Molecular dynamics simulations using a combined quantum mechanical and molecular mechanical (QM/MM) potential have been carried out to investigate the internal proton transfer equilibrium of the external aldimine species in L-dopa decarboxylase, and carbanion stabilization by the enzyme cofactor in the active site of alanine racemase. Solvent effects lower the free energy of the O-protonated PLP tautomer both in aqueous solution and in the active site, resulting a free energy difference of about - 1 kcal/mol relative to the N-protonated Schiff base in the enzyme. The external aldimine provides the dominant contribution to lowering the free energy barrier for the spontaneous decarboxylation of L-dopa in water, by a remarkable 16 kcal/mol, while the enzyme L-dopa decarboxylase further lowers the barrier by 8 kcal/mol. Kinetic isotope effects were also determined using a path integral free energy perturbation theory on the primary C-13 and the secondary H-2 substitutions. In the case of alanine racemase, if the pyridine ring is unprotonated as that in the active site, there is destabilizing contribution to the formation of the alpha-carbanion in the gas phase, although when the pyridine ring is protonated the contribution is stabilizing. In aqueous solution and in alanine racemase, the alpha-carbanion is stabilized both when the pyridine ring is protonated and unprotonated. The computational studies illustrated in this article show that combined QM/MM simulations can help provide a deeper understanding of the mechanisms of PLP-dependent enzymes. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology. (C) 2011 Published by Elsevier B.V.