Effect of Mg2+ on the structure and function of ribulose-1,5-bisphosphate carboxylase/oxygenase

Mg2+ in various concentrations was added to purified Rubisco in vitro to gain insight into the mechanism of molecular interactions between Mg2+ and Rubisco. The enzyme activity assays showed that the reaction between Rubisco and Mg2+ was two order, which means that the enhancement of Rubisco activity was accelerated by low concentration of Mg2+ and slowed by high concentration of Mg2+. The kinetics constant (K-m) and V-max was 1.91 mu M and 1.13 mu mol CO2 mg(-1) protein.min(-1), respectively, at a low concentration of Mg2+, and 3.45 mu M and 0.32 mu mol CO2.mg(-1) protein.min(-1), respectively, at a high concentration of Mg2+. By UV absorption and fluorescence spectroscopy assays, the Mg2+ was determined to be directly bound to Rubisco; the binding site of Mg2+ to Rubisco was 0.275, the binding constants (K-A) of the binding site were 6.33 x 10(4) and 5.5 x 10(4) l.mol(-1). Based on the analysis of the circular dichroism (CD) spectra, it was concluded that the binding of Mg2+ did not alter the secondary structure of Rubisco, suggesting that the observed enhancement of Rubisco carboxylase activity was caused by a subtle structural change in the active site through the formation of the complex with Mg2+.