text: 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. The substrates and cofactor bind in the following order: metal cofactor (either Co, Mg, or Mn), PEP, bicarbonate (HCO). The mechanism proceeds in two major steps, as described below and shown in figure 2:
  # The bicarbonate acts as a nucleophile to attack the phosphate group in PEP. This results in the splitting of PEP into a carboxyphosphate and the (very reactive) enolate form of pyruvate.
  # Proton transfer takes place at the carboxyphosphate. This is most likely modulated by a histidine (H138) residue that first deprotonates the carboxy side, and then, as an acid, protonates the phosphate part. The carboxyphosphate then exothermically decomposes into carbon dioxide and inorganic phosphate, at this point making this an irreversible reaction. Finally, after the decomposition, the carbon dioxide is attacked by the enolate to form oxaloacetate.
  The metal cofactor is necessary to coordinate the enolate and carbon dioxide intermediates; the CO molecule is only lost 3% of the time. The active site is hydrophobic to exclude water, since the carboxyphosphate intermediate is susceptible to hydrolysis.