Theoretical Studies of Mechanisms and Kinetic Isotope Effects on the Decarboxylation of Orotic Acid and Derivatives

Abstract

The mechanism of orotidine 5'-monophosphate decarboxylase was studied computationally by using the decarboxylation of orotic acid analogues as model systems. These calculations indicate that mechanisms involving proton transfer to the 2-oxygen or the 4-oxygen are energetically favorable, as compared to direct decarboxylation without proton transfer, for a series of model compounds where N1 is substituted with respectively H, CH(3), and a tetrahydrofuran moiety. Proton transfer to the 4-oxygen during decarboxylation is found to be energetically more favorable than 2-protonation, which is attributable to both the 4-oxygen site being more basic and an apparent intrinsic preference for the 4-protonation pathway. (15)N isotope effect calculations were also conducted, and compared to experimental (15)N isotope effects previously measured at N1 by Rishavy and Cleland (Biochemistry 2000, 39, 4569-4574). The theoretical isotope effects establish, for the first time, that the experimental (15)N isotope effect is consistent with decarboxylation without protonation, as well as with decarboxylation with protonation, at either O2 or at O4. Furthermore, we propose herein an isotope measurement that could potentially distinguish among mechanisms involving protonation from those that do not involve proton transfer.

The acidity of uracil and uracil analogs in the gas phase: Four surprisingly acidic sites and biological implications

“…The nature of the C6 anionic intermediate resulting from decarboxylation of orotate is of course the focus of mechanistic studies-how stable is it, and how does the enzyme catalyze the reaction? We have established, through earlier computational studies, that the C6 anion may garner special stability because of its resonance Structure 9, a carbene-ylide [7,49,60]. The C5 anion, with a calculated proton affinity of 376 kcal mol Ϫ1 , is comparable in proton affinity to the conjugate base of acrolein.…”

Molecular dynamic study of orotidine-5′-monophosphate decarboxylase in ground state and in intermediate state: A role of the 203–218 loop dynamics

“…Ab initio calculations have shown that O2-or O4-protonation of OMP lowers the activation barrier for decarboxylation by 22-24 kcal͞mol in the gas phase (5). Experimentally determined normal 15 N kinetic isotope effects (KIEs) at N1 suggested no change in the bond order of N1 before decarboxylation (6), but in the following computational study, calculated 15 N KIEs at N1 were rather insensitive to the bond order change (7). Recently, a mechanism involving an enamine protonation at C5 has been proposed as the first step in the decarboxylation reaction (8).…”

“…In gas phase the proton affinity of O4 is Ϸ15 kcal͞mol higher than that of O2, but the activation energies for decarboxylation are similar when starting from either protonated OMP (7). One might conclude that the reaction path via O2(H ϩ )I Ϫ might be favored in certain environments where O2 becomes basic and the proton source is activated.…”

Path‐integral calculations of heavy atom kinetic isotope effects in condensed phase reactions using higher‐order trotter factorizations

“…Herein, three possible carbonyl protonation states were investigated: deprotonated (1MO0), O2 protonated (1MO2), and O4 [24][25][26] Gas-phase model calculations…”

“…Singleton et al [24] and Phillips and Lee [25] obtained gas-phase decarboxylation potential energy barriers using density functional theory (DFT) and second-order perturbation theory. To validate the performance of AM1 for the molecules in Scheme 2, we calculate reaction energies for the orotate decarboxylation reactions using AM1 [37] and M06/6-31þG(d,p) [42] in the gas-phase and in DMSO continuum solvent.…”