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John P Richard - One of the best experts on this subject based on the ideXlab platform.

  • orotidine 5 monophosphate decarboxylase the operation of active site chains within and across protein subunits
    Biochemistry, 2020
    Co-Authors: Tiago A S Brandao, John P Richard
    Abstract:

    The D37 and T100' side chains of orotidine 5'-monophosphate decarboxylase (OMPDC) interact with the C-3' and C-2' ribosyl hydroxyl groups, respectively, of the bound substrate. We compare the intra-subunit interactions of D37 with the inter-subunit interactions of T100' by determining the effects of the D37G, D37A, T100'G, and T100'A substitutions on the following: (a) Kcat and Kcat/Km values for the OMPDC-catalyzed decarboxylations of OMP and 5-fluoroorotidine 5'-monophosphate (FOMP) and (b) the stability of dimeric OMPDC relative to the monomer. The D37G and T100'A substitutions resulted in 2 kcal mol-1 increases in ΔG† for Kcat/Km for the decarboxylation of OMP, while the D37A and T100'G substitutions resulted in larger 4 and 5 kcal mol-1 increases, respectively, in ΔG†. The D37G and T100'A substitutions both resulted in smaller 2 kcal mol-1 decreases in ΔG† for the decarboxylation of FOMP compared to that of OMP. These results show that the D37G and T100'A substitutions affect the barrier to the chemical decarboxylation step while the D37A and T100'G substitutions also affect the barrier to a slow, ligand-driven enzyme conformational change. Substrate binding induces the movement of an α-helix (G'98-S'106) toward the substrate C-2' ribosyl hydroxy bound at the main subunit. The T100'G substitution destabilizes the enzyme dimer by 3.5 kcal mol-1 compared to the monomer, which is consistent with the known destabilization of α-helices by the internal Gly side chains [Serrano, L., et al. (1992) Nature, 356, 453-455]. We propose that the T100'G substitution weakens the α-helical contacts at the dimer interface, which results in a decrease in the dimer stability and an increase in the barrier to the ligand-driven conformational change.

  • the organization of active site side chains of glycerol 3 phosphate dehydrogenase promotes efficient enzyme catalysis and rescue of variant enzymes
    Biochemistry, 2020
    Co-Authors: Judith R Cristobal, Archie C Reyes, John P Richard
    Abstract:

    A comparison of the values of Kcat/Km for reduction of dihydroxyacetone phosphate (DHAP) by NADH catalyzed by wild type and K120A/R269A variant glycerol-3-phosphate dehydrogenase from human liver (hlGPDH) shows that the transition state for enzyme-catalyzed hydride transfer is stabilized by 12.0 kcal/mol by interactions with the cationic K120 and R269 side chains. The transition state for the K120A/R269A variant-catalyzed reduction of DHAP is stabilized by 1.0 and 3.8 kcal/mol for reactions in the presence of 1.0 M EtNH3+ and guanidinium cation (Gua+), respectively, and by 7.5 kcal/mol for reactions in the presence of a mixture of each cation at 1.0 M, so that the transition state stabilization by the ternary E·EtNH3+·Gua+ complex is 2.8 kcal/mol greater than the sum of stabilization by the respective binary complexes. This shows that there is cooperativity between the paired activators in transition state stabilization. The effective molarities (EMs) of ∼50 M determined for the K120A and R269A side chains are ≪106 M, the EM for entropically controlled reactions. The unusually efficient rescue of the activity of hlGPDH-catalyzed reactions by the HPi/Gua+ pair and by the Gua+/EtNH3+ activator pair is due to stabilizing interactions between the protein and the activator pieces that organize the K120 and R269 side chains at the active site. This "preorganization" of side chains promotes effective catalysis by hlGPDH and many other enzymes. The role of the highly conserved network of side chains, which include Q295, R269, N270, N205, T264, K204, D260, and K120, in catalysis is discussed.

  • enzyme architecture breaking down the catalytic cage that activates orotidine 5 monophosphate decarboxylase for catalysis
    Journal of the American Chemical Society, 2018
    Co-Authors: Archie C Reyes, Tina L. Amyes, John A Gerlt, David C. Plache, Astrid P. Koudelka, John P Richard
    Abstract:

    We report the results of a study of the catalytic role of a network of four interacting amino acid side chains at yeast orotidine 5′-monophosphate decarboxylase (ScOMPDC), by the stepwise replacement of all four side chains. The H-bond, which links the −CH2OH side chain of S154 from the pyrimidine umbrella loop of ScOMPDC to the amide side chain of Q215 in the phosphodianion gripper loop, creates a protein cage for the substrate OMP. The role of this interaction in optimizing transition state stabilization from the dianion gripper side chains Q215, Y217, and R235 was probed by determining the kinetic parameter Kcat/Km for 16 enzyme variants, which include all combinations of single, double, triple, and quadruple S154A, Q215A, Y217F, and R235A mutations. The effects of consecutive Q215A, Y217F, and R235A mutations on ΔG⧧ for wild-type enzyme-catalyzed decarboxylation sum to 11.6 kcal/mol, but to only 7.6 kcal/mol when starting from S154A mutant. This shows that the S154A mutation results in a (11.6–7.6) = ...

