Phosphoenolpyruvate carboxykinase assayed at physiological concentrations of metal ions has a high affinity for CO2

The effect of Mn2+ / Mg2+ concentration on the activity of intact, homogeneous phosphoenolpyruvate carboxykinase (PEPCK) from leaves of the C-4 grass, Guinea grass (Panicum maximum), have been investigated. Assay conditions were optimized so that PEPCK activity could be measured at concentrations of Mn2+ / Mg(2+)similar to those found in the cytosol (low micromolar Mn2+, and millimolar Mg2+). PEPCK activity was totally dependent on Mn2+ and was activated at low micromolar concentrations of Mn2+ by millimolar concentrations of Mg2+. Therefore, at physiological concentrations of Mn2+, PEPCK has a requirement for Mg2+. Assay at physiological concentrations of Mn2+ / Mg(2+)led to a marked decrease in its affinity for ATP and a 13-fold increase in its affinity for CO2. The K-m (CO2) was further decreased by assay at physiological ATP to ADP ratios, reaching values as low as 20 mum CO2, comparable with the K, (CO2) of ribulose 1,5-bisphosphate carboxylase-oxygenase. This means that PEPCK will catalyze a reversible reaction and that it could operate as a carboxylase in vivo, a feature that could be particularly important in algal CO2-concentrating systems.