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

Mg(2+) in various concentrations was added to purified Rubisco in vitro to gain insight into the mechanism of molecular interactions between Mg(2+) and Rubisco. The enzyme activity assays showed that the reaction between Rubisco and Mg(2+) was two order, which means that the enhancement of Rubisco activity was accelerated by low concentration of Mg(2+) and slowed by high concentration of Mg(2+). The kinetics constant (K (m)) and V (max) was 1.91 microM and 1.13 micromol CO(2) mg(-1) protein.min(-1), respectively, at a low concentration of Mg(2+), and 3.45 microM and 0.32 micromol CO(2)mg(-1) protein.min(-1), respectively, at a high concentration of Mg(2+). By UV absorption and fluorescence spectroscopy assays, the Mg(2+) was determined to be directly bound to Rubisco; the binding site of Mg(2+) 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 Mg(2+) 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 Mg(2+).