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 nu(max) were 1.74 mu mol.L-1 and 0.42 mu mol CO2/(mg protein-min), respectively, at a low concentration of Pb2+, and 11.82 mu mol.L-1 and 0.28 mu 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 x 10(4) and 2.18 x 10(5) 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.