Phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (also known as PEP carboxylase, PEPCase, or PEPC; EC 4.1.1.31, 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: Phosphoenolpyruvate carboxylase (also known as PEP carboxylase, PEPCase, or PEPC; EC 4.1.1.31, 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: This reaction is used for carbon fixation in CAM (crassulacean acid metabolism) and C4 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. The overall enzyme exists as a dimer-of-dimers: two identical subunits closely interact to form a dimer through salt bridges between arginine (R438 - exact positions may vary depending on the origin of the gene) and glutamic acid (E433) residues. 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%), and have a mass of 106kDa each. 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. The mechanism of PEP carboxylase has been well studied. The enzymatic mechanism of forming oxaloacetate is very exothermic and thereby irreversible; the biological Gibbs free energy change (△G°’) is -30kJmol−1. The substrates and cofactor bind in the following order: metal cofactor (either Co2+, Mg2+, or Mn2+), PEP, bicarbonate (HCO3−). The mechanism proceeds in two major steps, as described below and shown in figure 2: 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. The three most important roles that PEP carboxylase plays in plants and bacteria metabolism are in the C4 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 (2x3 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, plants increase the local CO2 concentration in a process called the C4 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.