Molecular dynamics simulations of the intramolecular proton transfer and carbanion stabilization in the pyridoxal 5'-phosphate dependent enzymes L-dopa decarboxylase and alanine racemase.

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 (13)C and the secondary (2)H 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 α-carbanion in the gas phase, although when the pyridine ring is protonated the contribution is stabilizing. In aqueous solution and in alanine racemase, the α-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.