Binding Sites of Bicarbonate in PEP Carboxylase

PhosphoEnolPyruvate Carboxylase is used in plant metabolism for fruit maturation or seed development, as well as in the C4 and CAM mechanisms in photosynthesis where it is used for the capture of hydrated CO2 (bicarbonate). To find the yet unknown binding site of bicarbonate in this enzyme, we have first identified putative binding sites with non-equilibrium molecular dynamics simulations, and then ranked these sites with alchemical free energy calculations with corrections of computational artefacts. 14 pockets where bicarbonate could bind were identified, with three having realistic binding free energies with differences with the experimental value below 1 kcal/mol. One of these pockets is found far from the active site at 14 Å, whereas in the two others bicarbonate is in direct interaction with the magnesium ion. The study of mutant K606N allowed to discriminate between these two pockets and to identify the binding site of bicarbonate in PhosphoEnolPyruvate Carboxylase.