Plant Physiol, January 2002, Vol. 128, pp. 160-164

Phosphoenolpyruvate Carboxykinase Assayed at Physiological Concentrations of Metal Ions Has a High Affinity for CO₂¹

Robert Hill Institute and Department of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 2TN, United Kingdom

The effect of Mn²⁺/Mg²⁺ concentration on the activity of intact, homogeneous phosphoenolpyruvate carboxykinase (PEPCK) from leaves of the C₄ grass, Guinea grass (Panicum maximum), have been investigated. Assay conditions were optimized so that PEPCK activity could be measured at concentrations of Mn²⁺/Mg²⁺ 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⁺/Mg²⁺ 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 µM CO₂, comparable with the K_m (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.