Faculty of Chemistry, TU Kaiserslautern, Kaiserslautern, Germany.
Protein Sci. 2022 May;31(5):e4290. doi: 10.1002/pro.4290.
Peroxiredoxins use a variety of thiols to rapidly reduce hydroperoxides and peroxynitrite. While the oxidation kinetics of peroxiredoxins have been studied in great detail, enzyme-specific differences regarding peroxiredoxin reduction and the overall rate-limiting step under physiological conditions often remain to be deciphered. The 1-Cys peroxiredoxin 5 homolog PfAOP from the malaria parasite Plasmodium falciparum is an established model enzyme for glutathione/glutaredoxin-dependent peroxiredoxins. Here, we reconstituted the catalytic cycle of PfAOP in vitro and analyzed the reaction between oxidized PfAOP and reduced glutathione (GSH) using molecular docking and stopped-flow measurements. Molecular docking revealed that oxidized PfAOP has to adopt a locally unfolded conformation to react with GSH. Furthermore, we determined a second-order rate constant of 6 × 10 M s at 25°C and thermodynamic activation parameters ΔH , ΔS , and ΔG of 39.8 kJ/mol, -0.8 J/mol, and 40.0 kJ/mol, respectively. The gain-of-function mutant PfAOP had almost identical reaction parameters. Taking into account physiological hydroperoxide and GSH concentrations, we suggest (a) that the reaction between oxidized PfAOP and GSH might be even faster than the formation of the sulfenic acid in vivo, and (b) that conformational changes are likely rate limiting for PfAOP catalysis. In summary, we characterized and quantified the reaction between GSH and the model enzyme PfAOP, thus providing detailed insights regarding the reactivity of its sulfenic acid and the versatile chemistry of peroxiredoxins.
过氧化物酶利用各种硫醇迅速还原过氧化物和过氧亚硝酸盐。虽然过氧化物酶的氧化动力学已经得到了深入研究，但在生理条件下，关于过氧化物酶还原和整体限速步骤的酶特异性差异仍有待破译。疟原虫 Plasmodium falciparum 的 1-Cys 过氧化物酶 5 同源物 PfAOP 是谷胱甘肽/谷胱甘肽还原酶依赖的过氧化物酶的既定模型酶。在这里，我们在体外重建了 PfAOP 的催化循环，并使用分子对接和停流测量分析了氧化 PfAOP 与还原型谷胱甘肽 (GSH) 之间的反应。分子对接表明，氧化 PfAOP 必须采用局部展开的构象才能与 GSH 反应。此外，我们在 25°C 下确定了第二级反应速率常数为 6×10 M s ，热力学激活参数 ΔH 、ΔS 和 ΔG 分别为 39.8 kJ/mol、-0.8 J/mol 和 40.0 kJ/mol。功能获得突变体 PfAOP 的反应参数几乎相同。考虑到生理过氧化氢和 GSH 浓度，我们提出 (a) 氧化 PfAOP 与 GSH 之间的反应速度可能甚至快于体内亚磺酸的形成速度，以及 (b) 构象变化可能是 PfAOP 催化的限速步骤。总之，我们对 GSH 与模型酶 PfAOP 之间的反应进行了表征和量化，从而提供了关于其亚磺酸反应性和过氧化物酶化学多功能性的详细见解。