  • primary deuterium kinetic isotope effects a probe for the origin of the rate acceleration for hydride transfer catalyzed by glycerol 3 phosphate dehydrogenase
    Biochemistry, 2018
    Co-Authors: Archie C Reyes, Tina L. Amyes, John P Richard
    Abstract:

    Abstract. The following primary deuterium kinetic isotope effects (1o DKIEs) on Kcat/Km are reported for wildtype and mutant glycerol-3-phosphate dehydrogenase (GPDH)-catalyzed reduction of DHAP by NADL (L = H, D) over an 11.5 kcal/mol range of activation barriers: wildtype GPDH, ΔΔG‡ = 0 kcal/mol, 1o DKIE = 1.5; N270A, 5.6 kcal/mole, 3.1; R269A, 9.1 kcal/mol, 2.8; R269A + 1.0 M guanidine, 2.4 kcal/mol, 2.7; R269A/N270A, 11.5 kcal/mol, 2.4. Similar 1o DKIEs were observed on Kcat. The narrow range of 1o DKIEs (2.4 – 3.1) observed for a 9.1 kcal/mol change in reaction driving force provides strong evidence that these are intrinsic 1o DKIE on rate determining hydride transfer. Evidence is presented that the intrinsic DKIE on wildtype GPDH-catalyzed reduction of DHAP lies in this range. A similar range of 1o DKIE (2.4 – 2.9) on (Kcat/KGA, M -1 s -1 ) was reported for dianion activated hydride transfer from NADL to glycolaldehyde (GA) [J. Am. Chem. Soc. 2016, 138, 14526-14529]. These 1o DKIEs are much smaller ...

  • Enzyme Architecture: Breaking Down the Catalytic Cage that Activates Orotidine 5′-Monophosphate Decarboxylase for Catalysis
    2018
    Co-Authors: Archie C. Reyes, Tina L. Amyes, John A Gerlt, David C. Plache, Astrid P. Koudelka, John P Richard
    Abstract:

    We report the results of a study of the catalytic role of a network of four interacting amino acid side chains at yeast orotidine 5′-monophosphate decarboxylase (ScOMPDC), by the stepwise replacement of all four side chains. The H-bond, which links the −CH2OH side chain of S154 from the pyrimidine umbrella loop of ScOMPDC to the amide side chain of Q215 in the phosphodianion gripper loop, creates a protein cage for the substrate OMP. The role of this interaction in optimizing transition state stabilization from the dianion gripper side chains Q215, Y217, and R235 was probed by determining the kinetic parameter Kcat/Km for 16 enzyme variants, which include all combinations of single, double, triple, and quadruple S154A, Q215A, Y217F, and R235A mutations. The effects of consecutive Q215A, Y217F, and R235A mutations on ΔG⧧ for wild-type enzyme-catalyzed decarboxylation sum to 11.6 kcal/mol, but to only 7.6 kcal/mol when starting from S154A mutant. This shows that the S154A mutation results in a (11.6–7.6) = 4.0 kcal/mol decrease in transition state stabilization from interactions with Q215, Y217, and R235. Mutant cycles show that ca. 2 kcal/mol of this 4 kcal/mol effect is from the direct interaction between the S154 and Q215 side chains and that ca. 2 kcal/mol is from a tightening in the stabilizing interactions of the Y217 and R235 side chains. The sum of the effects of individual A154S, A215Q, F217Y and A235R substitutions at the quadruple mutant of ScOMPDC to give the corresponding triple mutants, 5.6 kcal/mol, is much smaller than 16.0 kcal/mol, the sum of the effects of the related four substitutions in wild-type ScOMPDC to give the respective single mutants. The small effect of substitutions at the quadruple mutant is consistent with a large entropic cost to holding the flexible loops of ScOMPDC in the active closed conformation

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

  • enzyme architecture breaking down the catalytic cage that activates orotidine 5 monophosphate decarboxylase for catalysis
    Journal of the American Chemical Society, 2018
    Co-Authors: Archie C Reyes, Tina L. Amyes, John A Gerlt, David C. Plache, Astrid P. Koudelka, John P Richard
    Abstract:

