Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5′-Monophosphate Decarboxylase

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

Research output: Contribution to journalArticle

Abstract

The caged complex between orotidine 5′-monophosphate decarboxylase (ScOMPDC) and 5-fluoroorotidine 5′-monophosphate (FOMP) undergoes decarboxylation 300 times faster than the caged complex between ScOMPDC and the physiological substrate, orotidine 5′-monophosphate (OMP). Consequently, the enzyme conformational changes required to lock FOMP at a protein cage and release product 5-fluorouridine 5′-monophosphate (FUMP) are kinetically significant steps. The caged form of ScOMPDC is stabilized by interactions between the side chains from Gln215, Tyr217, and Arg235 and the substrate phosphodianion. The control of these interactions over the barrier to the binding of FOMP and the release of FUMP was probed by determining the effect of all combinations of single, double, and triple Q215A, Y217F, and R235A mutations on kcat/Km and kcat for turnover of FOMP by wild-type ScOMPDC; its values are limited by the rates of substrate binding and product release, respectively. The Q215A and Y217F mutations each result in an increase in kcat and a decrease in kcat/Km, due to a weakening of the protein-phosphodianion interactions that favor fast product release and slow substrate binding. The Q215A/R235A mutation causes a large decrease in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP, which are limited by the rate of the decarboxylation step, but much smaller decreases in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of FOMP, which are limited by the rate of enzyme conformational changes. By contrast, the Y217A mutation results in large decreases in kcat/Km for ScOMPDC-catalyzed decarboxylation of both OMP and FOMP, because of the comparable effects of this mutation on rate-determining decarboxylation of enzyme-bound OMP and on the rate-determining enzyme conformational change for decarboxylation of FOMP. We propose that kcat = 8.2 s-1 for decarboxylation of FOMP by the Y217A mutant is equal to the rate constant for cage formation from the complex between FOMP and the open enzyme, that the tyrosyl phenol group stabilizes the closed form of ScOMPDC by hydrogen bonding to the substrate phosphodianion, and that the phenyl group of Y217 and F217 facilitates formation of the transition state for the rate-limiting conformational change. An analysis of kinetic data for mutant enzyme-catalyzed decarboxylation of OMP and FOMP provides estimates for the rate and equilibrium constants for the conformational change that traps FOMP at the enzyme active site.

Original languageEnglish (US)
Pages (from-to)4555-4564
Number of pages10
JournalBiochemistry
Volume54
Issue number29
DOIs
StatePublished - Jul 28 2015

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Decarboxylation
Carboxy-Lyases
Equilibrium constants
Catalysis
Rate constants
Enzymes
Substrates
Mutation
Kinetic parameters
orotidylic acid
Mutation Rate
Hydrogen Bonding
Phenol
Catalytic Domain
Hydrogen bonds
Proteins
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5′-Monophosphate Decarboxylase. / Goryanova, Bogdana; Goldman, Lawrence M.; Ming, Shonoi; Amyes, Tina L.; Gerlt, John Alan; Richard, John P.

In: Biochemistry, Vol. 54, No. 29, 28.07.2015, p. 4555-4564.

Research output: Contribution to journalArticle

Goryanova, Bogdana ; Goldman, Lawrence M. ; Ming, Shonoi ; Amyes, Tina L. ; Gerlt, John Alan ; Richard, John P. / Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5′-Monophosphate Decarboxylase. In: Biochemistry. 2015 ; Vol. 54, No. 29. pp. 4555-4564.
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abstract = "The caged complex between orotidine 5′-monophosphate decarboxylase (ScOMPDC) and 5-fluoroorotidine 5′-monophosphate (FOMP) undergoes decarboxylation 300 times faster than the caged complex between ScOMPDC and the physiological substrate, orotidine 5′-monophosphate (OMP). Consequently, the enzyme conformational changes required to lock FOMP at a protein cage and release product 5-fluorouridine 5′-monophosphate (FUMP) are kinetically significant steps. The caged form of ScOMPDC is stabilized by interactions between the side chains from Gln215, Tyr217, and Arg235 and the substrate phosphodianion. The control of these interactions over the barrier to the binding of FOMP and the release of FUMP was probed by determining the effect of all combinations of single, double, and triple Q215A, Y217F, and R235A mutations on kcat/Km and kcat for turnover of FOMP by wild-type ScOMPDC; its values are limited by the rates of substrate binding and product release, respectively. The Q215A and Y217F mutations each result in an increase in kcat and a decrease in kcat/Km, due to a weakening of the protein-phosphodianion interactions that favor fast product release and slow substrate binding. The Q215A/R235A mutation causes a large decrease in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP, which are limited by the rate of the decarboxylation step, but much smaller decreases in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of FOMP, which are limited by the rate of enzyme conformational changes. By contrast, the Y217A mutation results in large decreases in kcat/Km for ScOMPDC-catalyzed decarboxylation of both OMP and FOMP, because of the comparable effects of this mutation on rate-determining decarboxylation of enzyme-bound OMP and on the rate-determining enzyme conformational change for decarboxylation of FOMP. We propose that kcat = 8.2 s-1 for decarboxylation of FOMP by the Y217A mutant is equal to the rate constant for cage formation from the complex between FOMP and the open enzyme, that the tyrosyl phenol group stabilizes the closed form of ScOMPDC by hydrogen bonding to the substrate phosphodianion, and that the phenyl group of Y217 and F217 facilitates formation of the transition state for the rate-limiting conformational change. An analysis of kinetic data for mutant enzyme-catalyzed decarboxylation of OMP and FOMP provides estimates for the rate and equilibrium constants for the conformational change that traps FOMP at the enzyme active site.",
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T1 - Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5′-Monophosphate Decarboxylase

AU - Goryanova, Bogdana

AU - Goldman, Lawrence M.

