OMP decarboxylase: phosphodianion binding energy is used to stabilize a vinyl carbanion intermediate

Abstract

Orotidine 5'-monophosphate decarboxylase (OMPDC) catalyzes the exchange for deuterium from solvent D(2)O of the C-6 proton of 1-(β-d-erythrofuranosyl)-5-fluorouracil (FEU), a phosphodianion truncated product analog. The deuterium exchange reaction of FEU is accelerated 1.8 × 10(4)-fold by 1 M phosphite dianion (HPO(3)(2-)). This corresponds to a 5.8 kcal/mol stabilization of the vinyl carbanion-like transition state, which is similar to the 7.8 kcal/mol stabilization of the transition state for OMPDC-catalyzed decarboxylation of a truncated substrate analog by bound HPO(3)(2-). These results show that the intrinsic binding energy of phosphite dianion is used in the stabilization of the vinyl carbanion-like transition state common to the decarboxylation and deuterium exchange reactions.

Figure 1

¹⁹F NMR spectra obtained during the OMPDC-catalyzed deuterium exchange reaction of FEU (4.2 mM) in the presence of 4.7 mM phosphite dianion in D₂O at pD 8.1 and 25 °C.

Figure 2

The dependence of (k_cat/K_m)_obsd/k_o for the OMPDC-catalyzed deuterium exchange reaction of FEU on the concentration of phosphite dianion in D₂O at pD 8.1, 25 °C and I = 0.14 (NaCl).

Figure 3

Partial free energy profiles for the unactivated and phosphite-activated OMPDC-catalyzed deuterium exchange reactions of the truncated substrate h-FEU in D₂O. The slowest step for the enzyme-catalyzed reaction is deprotonation of substrate by the alkyl amino side chain of Lys-93 to form the enzyme bound carbanion. The carbanion-like transition state is stabilized by 5.8 kcal/mol by the binding of phosphite dianion. In this figure, the barrier to formation of the enzyme-bound carbanion also includes rotation of the CH₂-ND₂H⁺ bond at Lys-93 that is required to exchange the positions of the substrate derived –H and the solvent derived –D prior to hydron transfer to form d-FEU.^,,

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