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 Alan Gerlt, Tina L. Amyes, John P. Richard

Research output: Contribution to journalArticle

Abstract

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 1 H 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 × 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-catalyzed 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.

Original languageEnglish (US)
Pages (from-to)14580-14594
Number of pages15
JournalJournal of the American Chemical Society
Volume134
Issue number35
DOIs
StatePublished - Sep 5 2012

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Uridine Monophosphate
Proton transfer
Carboxy-Lyases
deuterium
Deuterium
Protons
Enzymes
enzyme
Nucleotides
stabilization
Stabilization
Rate constants
Ion exchange
fold
decarboxylation
Cats
Equilibrium constants
orotidylic acid
effect
Decarboxylation

ASJC Scopus subject areas

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

Cite this

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

In: Journal of the American Chemical Society, Vol. 134, No. 35, 05.09.2012, p. 14580-14594.

Research output: Contribution to journalArticle

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abstract = "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 1 H 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 × 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-catalyzed 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.",
author = "Tsang, {Wing Yin} and Wood, {B. Mc Kay} and Wong, {Freeman M.} and Weiming Wu and Gerlt, {John Alan} and Amyes, {Tina L.} and Richard, {John P.}",
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T1 - Proton transfer from C-6 of uridine 5′-monophosphate catalyzed by orotidine 5′-monophosphate decarboxylase

T2 - Formation and stability of a vinyl carbanion intermediate and the effect of a 5-fluoro substituent

AU - Tsang, Wing Yin

AU - Wood, B. Mc Kay

AU - Wong, Freeman M.

AU - Wu, Weiming

AU - Gerlt, John Alan

AU - Amyes, Tina L.

AU - Richard, John P.

PY - 2012/9/5

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N2 - 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 1 H 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 × 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-catalyzed 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.

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