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  • Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5'-Monophosphate Decarboxylase.
    Biochemistry, 2015
    Co-Authors: Bogdana Goryanova, Tina L. Amyes, John A. Gerlt, Shonoi A. Ming, Lawrence M. Goldman, John P Richard
    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...

  • Catalysis by Orotidine 5'-Monophosphate Decarboxylase: Effect of 5-Fluoro and 4'-Substituents on the Decarboxylation of Two-
    2014
    Co-Authors: Bogdana Goryanova, Krisztina Spong, Tina L. Amyes, John P Richard
    Abstract:

    The syntheses of two novel truncated analogs of the natural substrate Orotidine 5′-monophosphate (OMP) for Orotidine 5′-monophosphate decarboxylase (OMPDC) with enhanced reactivity towards decarboxylation are reported: 1-(β-D-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5′deoxy-5-fluoroOrotidine (5′-dFO). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of FEO (10 M −1 s −1 ) and 1-(β-D-erythrofuranosyl)orotic acid (EO, 0.026 M −1 s −1 ) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of FEO. The OMPDC-catalyzed decarboxylations of FEO and EO are both activated by exogenous phosphite dianion (HPO3 2− ), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of FEO. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of FEO is not limited by the chemical step of decarboxylation of the enzyme-bound substrate. Evidence is presented that there is a change in rate-limiting step from the chemical step of decarboxylation for the phosphite-activated reaction of EO, to closure of the phosphate gripper loop and an enzyme conformational change at the ternary E·FEO·HPO3 2− complex for the reaction of FEO. The 4′-CH3 and 4′-CH2OH groups of 5′-dFO and Orotidine, respectively, result in identical destabilizations of the transition state for the unactivated decarboxylation of 2.9 kcal/mol. By contrast, the 4′-CH3 group of 5′-dFO and the 4′-CH2OH group of Orotidine result in very different 4.7 and 8.3 kcal/mol destabilizations of the transition state for the phosphite-activated decarboxylation. Here, the destabilizing effect of the 4′-CH3 substituent at 5′-dFO is masked by the rate-limiting conformational change that depresses the third-order rate constant for the phosphite-activated reaction of the parent substrate FEO.

  • Catalysis by Orotidine 5'-monophosphate decarboxylase: effect of 5-fluoro and 4'-substituents on the decarboxylation of two-part substrates.
    Biochemistry, 2013
    Co-Authors: Bogdana Goryanova, Krisztina Spong, Tina L. Amyes, John P Richard
    Abstract:

    The syntheses of two novel truncated analogs of the natural substrate Orotidine 5′-monophosphate (OMP) for Orotidine 5′-monophosphate decarboxylase (OMPDC) with enhanced reactivity toward decarboxylation are reported: 1-(β-d-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5′-deoxy-5-fluoroOrotidine (5′-dFO). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of FEO (10 M–1 s–1) and 1-(β-d-erythrofuranosyl)orotic acid (EO, 0.026 M–1 s–1) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of FEO. The OMPDC-catalyzed decarboxylations of FEO and EO are both activated by exogenous phosphite dianion (HPO32–), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of FEO. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of FEO is not limited by the chemical step of decarboxylation of the enzyme-boun...

  • Catalysis by Orotidine 5'-monophosphate decarboxylase: effect of 5-fluoro and 4'-substituents on the decarboxylation of two-part substrates.
    Biochemistry, 2013
    Co-Authors: Bogdana Goryanova, Krisztina Spong, Tina L. Amyes, John P Richard
    Abstract:

    The syntheses of two novel truncated analogs of the natural substrate Orotidine 5'-monophosphate (OMP) for Orotidine 5'-monophosphate decarboxylase (OMPDC) with enhanced reactivity toward decarboxylation are reported: 1-(β-d-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5'-deoxy-5-fluoroOrotidine (5'-dFO). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of FEO (10 M⁻¹ s⁻¹) and 1-(β-d-erythrofuranosyl)orotic acid (EO, 0.026 M⁻¹ s⁻¹) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of FEO. The OMPDC-catalyzed decarboxylations of FEO and EO are both activated by exogenous phosphite dianion (HPO₃²⁻), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of FEO. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of FEO is not limited by the chemical step of decarboxylation of the enzyme-bound substrate. Evidence is presented that there is a change in the rate-limiting step from the chemical step of decarboxylation for the phosphite-activated reaction of EO, to closure of the phosphate gripper loop and an enzyme conformational change at the ternary E•FEO•HPO₃²⁻ complex for the reaction of FEO. The 4'-CH₃ and 4'-CH₂OH groups of 5'-dFO and Orotidine, respectively, result in identical destabilizations of the transition state for the unactivated decarboxylation of 2.9 kcal/mol. By contrast, the 4'-CH₃ group of 5'-dFO and the 4'-CH₂OH group of Orotidine result in very different 4.7 and 8.3 kcal/mol destabilizations of the transition state for the phosphite-activated decarboxylation. Here, the destabilizing effect of the 4'-CH₃ substituent at 5'-dFO is masked by the rate-limiting conformational change that depresses the third-order rate constant for the phosphite-activated reaction of the parent substrate FEO.

