Role of a guanidinium cation-phosphodianion pair in stabilizing the vinyl carbanion intermediate of orotidine 5′-phosphate decarboxylase-catalyzed reactions

Bogdana Goryanova, Lawrence M. Goldman, Tina L. Amyes, John Alan Gerlt, John P. Richard

Research output: Contribution to journalArticle

Abstract

The side chain cation of Arg235 provides a 5.6 and 2.6 kcal/mol stabilization of the transition states for orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC) from Saccharomyces cerevisiae catalyzed reactions of OMP and 5-fluoroorotidine 5′-monophosphate (FOMP), respectively, a 7.2 kcal/mol stabilization of the vinyl carbanion-like transition state for enzyme-catalyzed exchange of the C-6 proton of 5-fluorouridine 5′-monophosphate (FUMP), but no stabilization of the transition states for enzyme-catalyzed decarboxylation of truncated substrates 1-(β-d- erythrofuranosyl)orotic acid and 1-(β-d-erythrofuranosyl) 5-fluorouracil. These observations show that the transition state stabilization results from formation of a protein cation-phosphodianion pair, and that there is no detectable stabilization from an interaction between the side chain and the pyrimidine ring of substrate. The 5.6 kcal/mol side chain interaction with the transition state for the decarboxylation reaction is 50% of the total 11.2 kcal/mol transition state stabilization by interactions with the phosphodianion of OMP, whereas the 7.2 kcal/mol side chain interaction with the transition state for the deuterium exchange reaction is a larger 78% of the total 9.2 kcal/mol transition state stabilization by interactions with the phosphodianion of FUMP. The effect of the R235A mutation on the enzyme-catalyzed deuterium exchange is expressed predominantly as a change in the turnover number k ex, whereas the effect on the enzyme-catalyzed decarboxylation of OMP is expressed predominantly as a change in the Michaelis constant Km. These results are rationalized by a mechanism in which the binding of OMP, compared with that for FUMP, provides a larger driving force for conversion of OMPDC from an inactive open conformation to a productive, active, closed conformation.

Original languageEnglish (US)
Pages (from-to)7500-7511
Number of pages12
JournalBiochemistry
Volume52
Issue number42
DOIs
StatePublished - Nov 6 2013

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Orotidine-5'-Phosphate Decarboxylase
Guanidine
Cations
Stabilization
Decarboxylation
Deuterium
Enzymes
Orotic Acid
Conformations
Carboxy-Lyases
Fluorouracil
Saccharomyces cerevisiae
Protons
Substrates
orotidylic acid
Yeast
Mutation
5-fluorouridine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Role of a guanidinium cation-phosphodianion pair in stabilizing the vinyl carbanion intermediate of orotidine 5′-phosphate decarboxylase-catalyzed reactions. / Goryanova, Bogdana; Goldman, Lawrence M.; Amyes, Tina L.; Gerlt, John Alan; Richard, John P.

In: Biochemistry, Vol. 52, No. 42, 06.11.2013, p. 7500-7511.

Research output: Contribution to journalArticle

Goryanova, Bogdana ; Goldman, Lawrence M. ; Amyes, Tina L. ; Gerlt, John Alan ; Richard, John P. / Role of a guanidinium cation-phosphodianion pair in stabilizing the vinyl carbanion intermediate of orotidine 5′-phosphate decarboxylase-catalyzed reactions. In: Biochemistry. 2013 ; Vol. 52, No. 42. pp. 7500-7511.
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abstract = "The side chain cation of Arg235 provides a 5.6 and 2.6 kcal/mol stabilization of the transition states for orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC) from Saccharomyces cerevisiae catalyzed reactions of OMP and 5-fluoroorotidine 5′-monophosphate (FOMP), respectively, a 7.2 kcal/mol stabilization of the vinyl carbanion-like transition state for enzyme-catalyzed exchange of the C-6 proton of 5-fluorouridine 5′-monophosphate (FUMP), but no stabilization of the transition states for enzyme-catalyzed decarboxylation of truncated substrates 1-(β-d- erythrofuranosyl)orotic acid and 1-(β-d-erythrofuranosyl) 5-fluorouracil. These observations show that the transition state stabilization results from formation of a protein cation-phosphodianion pair, and that there is no detectable stabilization from an interaction between the side chain and the pyrimidine ring of substrate. The 5.6 kcal/mol side chain interaction with the transition state for the decarboxylation reaction is 50{\%} of the total 11.2 kcal/mol transition state stabilization by interactions with the phosphodianion of OMP, whereas the 7.2 kcal/mol side chain interaction with the transition state for the deuterium exchange reaction is a larger 78{\%} of the total 9.2 kcal/mol transition state stabilization by interactions with the phosphodianion of FUMP. The effect of the R235A mutation on the enzyme-catalyzed deuterium exchange is expressed predominantly as a change in the turnover number k ex, whereas the effect on the enzyme-catalyzed decarboxylation of OMP is expressed predominantly as a change in the Michaelis constant Km. These results are rationalized by a mechanism in which the binding of OMP, compared with that for FUMP, provides a larger driving force for conversion of OMPDC from an inactive open conformation to a productive, active, closed conformation.",
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T1 - Role of a guanidinium cation-phosphodianion pair in stabilizing the vinyl carbanion intermediate of orotidine 5′-phosphate decarboxylase-catalyzed reactions

AU - Goryanova, Bogdana

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AU - Amyes, Tina L.

AU - Gerlt, John Alan

AU - Richard, John P.

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AB - The side chain cation of Arg235 provides a 5.6 and 2.6 kcal/mol stabilization of the transition states for orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC) from Saccharomyces cerevisiae catalyzed reactions of OMP and 5-fluoroorotidine 5′-monophosphate (FOMP), respectively, a 7.2 kcal/mol stabilization of the vinyl carbanion-like transition state for enzyme-catalyzed exchange of the C-6 proton of 5-fluorouridine 5′-monophosphate (FUMP), but no stabilization of the transition states for enzyme-catalyzed decarboxylation of truncated substrates 1-(β-d- erythrofuranosyl)orotic acid and 1-(β-d-erythrofuranosyl) 5-fluorouracil. These observations show that the transition state stabilization results from formation of a protein cation-phosphodianion pair, and that there is no detectable stabilization from an interaction between the side chain and the pyrimidine ring of substrate. The 5.6 kcal/mol side chain interaction with the transition state for the decarboxylation reaction is 50% of the total 11.2 kcal/mol transition state stabilization by interactions with the phosphodianion of OMP, whereas the 7.2 kcal/mol side chain interaction with the transition state for the deuterium exchange reaction is a larger 78% of the total 9.2 kcal/mol transition state stabilization by interactions with the phosphodianion of FUMP. The effect of the R235A mutation on the enzyme-catalyzed deuterium exchange is expressed predominantly as a change in the turnover number k ex, whereas the effect on the enzyme-catalyzed decarboxylation of OMP is expressed predominantly as a change in the Michaelis constant Km. These results are rationalized by a mechanism in which the binding of OMP, compared with that for FUMP, provides a larger driving force for conversion of OMPDC from an inactive open conformation to a productive, active, closed conformation.

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