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

摘要

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 ℃ was monitored by 'H NMR spectroscopy. Deuterium exchange proceeds by proton transfer from C-6 of the bound nudeotide 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 × 10~9-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-cataryzed deuterium exchange of 3 × 10~(10) 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.