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

Wing Yin Tsang, B. Mc Kay Wood, Freeman M. Wong, Weiming Wu, John A. Gerlt, Tina L. Amyes, John P. Richard

Research output: Contribution to journalArticle


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 kcat 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 pKa 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 pKa values for proton transfer from C-6 of uridine (pKCH = 28.8) and 5-fluorouridine (pKCH = 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 kcat/Km 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.

Original languageEnglish (US)
Pages (from-to)14580-14594
Number of pages15
JournalJournal of the American Chemical Society
Issue number35
StatePublished - Sep 5 2012


ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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