Background

The enzyme phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) catalyzes the essentially irreversible reaction between bicarbonate and phosphoenolpyruvate (PEP) to form oxaloacetate and inorganic phosphate. This reaction is a critical step in carbon metabolism in plants and bacteria, but the enzyme is most widely studied in the context of its critical role in C₄ photosynthesis, where it is responsible for primary carbon fixation. In typical PEPC assays, the enzyme is coupled to malate dehydrogenase (MDH), which converts oxaloacetate to malate, consuming NADH, and leading to a decrease in absorbance at 340 nm. This reliable coupled assay has been used extensively to compare PEPCs from different species and to understand the consequences of post-translational modification on inhibitor binding and affinity for PEP (Janc et al., 1992; Duff et al., 1995; Blasing et al., 2000; Jacobs et al., 2008; Paulus et al., 2013). Studies where the concentration of bicarbonate is varied are more challenging, as background bicarbonate, arising from equilibration with atmospheric CO₂, is found at concentrations above the K_m for bicarbonate (often less than 100 µM), preventing accurate kinetic measurements. Measuring the rate of the PEPC reaction at low bicarbonate concentrations requires that background bicarbonate be removed as much as possible, and careful gas-tight assay procedures. Accurate kinetic measurements also require determining the concentration of residual bicarbonate, allowing calculation of the correct concentration of substrate.

We describe here a set of methods to remove most background bicarbonate, quantify the remaining amount, and reliably assay the enzyme without significant contamination from atmospheric CO₂. Using these methods, we can reliably reduce background bicarbonate to less than 50 µM.

Overview: The methods described here are simple and repeatable. The major assay components, buffer and water, are sparged with nitrogen gas to reduce the background bicarbonate concentration. Assays are constructed and sealed under nitrogen to minimize contamination from atmospheric carbon dioxide. Assays are then initiated by the addition of PEPC, delivered with a gastight syringe. Background bicarbonate is determined using an endpoint assay.

Limitations: While we have found these methods suitable for measuring the properties of a wide range of PEPC enzymes (Bauwe, 1986; Phansopa et al., 2020), they are not universally applicable. For PEPC enzymes with K_m^bicarbonate much below the residual background bicarbonate, the experimental design and data analysis described here are not suitable, so an alternative approach using the integrated Michaelis-Menten equation is recommended. This alternative approach requires additional controls to overcome problems associated with product inhibition, enzyme instability, and product instability leading to regeneration of CO₂ in solution; these are not described here and the interested reader should consult the careful work of Bauwe (1986), and DiMario and Cousins (2019).

Also, as the product oxaloacetate is converted into malate in the assay described here, it is not possible to carry out product inhibition studies using oxaloacetate. This limitation could be resolved by detecting a substrate or the other product. An elegant alternative detection system for gaseous CO₂, coupled to the substrate concentration through carbonic anhydrase, has been described using membrane-inlet mass spectrometry (DiMario and Cousins, 2019). Our attempts to develop an alternative assay to monitor the production of inorganic phosphate using the purine nucleotide phosphorylase assay (Webb, 1992) were unsuccessful, due to inhibition of PEPC by components of the coupling system (unpublished work).