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

• Published in: Biochimica et biophysica acta Vol. 1814; no. 11; pp. 1438 - 1446
• Main Authors: Lin, Yen-Lin, Gao, Jiali, Rubinstein, Amir, Major, Dan Thomas
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-11-2011
• Subjects: active sites; alanine; Alanine Racemase - metabolism; Anions; aqueous solutions; Carbanion stabilization; carbon; decarboxylation; deuterium; Dopa Decarboxylase - metabolism; energy; Enzymatic decarboxylation; enzymes; enzymology; gases; Internal proton transfer of PLP; L-dopa; molecular dynamics; Molecular Dynamics Simulation; Protons; pyridoxal; pyridoxal phosphate; Pyridoxal Phosphate - chemistry; Pyridoxal Phosphate - metabolism; QM/MM simulation; Quantum Theory; Racemization; schiff bases; solvents; stable isotopes; Internal proton transfer of PLP; DDC; AlaR; NQE; AM1; KIE; Enzymatic decarboxylation; Racemization; QM/MM; Carbanion stabilization; PLP; QM/MM simulation; PSB; PI-FEP/UM
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2011.05.002

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
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.
► The O-protonated hydroxyimine tautomer is preferred in
l-dopa decarboxylase. ► Solvent effects play a major role in the carbanion stabilization in water and in alanine decarboxylase. ► Computed kinetic isotope effects provide insight into
l-dopa decarboxylation. ► Pyridoxal 5′-phosphate cofactor significantly lower the barrier of amino acid decarboxylation.