a)、稳态动力学参数和热力学稳定性。通过将中心脯氨酸替换为苏氨酸/丝氨酸，没有引入与亲核半胱氨酸的硫形成的额外氢键。唯一的结构差异是亲核半胱氨酸的直接化学环境变得更亲水。亲核半胱氨酸的pK(a)值下降约一个pH单位，其氧化还原电位增加30 mV。硫氧还蛋白变得更具氧化性，催化底物还原的效率（k(cat)/K(M)）相对于野生型Sa_Trx降低了七倍。在整个温度范围内，野生型Sa_Trx的氧化形式比还原形式稳定得多。还原底物蛋白的驱动力是氧化形式与还原形式的相对稳定性Delta(T(1/2))(ox/red)。在Sa_Trx P31T突变体中，这种驱动力降低。Delta(T(1/2))(ox/red)从15.5摄氏度（野生型）降至5.8摄氏度（P31T突变体）。总之，硫氧还蛋白中的活性位点脯氨酸决定了底物还原的驱动潜力。
