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

Abstract

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.

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity

“…7,23−25 Several groups have put forward the hypothesis that in certain cases the intermediate that forms is not a true quinonoid, but rather a carbanionic species—the chief distinction again being whether the pyridine ring nitrogen is protonated and can thus support significant quinone character. 7,26−28 This hypothesis is supported by structural studies of several PLP-dependent enzymes that find hydrogen-bonding interactions between the pyridine ring nitrogen and side chains of polar, nonacidic residues, such as serine, and positively charged residues, such as arginine.…”

Enzymatic Kinetic Isotope Effects from Path-Integral Free Energy Perturbation Theory

“…This latter point is especially important to enhance convergence by avoiding being trapped in a local minimum of the classical potential in the path-integral sampling. Using this sampling technique, the PI-FEP method has been applied to a range of condensed-phase and enzymatic systems (Fan, Cembran, Ma, & Gao, 2013; Gao, Wong, & Major, 2008; Lin, Gao, Rubinstein, & Major, 2011; Major & Gao, 2005, 2007; Major et al, 2006, 2009, 2005). …”

Nuclear quantum effects and kinetic isotope effects in enzyme reactions

“…This greatly improved the accuracy of the computed KIE, and has been used extensively since [8,42,43,[88][89][90]92,[100][101][102]. Others have also adopted mass-perturbation techniques for PI KIE in the study of small molecules [103,104].…”

“…A non-exhaustive list of such enzyme studies includes dihydrofolate reductase (DHFR) [41,46,56,90,102,[110][111][112][113][114], alcohol dehydrogenase (ADH) [50,83,115,116], lipoxygenase [55,59,[117][118][119], methylamine dehydrogenase [115,[120][121][122][123][124], thymidylate synthase [125,126], chorismate mutase [127][128][129], several decarboxylases [43,100,101,130], methylmalonyl-CoA mutase [131,132], alanine racemase [39,100,133], formate dehydrogenase [42,134], nitroalkane oxidase [8,108,135], morphinone reductase [116], and glyoxalase [134,136]. In the following we will focus on some examples of work performed in our group, with which ...…”