AU - Ming, Shonoi

AU - Amyes, Tina L.

AU - Gerlt, John Alan

AU - Richard, John P.

PY - 2015/7/28

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N2 - The caged complex between orotidine 5′-monophosphate decarboxylase (ScOMPDC) and 5-fluoroorotidine 5′-monophosphate (FOMP) undergoes decarboxylation 300 times faster than the caged complex between ScOMPDC and the physiological substrate, orotidine 5′-monophosphate (OMP). Consequently, the enzyme conformational changes required to lock FOMP at a protein cage and release product 5-fluorouridine 5′-monophosphate (FUMP) are kinetically significant steps. The caged form of ScOMPDC is stabilized by interactions between the side chains from Gln215, Tyr217, and Arg235 and the substrate phosphodianion. The control of these interactions over the barrier to the binding of FOMP and the release of FUMP was probed by determining the effect of all combinations of single, double, and triple Q215A, Y217F, and R235A mutations on kcat/Km and kcat for turnover of FOMP by wild-type ScOMPDC; its values are limited by the rates of substrate binding and product release, respectively. The Q215A and Y217F mutations each result in an increase in kcat and a decrease in kcat/Km, due to a weakening of the protein-phosphodianion interactions that favor fast product release and slow substrate binding. The Q215A/R235A mutation causes a large decrease in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP, which are limited by the rate of the decarboxylation step, but much smaller decreases in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of FOMP, which are limited by the rate of enzyme conformational changes. By contrast, the Y217A mutation results in large decreases in kcat/Km for ScOMPDC-catalyzed decarboxylation of both OMP and FOMP, because of the comparable effects of this mutation on rate-determining decarboxylation of enzyme-bound OMP and on the rate-determining enzyme conformational change for decarboxylation of FOMP. We propose that kcat = 8.2 s-1 for decarboxylation of FOMP by the Y217A mutant is equal to the rate constant for cage formation from the complex between FOMP and the open enzyme, that the tyrosyl phenol group stabilizes the closed form of ScOMPDC by hydrogen bonding to the substrate phosphodianion, and that the phenyl group of Y217 and F217 facilitates formation of the transition state for the rate-limiting conformational change. An analysis of kinetic data for mutant enzyme-catalyzed decarboxylation of OMP and FOMP provides estimates for the rate and equilibrium constants for the conformational change that traps FOMP at the enzyme active site.

AB - The caged complex between orotidine 5′-monophosphate decarboxylase (ScOMPDC) and 5-fluoroorotidine 5′-monophosphate (FOMP) undergoes decarboxylation 300 times faster than the caged complex between ScOMPDC and the physiological substrate, orotidine 5′-monophosphate (OMP). Consequently, the enzyme conformational changes required to lock FOMP at a protein cage and release product 5-fluorouridine 5′-monophosphate (FUMP) are kinetically significant steps. The caged form of ScOMPDC is stabilized by interactions between the side chains from Gln215, Tyr217, and Arg235 and the substrate phosphodianion. The control of these interactions over the barrier to the binding of FOMP and the release of FUMP was probed by determining the effect of all combinations of single, double, and triple Q215A, Y217F, and R235A mutations on kcat/Km and kcat for turnover of FOMP by wild-type ScOMPDC; its values are limited by the rates of substrate binding and product release, respectively. The Q215A and Y217F mutations each result in an increase in kcat and a decrease in kcat/Km, due to a weakening of the protein-phosphodianion interactions that favor fast product release and slow substrate binding. The Q215A/R235A mutation causes a large decrease in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP, which are limited by the rate of the decarboxylation step, but much smaller decreases in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of FOMP, which are limited by the rate of enzyme conformational changes. By contrast, the Y217A mutation results in large decreases in kcat/Km for ScOMPDC-catalyzed decarboxylation of both OMP and FOMP, because of the comparable effects of this mutation on rate-determining decarboxylation of enzyme-bound OMP and on the rate-determining enzyme conformational change for decarboxylation of FOMP. We propose that kcat = 8.2 s-1 for decarboxylation of FOMP by the Y217A mutant is equal to the rate constant for cage formation from the complex between FOMP and the open enzyme, that the tyrosyl phenol group stabilizes the closed form of ScOMPDC by hydrogen bonding to the substrate phosphodianion, and that the phenyl group of Y217 and F217 facilitates formation of the transition state for the rate-limiting conformational change. An analysis of kinetic data for mutant enzyme-catalyzed decarboxylation of OMP and FOMP provides estimates for the rate and equilibrium constants for the conformational change that traps FOMP at the enzyme active site.

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