Orotic acid decarboxylation in water and nonpolar solvents: a potential role for desolvation in the action of OMP decarboxylase

Abstract

OMP decarboxylase (ODCase) generates a very large rate enhancement without the assistance of metals or other cofactors. The uncatalyzed decarboxylation of 1-methylorotate in water is shown to involve the monoanion, although uncharged 1-methylorotic acid is decarboxylated at a similar rate. To measure the extent to which the rate of the nonenzymatic decarboxylation of orotate derivatives might be enhanced by their removal from solvent water, the 1-phosphoribosyl moiety of OMP was replaced with 1-substituents that would allow it to enter less polar solvents. When the tetrabutylammonium salt of 1-cyclohexylorotate was transferred from water to a series of dipolar aprotic solvents, its rate of decarboxylation increased markedly, varying with the relative ability of each solvent to release the substrate in the ground state from stabilization by solvent water acting as a proton donor. These findings are consistent with the view that separation of the substrate from solvent water may contribute, at least to a limited extent, to the rate enhancement produced by ODCase. This enzyme's active site, like that of another cofactorless enzyme recently shown to produce a rate enhancement similar in magnitude (uroporphyrinogen decarboxylase), is equipped with an ammonium group positioned in such a way as to balance the electrostatic charge of the carboxylate group of the substrate and later supply a proton to the incipient carbanion in a relatively waterless environment.

Figure 1

Effects of pH on log k (s⁻¹) for decarboxylation of 1-MeO at 160°C in various buffers (see text). Data were obtained using quartz tubes or Teflon cups in steel bombs . The solid line is calculated for the equation: $$\log \mathrm{k}=5.1\left[1\text{-}{\mathrm{MeO}}^{-}\right]-6.3\left[1\text{-}{\mathrm{MeO}}^{-2}\right]$$ where the 1-MeO⁻ is the monoanion and 1-MeO⁻² is the dianion of 1-methylorotate and the pK_a value of 1-MeO is 9.8.

Figure 2

Rate constants (log k, s⁻¹) for the decarboxylation of orotic acid derivatives in potassium phosphate buffer (0.1 M, pH 7.0), plotted as a function of the reciprocal of absolute temperature.

Figure 3

Rate constants (log k, s⁻¹) for the decarboxylation of 1-ChxO - TBA salt in various solvents, compared with 1-ChxO in potassium phosphate buffer (0.1 M, pH 7), plotted as a function of the reciprocal of absolute temperature.

Figure 4

¹H NMR spectra of the C6 and C5 protons of 1-ChxU arising from the decarboxylation of 1-ChxO conducted in three solvents. The top three samples were diluted in D₂O, while the fourth was diluted in DMSO-d₆.When traces of H₂O were present in these reactions, two doublets (J = 7.9 Hz) were produced when the C6 carbanion extracted a proton from water. In acetone-d₆, the C6-carbanion extracted a deuteron from the solvent, leaving only the C5 proton resonance as a singlet. Acetonitrile-d₃ and “dry” DMSO showed evidence of both reactions. In DMSO-d₆, the resonances were shifted upfield from their positions in D₂O.

Figure 5

The decarboxylation of 1-methylorotate , the decarboxylation of 3-carboxybenzisoxazole and the reaction of NaN₃ with 4-fluoro-nitrobenzene. These reactions are characterized by delocalization of charge in the transition state.

Figure 6

Proposed similarity of interaction of the ammonium groups of Lys-93 in ODCase and the Arg-37 of Urogen III Decarboxylase with the carboxylate groups of their respective OMP and urogen III substrates.