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 C(4) compound oxaloacetate. This enzyme is particularly important in the context of C(4) 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 CO(2). We describe here a simple, repeatable, and gas-tight assay system for PEPC that allows bicarbonate concentrations to be controlled above ca. 50 µM.