  • Orotidine 5′-Monophosphate Decarboxylase: Transition State Stabilization from Remote Protein–Phosphodianion Interactions
    Biochemistry, 2012
    Co-Authors: Tina L. Amyes, John A. Gerlt, Shonoi A. Ming, Lawrence M. Goldman, B. Mc Kay Wood, Bijoy J. Desai, John P Richard
    Abstract:

    Mutants of Orotidine 5′-monophosphate decarboxylase containing all possible single (Q215A, Y217F, and R235A), double, and triple substitutions of the side chains that interact with the phosphodianion group of the substrate Orotidine 5′-monophosphate have been prepared. Essentially the entire effect of these mutations on the decarboxylation of the truncated neutral substrate 1-(β-d-erythrofuranosyl)orotic acid that lacks a phosphodianion group is expressed as a decrease in the third-order rate constant for activation by phosphite dianion. The results are consistent with a model in which phosphodianion binding interactions are utilized to stabilize a rare closed enzyme form that exhibits a high catalytic activity for decarboxylation.

Tina L. Amyes - One of the best experts on this subject based on the ideXlab platform.

  • Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5'-Monophosphate Decarboxylase.
    Biochemistry, 2015
    Co-Authors: Bogdana Goryanova, Tina L. Amyes, John A. Gerlt, Shonoi A. Ming, Lawrence M. Goldman, John P Richard
    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...

  • Catalysis by Orotidine 5'-Monophosphate Decarboxylase: Effect of 5-Fluoro and 4'-Substituents on the Decarboxylation of Two-
    2014
    Co-Authors: Bogdana Goryanova, Krisztina Spong, Tina L. Amyes, John P Richard
    Abstract:

    The syntheses of two novel truncated analogs of the natural substrate Orotidine 5′-monophosphate (OMP) for Orotidine 5′-monophosphate decarboxylase (OMPDC) with enhanced reactivity towards decarboxylation are reported: 1-(β-D-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5′deoxy-5-fluoroOrotidine (5′-dFO). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of FEO (10 M −1 s −1 ) and 1-(β-D-erythrofuranosyl)orotic acid (EO, 0.026 M −1 s −1 ) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of FEO. The OMPDC-catalyzed decarboxylations of FEO and EO are both activated by exogenous phosphite dianion (HPO3 2− ), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of FEO. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of FEO is not limited by the chemical step of decarboxylation of the enzyme-bound substrate. Evidence is presented that there is a change in rate-limiting step from the chemical step of decarboxylation for the phosphite-activated reaction of EO, to closure of the phosphate gripper loop and an enzyme conformational change at the ternary E·FEO·HPO3 2− complex for the reaction of FEO. The 4′-CH3 and 4′-CH2OH groups of 5′-dFO and Orotidine, respectively, result in identical destabilizations of the transition state for the unactivated decarboxylation of 2.9 kcal/mol. By contrast, the 4′-CH3 group of 5′-dFO and the 4′-CH2OH group of Orotidine result in very different 4.7 and 8.3 kcal/mol destabilizations of the transition state for the phosphite-activated decarboxylation. Here, the destabilizing effect of the 4′-CH3 substituent at 5′-dFO is masked by the rate-limiting conformational change that depresses the third-order rate constant for the phosphite-activated reaction of the parent substrate FEO.

