Phosphoenolpyruvate carboxylase

Class of enzymes

Phosphoenolpyruvate carboxylase (also known as PEP carboxylase, PEPCase, or PEPC; , PDB ID: 3ZGE) is an enzyme in the family of carboxy lyases found in plants and some bacteria that catalyzes the addition of bicarbonate (HCO3−) to phosphoenolpyruvate (PEP) to form the four carbon compound oxaloacetate and inorganic phosphate:

PEP + HCO3− → oxaloacetate + Pi

This reaction is used for carbon fixation in CAM (crassulacean acid metabolism) and organisms, as well as to regulate flux through the citric acid cycle (also known as Krebs or TCA cycle) in bacteria and plants. The enzyme structure and its two step catalytic, irreversible mechanism have been well studied. PEP carboxylase is highly regulated, both by phosphorylation and allostery.

The PEP carboxylase enzyme is present in plants and some types of bacteria, but not in fungi or animals (including humans). The genes vary between organisms, but are strictly conserved around the active and allosteric sites discussed in the mechanism and regulation sections. Tertiary structure of the enzyme is also conserved. The crystal structure of PEP carboxylase in multiple organisms, including Zea mays (maize), and Escherichia coli has been determined. This dimer assembles (more loosely) with another of its kind to form the four subunit complex. The monomer subunits are mainly composed of alpha helices (65%), The sequence length is about 966 amino acids. The enzyme active site is not completely characterized. It includes a conserved aspartic acid (D564) and a glutamic acid (E566) residue that non covalently bind a divalent metal cofactor ion through the carboxylic acid functional groups. The metal cofactor is necessary to coordinate the enolate and carbon dioxide intermediates; the CO2 molecule is only lost 3% of the time. PEP carboxylase plays the key role of binding CO2 in the form of bicarbonate with PEP to create oxaloacetate in the mesophyll tissue. This is then converted back to pyruvate (through a malate intermediate), to release the CO2 in the deeper layer of bundle sheath cells for carbon fixation by RuBisCO and the Calvin cycle. Pyruvate is converted back to PEP in the mesophyll cells, and the cycle begins again, thus actively pumping CO2. The second important and very similar biological significance of PEP carboxylase is in the CAM cycle. This cycle is common in organisms living in arid habitats. Plants cannot afford to open stomata during the day to take in CO2, as they would lose too much water by transpiration. Instead, stomata open at night, when water evaporation is minimal, and take in CO2 by fixing with PEP to form oxaloacetate though PEP carboxylase. Oxaloacetate is converted to malate by malate dehydrogenase, and stored for use during the day when the light dependent reaction generates energy (mainly in the form of ATP) and reducing equivalents such as NADPH to run the Calvin cycle.

PEP carboxylase is mainly subject to two levels of regulation: phosphorylation and allostery. Figure 3 shows a schematic of the regulatory mechanism. Phosphorylation by phosphoenolpyruvate carboxylase kinase turns the enzyme on, whereas phosphoenolpyruvate carboxylase phosphatase turns it back off. Both kinase and phosphatase are regulated by transcription. It is further believed that malate acts as a feedback inhibitor of kinase expression levels, and as an activator for phosphatase expression (transcription). Since oxaloacetate is converted to malate in CAM and organisms, high concentrations of malate activate phosphatase expression the phosphatase subsequently de phosphorylates and thus de actives PEP carboxylase, leading to no further accumulation of oxaloacetate and thus no further conversion of oxaloacetate to malate. Hence malate production is down regulated. and fructose 1,6 bisphosphate (F 1,6 BP). Studies have shown that energy equivalents such as AMP, ADP and ATP have no significant effect on PEP carboxylase. The magnitudes of the allosteric effects of these different molecules on PEP carboxylase activity depend on individual organisms.