An in vitro Coupled Assay for PEPC with Control of Bicarbonate Concentration

Phosphoenolpyruvate carboxylase (PEPC) catalyzes a critical step in carbon metabolism in plants and bacteria, the irreversible reaction between bicarbonate and phosphoenolpyruvate to produce the C₄ compound oxaloacetate. This enzyme is particularly important in the context of C₄ photosynthesis, where it is the initial carbon-fixing enzyme. Many studies have used kinetic approaches to characterize the properties of PEPCs from different species, different post-translational states, and after mutagenesis. Most of these studies have worked at a fixed saturating concentration of bicarbonate. Controlling the concentration of bicarbonate is difficult at low concentrations because of equilibration with atmospheric CO₂. We describe here a simple, repeatable, and gas-tight assay system for PEPC that allows bicarbonate concentrations to be controlled above ca. 50 µM.