  • Catalysis by Orotidine 5'-monophosphate decarboxylase: effect of 5-fluoro and 4'-substituents on the decarboxylation of two-part substrates.
    Biochemistry, 2013
    Co-Authors: Bogdana Goryanova, Krisztina Spong, Tina L. Amyes, John P Richard
    Abstract:

    The syntheses of two novel truncated analogs of the natural substrate Orotidine 5′-monophosphate (OMP) for Orotidine 5′-monophosphate decarboxylase (OMPDC) with enhanced reactivity toward decarboxylation are reported: 1-(β-d-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5′-deoxy-5-fluoroOrotidine (5′-dFO). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of FEO (10 M–1 s–1) and 1-(β-d-erythrofuranosyl)orotic acid (EO, 0.026 M–1 s–1) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of FEO. The OMPDC-catalyzed decarboxylations of FEO and EO are both activated by exogenous phosphite dianion (HPO32–), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of FEO. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of FEO is not limited by the chemical step of decarboxylation of the enzyme-boun...

  • Catalysis by Orotidine 5'-monophosphate decarboxylase: effect of 5-fluoro and 4'-substituents on the decarboxylation of two-part substrates.
    Biochemistry, 2013
    Co-Authors: Bogdana Goryanova, Krisztina Spong, Tina L. Amyes, John P Richard
    Abstract:

    The syntheses of two novel truncated analogs of the natural substrate Orotidine 5'-monophosphate (OMP) for Orotidine 5'-monophosphate decarboxylase (OMPDC) with enhanced reactivity toward decarboxylation are reported: 1-(β-d-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5'-deoxy-5-fluoroOrotidine (5'-dFO). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of FEO (10 M⁻¹ s⁻¹) and 1-(β-d-erythrofuranosyl)orotic acid (EO, 0.026 M⁻¹ s⁻¹) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of FEO. The OMPDC-catalyzed decarboxylations of FEO and EO are both activated by exogenous phosphite dianion (HPO₃²⁻), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of FEO. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of FEO is not limited by the chemical step of decarboxylation of the enzyme-bound substrate. Evidence is presented that there is a change in the rate-limiting step from the chemical step of decarboxylation for the phosphite-activated reaction of EO, to closure of the phosphate gripper loop and an enzyme conformational change at the ternary E•FEO•HPO₃²⁻ complex for the reaction of FEO. The 4'-CH₃ and 4'-CH₂OH groups of 5'-dFO and Orotidine, respectively, result in identical destabilizations of the transition state for the unactivated decarboxylation of 2.9 kcal/mol. By contrast, the 4'-CH₃ group of 5'-dFO and the 4'-CH₂OH group of Orotidine result in very different 4.7 and 8.3 kcal/mol destabilizations of the transition state for the phosphite-activated decarboxylation. Here, the destabilizing effect of the 4'-CH₃ substituent at 5'-dFO is masked by the rate-limiting conformational change that depresses the third-order rate constant for the phosphite-activated reaction of the parent substrate FEO.

  • Orotidine 5′-Monophosphate Decarboxylase: Transition State Stabilization from Remote Protein–Phosphodianion Interactions
    Biochemistry, 2012
    Co-Authors: Tina L. Amyes, John A. Gerlt, Shonoi A. Ming, Lawrence M. Goldman, B. Mc Kay Wood, Bijoy J. Desai, John P Richard
    Abstract:

    Mutants of Orotidine 5′-monophosphate decarboxylase containing all possible single (Q215A, Y217F, and R235A), double, and triple substitutions of the side chains that interact with the phosphodianion group of the substrate Orotidine 5′-monophosphate have been prepared. Essentially the entire effect of these mutations on the decarboxylation of the truncated neutral substrate 1-(β-d-erythrofuranosyl)orotic acid that lacks a phosphodianion group is expressed as a decrease in the third-order rate constant for activation by phosphite dianion. The results are consistent with a model in which phosphodianion binding interactions are utilized to stabilize a rare closed enzyme form that exhibits a high catalytic activity for decarboxylation.

J A Gerlt - One of the best experts on this subject based on the ideXlab platform.

  • Orotidine 5 monophosphate decarboxylase transition state stabilization from remote protein phosphodianion interactions
    Biochemistry, 2012
    Co-Authors: Tina L. Amyes, Lawrence M. Goldman, Bijoy J. Desai, Shonoi Ming, Mc Kay B Wood, J A Gerlt, John P Richard
    Abstract:

    Mutants of Orotidine 5′-monophosphate decarboxylase containing all possible single (Q215A, Y217F, and R235A), double, and triple substitutions of the side chains that interact with the phosphodianion group of the substrate Orotidine 5′-monophosphate have been prepared. Essentially the entire effect of these mutations on the decarboxylation of the truncated neutral substrate 1-(β-d-erythrofuranosyl)orotic acid that lacks a phosphodianion group is expressed as a decrease in the third-order rate constant for activation by phosphite dianion. The results are consistent with a model in which phosphodianion binding interactions are utilized to stabilize a rare closed enzyme form that exhibits a high catalytic activity for decarboxylation.

