Phosphoenolpyruvate carboxylase

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. This metal ion can be magnesium, manganese or cobalt depending on the organism, and its role is to coordinate the phosphoenolpyruvate molecule as well as the reaction intermediates. A histidine (H138) residue at the active site is believed to facilitate proton transfer during the catalytic mechanism. ==Enzyme mechanism==

The mechanism of PEP carboxylase has been well studied. The enzymatic mechanism of forming oxaloacetate is very exergonic, and thereby irreversible, in biochemical standard conditions; the biological standard Gibbs free energy change (∆G°’) is −30 kJ⋅mol−1. The metal cofactor is necessary to coordinate the enolate and carbon dioxide intermediates; the CO2 molecule is only lost 3% of the time. The active site is hydrophobic to exclude water, since the carboxyphosphate intermediate is susceptible to hydrolysis. == Function ==

The three most important roles that PEP carboxylase plays in plants and bacteria metabolism are in the cycle, the CAM cycle, and the citric acid cycle biosynthesis flux. The primary mechanism of carbon dioxide assimilation in plants is through the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (also known as RuBisCO), that adds CO2 to ribulose-1,5-bisphosphate (a 5 carbon sugar), to form two molecules of 3-phosphoglycerate (2×3 carbon sugars). However, at higher temperatures and lower CO2 concentrations, RuBisCO adds oxygen instead of carbon dioxide, to form the unusable product glycolate in a process called photorespiration. To prevent this wasteful process, some plants increase the local CO2 concentration in a process called the cycle. 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. ==Regulation==

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. == References ==