Proton transfer from C-6 of uridine 5′-monophosphate catalyzed by orotidine 5′-monophosphate decarboxylase: Formation and stability of a vinyl carbanion intermediate and the effect of a 5-fluoro substituent

The exchange for deuterium of the C-6 protons of uridine 5′-monophosphate (UMP) and 5-fluorouridine 5′-monophosphate (F-UMP) catalyzed by yeast orotidine 5′-monophosphate decarboxylase (ScOMPDC) at pD 6.5-9.3 and 25 °C was monitored by ¹H NMR spectroscopy. Deuterium exchange proceeds by proton transfer from C-6 of the bound nucleotide to the deprotonated side chain of Lys-93 to give the enzyme-bound vinyl carbanion. The pD-rate profiles for k_cat give turnover numbers for deuterium exchange into enzyme-bound UMP and F-UMP of 1.2 × 10 ^-5 and 0.041 s^-1, respectively, so that the 5-fluoro substituent results in a 3400-fold increase in the first-order rate constant for deuterium exchange. The binding of UMP and F-UMP to ScOMPDC results in 0.5 and 1.4 unit decreases, respectively, in the pK_a of the side chain of the catalytic base Lys-93, showing that these nucleotides bind preferentially to the deprotonated enzyme. We also report the first carbon acid pK_a values for proton transfer from C-6 of uridine (pK_CH = 28.8) and 5-fluorouridine (pK_CH = 25.1) in aqueous solution. The stabilizing effects of the 5-fluoro substituent on C-6 carbanion formation in solution (5 kcal/mol) and at ScOMPDC (6 kcal/mol) are similar. The binding of UMP and F-UMP to ScOMPDC results in a greater than 5 × 10⁹-fold increase in the equilibrium constant for proton transfer from C-6, so that ScOMPDC stabilizes the bound vinyl carbanions, relative to the bound nucleotides, by at least 13 kcal/mol. The pD-rate profile for k_cat/K_m for deuterium exchange into F-UMP gives the intrinsic second-order rate constant for exchange catalyzed by the deprotonated enzyme as 2300 M^-1 s ^-1. This was used to calculate a total rate acceleration for ScOMPDC-catalyzed deuterium exchange of 3 × 10¹⁰ M ^-1, which corresponds to a transition-state stabilization for deuterium exchange of 14 kcal/mol. We conclude that a large portion of the total transition-state stabilization for the decarboxylation of orotidine 5′-monophosphate can be accounted for by stabilization of the enzyme-bound vinyl carbanion intermediate of the stepwise reaction.