  • omp decarboxylase phosphodianion binding energy is used to stabilize a vinyl carbanion intermediate
    Journal of the American Chemical Society, 2011
    Co-Authors: Bogdana Goryanova, Tina L. Amyes, J A Gerlt, John P Richard
    Abstract:

    Orotidine 5′-monophosphate decarboxylase (OMPDC) catalyzes the exchange for deuterium from solvent D2O of the C-6 proton of 1-(β-d-erythrofuranosyl)-5-fluorouracil (FEU), a phosphodianion truncated...

  • activation of r235a mutant Orotidine 5 monophosphate decarboxylase by the guanidinium cation effective molarity of the cationic side chain of arg 235
    Biochemistry, 2010
    Co-Authors: Shonoi A. Barnett, Tina L. Amyes, Mc Kay B Wood, J A Gerlt, John P Richard
    Abstract:

    The R235A mutation at yeast Orotidine 5′-monophosphate decarboxylase (OMPDC) results in a 1300-fold increase in Km and a 14-fold decrease in kcat for decarboxylation of Orotidine 5′-monophosphate, corresponding to a 5.8 kcal/mol destabilization of the transition state. There is strong activation of this mutant enzyme by added guanidinium cation (Gua+): 1 M Gua+ stabilizes the transition state by ca. 3 kcal/mol. This stabilization is due to the binding of Gua+ to the binary Emut·OMP complex, with a Kd of 50 mM, to form the 9-fold more reactive ternary Emut·OMP·Gua+ complex. The “effective molarity” of the cationic side chain of Arg-235 at the wild-type enzyme is calculated to be 160 M.

  • an examination of the relationship between active site loop size and thermodynamic activation parameters for Orotidine 5 monophosphate decarboxylase from mesophilic and thermophilic organisms
    Biochemistry, 2009
    Co-Authors: Krisztina Toth, Tina L. Amyes, Mc Kay B Wood, J A Gerlt, Kui K Chan, John P Richard
    Abstract:

    Closure of the active site phosphate gripper loop of Orotidine 5′-monophosphate decarboxylase from Saccharomyces cerevisiae (ScOMPDC) over the bound substrate Orotidine 5′-monophosphate (OMP) activates the bound substrate for decarboxylation by at least 104-fold [Amyes, T. L., Richard, J. P., and Tait, J. J. (2005) J. Am. Chem. Soc. 127, 15708−15709]. The 19-residue phosphate gripper loop of the mesophilic ScOMPDC is much larger than the nine-residue loop at the ortholog from the thermophile Methanothermobacter thermautotrophicus (MtOMPDC). This difference in loop size results in a small decrease in the total intrinsic phosphate binding energy of the phosphodianion group of OMP from 11.9 to 11.6 kcal/mol, along with a modest decrease in the extent of activation by phosphite dianion of decarboxylation of the truncated substrate 1-(β-d-erythrofuranosyl)orotic acid. The activation parameters ΔH⧧ and ΔS⧧ for kcat for decarboxylation of OMP are 3.6 kcal/mol and 10 cal K−1 mol−1 more positive, respectively, for...

  • dissecting the total transition state stabilization provided by amino acid side chains at Orotidine 5 monophosphate decarboxylase a two part substrate approach
    Biochemistry, 2008
    Co-Authors: Shonoi A. Barnett, Tina L. Amyes, Bryant M. Wood, J A Gerlt, John P Richard
    Abstract:

    Kinetic analysis of decarboxylation catalyzed by S154A, Q215A, and S154A/Q215A mutant yeast Orotidine 5′-monophosphate decarboxylases with Orotidine 5′-monophosphate (OMP) and with a truncated nucleoside substrate (EO) activated by phosphite dianion shows (1) the side chain of Ser-154 stabilizes the transition state through interactions with the pyrimidine rings of OMP or EO, (2) the side chain of Gln-215 interacts with the phosphodianion group of OMP or with phosphite dianion, and (3) the interloop hydrogen bond between the side chains of Ser-154 and Gln-215 orients the amide side chain of Gln-215 to interact with the phosphodianion group of OMP or with phosphite dianion.

