Effect of Pb2＋ on the Kinetic and Spectral Characterization of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

本文利用各种光谱手段在体外研究了各种浓度的Pb2+对菠菜Rubisco活性影响的机制。 结果表明，Rubisco活性随着Pb2+处理浓度的增加而逐渐下降，低浓度Pb2+下Rubisco的动力学常数和最大反应速率分别为1.74 µM 和 0.42 µmol CO2/mg protein∙min，高浓度Pb2+下Rubisco的动力学常数和最大反应速率分别为11.82 µM and 0.28 µmol CO2/mg protein∙min。光谱学分析证实Pb2+可直接结合到Rubisco上, 其结合位点数为1.1个，结合常数分别为8.63×104 和 2.18×105 L/mol。ICP-MS和圆二色谱分析证实Pb2+取代了酶活性中心的Mg2+ 并改变了酶的构象。

Effect of Pb2+ on the Kinetic and Spectral Characterization of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

Pb²⁺ at various concentrations was added to the purified ribulose‐1,5‐bisphosphate carboxylase/oxygenase (rubisco, E.C.4.1.1.39) in vitro to gain insight into the mechanism of molecular interactions between Pb²⁺ and rubisco using spectral methods. It was found that the carboxylase activity of rubisco gradually decreased with increasing concentrations of Pb²⁺. The kinetics constant (K_m) and v_max were 1.74 mol·L^?1 and 0.42 mol CO₂/(mg protein·min), respectively, at a low concentration of Pb²⁺, and 11.82 mol·L^?1 and 0.28 mol CO₂/(mg protein·min), respectively, at a high concentration of Pb²⁺. The spectroscopy assays suggested that the Pb²⁺ was determined to be directly bound to rubisco; the binding site of Pb²⁺ to rubisco was 1.1 and the binding constants were 8.63×10⁴ and 2.18×10⁵ L/mol. Based on the analysis of inductively coupled plasma‐mass spectrometry (ICP‐MS) and the circular dichroism (CD) spectra, it was concluded that Pb²⁺ replaced Mg²⁺ from the catalytic center in rubisco and the binding of Pb²⁺ entirely altered the primary conformation of rubisco, implying that the Pb²⁺ coordination created a new metal ion‐active site form of rubisco, thus leading to a reduction in the carboxylase activity of rubisco.