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

Yen-Lin Lin, Jiali Gao, Amir Rubinstein, Dan Thomas Major
Biochimica et biophysica acta 2011 v.1814 no.11 pp. 1438-1446
L-dopa, active sites, alanine, aqueous solutions, carbon, decarboxylation, deuterium, energy, enzymes, enzymology, gases, molecular dynamics, pyridoxal, pyridoxal phosphate, schiff bases, solvents, stable isotopes
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 −1kcal/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 16kcal/mol, while the enzyme l-dopa decarboxylase further lowers the barrier by 8kcal/mol. Kinetic isotope effects were also determined using a path integral free energy perturbation theory on the primary ¹³C and the secondary ²H 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.