Bryant M. Wood - One of the best experts on this subject based on the ideXlab platform.

  • dissecting the total transition state stabilization provided by amino acid side chains at Orotidine 5 monophosphate decarboxylase a two part substrate approach
    Biochemistry, 2008
    Co-Authors: Shonoi A. Barnett, Tina L. Amyes, Bryant M. Wood, J A Gerlt, John P Richard
    Abstract:

    Kinetic analysis of decarboxylation catalyzed by S154A, Q215A, and S154A/Q215A mutant yeast Orotidine 5′-monophosphate decarboxylases with Orotidine 5′-monophosphate (OMP) and with a truncated nucleoside substrate (EO) activated by phosphite dianion shows (1) the side chain of Ser-154 stabilizes the transition state through interactions with the pyrimidine rings of OMP or EO, (2) the side chain of Gln-215 interacts with the phosphodianion group of OMP or with phosphite dianion, and (3) the interloop hydrogen bond between the side chains of Ser-154 and Gln-215 orients the amide side chain of Gln-215 to interact with the phosphodianion group of OMP or with phosphite dianion.

  • Dissecting the Total Transition State Stabilization Provided by Amino Acid Side Chains at Orotidine 5′-Monophosphate Decarboxylase: A Two-Part Substrate Approach†
    Biochemistry, 2008
    Co-Authors: Shonoi A. Barnett, Tina L. Amyes, Bryant M. Wood, John A. Gerlt, John P Richard
    Abstract:

    Kinetic analysis of decarboxylation catalyzed by S154A, Q215A, and S154A/Q215A mutant yeast Orotidine 5′-monophosphate decarboxylases with Orotidine 5′-monophosphate (OMP) and with a truncated nucleoside substrate (EO) activated by phosphite dianion shows (1) the side chain of Ser-154 stabilizes the transition state through interactions with the pyrimidine rings of OMP or EO, (2) the side chain of Gln-215 interacts with the phosphodianion group of OMP or with phosphite dianion, and (3) the interloop hydrogen bond between the side chains of Ser-154 and Gln-215 orients the amide side chain of Gln-215 to interact with the phosphodianion group of OMP or with phosphite dianion.

  • formation and stability of a vinyl carbanion at the active site of Orotidine 5 monophosphate decarboxylase pka of the c 6 proton of enzyme bound ump
    Journal of the American Chemical Society, 2008
    Co-Authors: Tina L. Amyes, Bryant M. Wood, J A Gerlt, Kui K Chan, John P Richard
    Abstract:

    We report that Orotidine 5‘-monophosphate decarboxylase (OMPDC) catalyzes exchange of the C-6 proton of uridine 5‘-monophosphate (UMP) for deuterium from solvent in D2O at 25 °C and pD 7.0−9.3. Kinetic analysis of deuterium exchange gives pKa ≤ 22 for carbon deprotonation of enzyme-bound UMP, which is at least 10 units lower than that for deprotonation of an analogue of UMP in water. The observation of enzyme-catalyzed deuterium exchange via a stabilized carbanion provides convincing evidence for the decarboxylation of Orotidine 5‘-monophosphate (OMP) by OMPDC to give the same carbanion intermediate. The data show that yeast OMPDC stabilizes the bound vinyl carbanion by at least 14 kcal/mol. We conclude that OMPDC also provides substantial stabilization of the late carbanion-like transition state for the decarboxylation of OMP, and that this transition state stabilization constitutes a large fraction, but probably not all, of the enormous 1017-fold enzymatic rate acceleration.

  • product deuterium isotope effect for Orotidine 5 monophosphate decarboxylase evidence for the existence of a short lived carbanion intermediate
    Journal of the American Chemical Society, 2007
    Co-Authors: Krisztina Toth, Tina L. Amyes, Bryant M. Wood, J A Gerlt, Kui K Chan, John P Richard
    Abstract:

    A product isotope effect (PIE) of 1.0 was obtained from 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) H2O/D2O catalyzed by Orotidine 5‘-monophosphate decarboxylase. This unitary product isotope effect 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. The complete lack of selectivity for the reaction of solvent H and D that is implied by the value of PIE = 1.0 may be enforced by restricted motion of the NL3+ group of the side-chain of Lys-93 that has been proposed to protonate a vinyl carbanion intermediate.

Shonoi A. Barnett - One of the best experts on this subject based on the ideXlab platform.