Product deuterium isotope effects for orotidine 5′-monophosphate decarboxylase

Effect of changing substrate and enzyme structure on the partitioning of the vinyl carbanion reaction intermediate

Krisztina Toth, Tina L. Amyes, Bryant M. Wood, Kui Chan, John Alan Gerlt, John P. Richard

Research output: Contribution to journalArticle

Abstract

A product deuterium isotope effect (PIE) of 1.0 was determined as the ratio of the yields of [6-1H]-uridine 5′-monophosphate (50%) and [6-2H]-uridine 5′-monophosphate (50%) from the decarboxylation of orotidine 5′-monophosphate (OMP) in 50/50 (v/v) HOH/DOD catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) from Saccharomyces cerevisiae, Methanothermobacter thermautotrophicus, and Escherichia coli. This unitary PIE eliminates a proposed mechanism for enzyme-catalyzed decarboxylation in which proton transfer from Lys-93 to C-6 of OMP provides electrophilic push to the loss of CO2 in a concerted reaction. We propose that the complete lack of selectivity for the reaction of solvent H and D, which is implied by the value of PIE = 1.0, is enforced by restricted C-N bond rotation of the -CH2-NL3 + group of the side chain of Lys-93. A smaller PIE of 0.93 was determined as the ratio of the product yields for OMPDC-catalyzed decarboxylation of 5-fluoroorotidine 5′-monophosphate (5-FOMP) in 50/50 (v/v) HOH/DOD. Mutations on the following important active-site residues of OMPDC from S. cerevisiae have no effect on the PIE on OMPDC-catalyzed decarboxylation of OMP or decarboxylation of 5-FOMP: R235A, Y217A, Q215A, S124A, and S154A/Q215A.

Original languageEnglish (US)
Pages (from-to)7018-7024
Number of pages7
JournalJournal of the American Chemical Society
Volume132
Issue number20
DOIs
StatePublished - May 26 2010

Fingerprint

Reaction intermediates
Carboxy-Lyases
Deuterium
deuterium
decarboxylation
Isotopes
Decarboxylation
partitioning
Enzymes
isotope
enzyme
substrate
Substrates
Uridine Monophosphate
Yeast
Saccharomyces cerevisiae
Proton transfer
Methanobacteriaceae
Escherichia coli
orotidylic acid

ASJC Scopus subject areas

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

Cite this

Product deuterium isotope effects for orotidine 5′-monophosphate decarboxylase : Effect of changing substrate and enzyme structure on the partitioning of the vinyl carbanion reaction intermediate. / Toth, Krisztina; Amyes, Tina L.; Wood, Bryant M.; Chan, Kui; Gerlt, John Alan; Richard, John P.

In: Journal of the American Chemical Society, Vol. 132, No. 20, 26.05.2010, p. 7018-7024.

Research output: Contribution to journalArticle

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abstract = "A product deuterium isotope effect (PIE) of 1.0 was determined as the ratio of the yields of [6-1H]-uridine 5′-monophosphate (50{\%}) and [6-2H]-uridine 5′-monophosphate (50{\%}) from the decarboxylation of orotidine 5′-monophosphate (OMP) in 50/50 (v/v) HOH/DOD catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) from Saccharomyces cerevisiae, Methanothermobacter thermautotrophicus, and Escherichia coli. This unitary PIE eliminates a proposed mechanism for enzyme-catalyzed decarboxylation in which proton transfer from Lys-93 to C-6 of OMP provides electrophilic push to the loss of CO2 in a concerted reaction. We propose that the complete lack of selectivity for the reaction of solvent H and D, which is implied by the value of PIE = 1.0, is enforced by restricted C-N bond rotation of the -CH2-NL3 + group of the side chain of Lys-93. A smaller PIE of 0.93 was determined as the ratio of the product yields for OMPDC-catalyzed decarboxylation of 5-fluoroorotidine 5′-monophosphate (5-FOMP) in 50/50 (v/v) HOH/DOD. Mutations on the following important active-site residues of OMPDC from S. cerevisiae have no effect on the PIE on OMPDC-catalyzed decarboxylation of OMP or decarboxylation of 5-FOMP: R235A, Y217A, Q215A, S124A, and S154A/Q215A.",
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AU - Richard, John P.

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