    We report the results of a study of the catalytic role of a network of four interacting amino acid side chains at yeast orotidine 5′-monophosphate decarboxylase (ScOMPDC), by the stepwise replacement of all four side chains. The H-bond, which links the −CH2OH side chain of S154 from the pyrimidine umbrella loop of ScOMPDC to the amide side chain of Q215 in the phosphodianion gripper loop, creates a protein cage for the substrate OMP. The role of this interaction in optimizing transition state stabilization from the dianion gripper side chains Q215, Y217, and R235 was probed by determining the kinetic parameter Kcat/Km for 16 enzyme variants, which include all combinations of single, double, triple, and quadruple S154A, Q215A, Y217F, and R235A mutations. The effects of consecutive Q215A, Y217F, and R235A mutations on ΔG⧧ for wild-type enzyme-catalyzed decarboxylation sum to 11.6 kcal/mol, but to only 7.6 kcal/mol when starting from S154A mutant. This shows that the S154A mutation results in a (11.6–7.6) = ...

  • primary deuterium kinetic isotope effects a probe for the origin of the rate acceleration for hydride transfer catalyzed by glycerol 3 phosphate dehydrogenase
    Biochemistry, 2018
    Co-Authors: Archie C Reyes, Tina L. Amyes, John P Richard
    Abstract:

    Abstract. The following primary deuterium kinetic isotope effects (1o DKIEs) on Kcat/Km are reported for wildtype and mutant glycerol-3-phosphate dehydrogenase (GPDH)-catalyzed reduction of DHAP by NADL (L = H, D) over an 11.5 kcal/mol range of activation barriers: wildtype GPDH, ΔΔG‡ = 0 kcal/mol, 1o DKIE = 1.5; N270A, 5.6 kcal/mole, 3.1; R269A, 9.1 kcal/mol, 2.8; R269A + 1.0 M guanidine, 2.4 kcal/mol, 2.7; R269A/N270A, 11.5 kcal/mol, 2.4. Similar 1o DKIEs were observed on Kcat. The narrow range of 1o DKIEs (2.4 – 3.1) observed for a 9.1 kcal/mol change in reaction driving force provides strong evidence that these are intrinsic 1o DKIE on rate determining hydride transfer. Evidence is presented that the intrinsic DKIE on wildtype GPDH-catalyzed reduction of DHAP lies in this range. A similar range of 1o DKIE (2.4 – 2.9) on (Kcat/KGA, M -1 s -1 ) was reported for dianion activated hydride transfer from NADL to glycolaldehyde (GA) [J. Am. Chem. Soc. 2016, 138, 14526-14529]. These 1o DKIEs are much smaller ...

  • Enzyme Architecture: Breaking Down the Catalytic Cage that Activates Orotidine 5′-Monophosphate Decarboxylase for Catalysis
    2018
    Co-Authors: Archie C. Reyes, Tina L. Amyes, John A Gerlt, David C. Plache, Astrid P. Koudelka, John P Richard
    Abstract:

    We report the results of a study of the catalytic role of a network of four interacting amino acid side chains at yeast orotidine 5′-monophosphate decarboxylase (ScOMPDC), by the stepwise replacement of all four side chains. The H-bond, which links the −CH2OH side chain of S154 from the pyrimidine umbrella loop of ScOMPDC to the amide side chain of Q215 in the phosphodianion gripper loop, creates a protein cage for the substrate OMP. The role of this interaction in optimizing transition state stabilization from the dianion gripper side chains Q215, Y217, and R235 was probed by determining the kinetic parameter Kcat/Km for 16 enzyme variants, which include all combinations of single, double, triple, and quadruple S154A, Q215A, Y217F, and R235A mutations. The effects of consecutive Q215A, Y217F, and R235A mutations on ΔG⧧ for wild-type enzyme-catalyzed decarboxylation sum to 11.6 kcal/mol, but to only 7.6 kcal/mol when starting from S154A mutant. This shows that the S154A mutation results in a (11.6–7.6) = 4.0 kcal/mol decrease in transition state stabilization from interactions with Q215, Y217, and R235. Mutant cycles show that ca. 2 kcal/mol of this 4 kcal/mol effect is from the direct interaction between the S154 and Q215 side chains and that ca. 2 kcal/mol is from a tightening in the stabilizing interactions of the Y217 and R235 side chains. The sum of the effects of individual A154S, A215Q, F217Y and A235R substitutions at the quadruple mutant of ScOMPDC to give the corresponding triple mutants, 5.6 kcal/mol, is much smaller than 16.0 kcal/mol, the sum of the effects of the related four substitutions in wild-type ScOMPDC to give the respective single mutants. The small effect of substitutions at the quadruple mutant is consistent with a large entropic cost to holding the flexible loops of ScOMPDC in the active closed conformation

  • enzyme architecture erection of active orotidine 5 monophosphate decarboxylase by substrate induced conformational changes
    Journal of the American Chemical Society, 2017
    Co-Authors: Archie C Reyes, Tina L. Amyes, John P Richard
    Abstract:

    Orotidine 5′-monophosphate decarboxylase (OMPDC) catalyzes the decarboxylation of 5-fluoroorotate (FO) with Kcat/Km = 1.4 × 10–7 M–1 s–1. Combining this and related kinetic parameters shows that the 31 kcal/mol stabilization of the transition state for decarboxylation of OMP provided by OMPDC represents the sum of 11.8 and 10.6 kcal/mol stabilization by the substrate phosphodianion and the ribosyl ring, respectively, and an 8.6 kcal/mol stabilization from the orotate ring. The transition state for OMPDC-catalyzed decarboxylation of FO is stabilized by 5.2, 7.2, and 9.0 kcal/mol, respectively, by 1.0 M phosphite dianion, d-glycerol 3-phosphate and d-erythritol 4-phosphate. The stabilization is due to the utilization of binding interactions of the substrate fragments to drive an enzyme conformational change, which locks the orotate ring of the whole substrate, or the substrate pieces in a caged complex. We propose that enzyme-activation is a possible, and perhaps probable, consequence of any substrate-induc...

  • Enzyme Architecture: Erection of Active Orotidine 5′-Monophosphate Decarboxylase by Substrate-Induced Conformational Changes
    2017
    Co-Authors: Archie C. Reyes, Tina L. Amyes, John P Richard
    Abstract:

    Orotidine 5′-monophosphate decarboxylase (OMPDC) catalyzes the decarboxylation of 5-fluoroorotate (FO) with Kcat/Km = 1.4 × 10–7 M–1 s–1. Combining this and related kinetic parameters shows that the 31 kcal/mol stabilization of the transition state for decarboxylation of OMP provided by OMPDC represents the sum of 11.8 and 10.6 kcal/mol stabilization by the substrate phosphodianion and the ribosyl ring, respectively, and an 8.6 kcal/mol stabilization from the orotate ring. The transition state for OMPDC-catalyzed decarboxylation of FO is stabilized by 5.2, 7.2, and 9.0 kcal/mol, respectively, by 1.0 M phosphite dianion, d-glycerol 3-phosphate and d-erythritol 4-phosphate. The stabilization is due to the utilization of binding interactions of the substrate fragments to drive an enzyme conformational change, which locks the orotate ring of the whole substrate, or the substrate pieces in a caged complex. We propose that enzyme-activation is a possible, and perhaps probable, consequence of any substrate-induced enzyme conformational change

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

  • enzyme architecture breaking down the catalytic cage that activates orotidine 5 monophosphate decarboxylase for catalysis
    Journal of the American Chemical Society, 2018
    Co-Authors: Archie C Reyes, Tina L. Amyes, John A Gerlt, David C. Plache, Astrid P. Koudelka, John P Richard
    Abstract:

    We report the results of a study of the catalytic role of a network of four interacting amino acid side chains at yeast orotidine 5′-monophosphate decarboxylase (ScOMPDC), by the stepwise replacement of all four side chains. The H-bond, which links the −CH2OH side chain of S154 from the pyrimidine umbrella loop of ScOMPDC to the amide side chain of Q215 in the phosphodianion gripper loop, creates a protein cage for the substrate OMP. The role of this interaction in optimizing transition state stabilization from the dianion gripper side chains Q215, Y217, and R235 was probed by determining the kinetic parameter Kcat/Km for 16 enzyme variants, which include all combinations of single, double, triple, and quadruple S154A, Q215A, Y217F, and R235A mutations. The effects of consecutive Q215A, Y217F, and R235A mutations on ΔG⧧ for wild-type enzyme-catalyzed decarboxylation sum to 11.6 kcal/mol, but to only 7.6 kcal/mol when starting from S154A mutant. This shows that the S154A mutation results in a (11.6–7.6) = ...

  • Enzyme Architecture: Breaking Down the Catalytic Cage that Activates Orotidine 5′-Monophosphate Decarboxylase for Catalysis
    2018
    Co-Authors: Archie C. Reyes, Tina L. Amyes, John A Gerlt, David C. Plache, Astrid P. Koudelka, John P Richard
    Abstract:

    We report the results of a study of the catalytic role of a network of four interacting amino acid side chains at yeast orotidine 5′-monophosphate decarboxylase (ScOMPDC), by the stepwise replacement of all four side chains. The H-bond, which links the −CH2OH side chain of S154 from the pyrimidine umbrella loop of ScOMPDC to the amide side chain of Q215 in the phosphodianion gripper loop, creates a protein cage for the substrate OMP. The role of this interaction in optimizing transition state stabilization from the dianion gripper side chains Q215, Y217, and R235 was probed by determining the kinetic parameter Kcat/Km for 16 enzyme variants, which include all combinations of single, double, triple, and quadruple S154A, Q215A, Y217F, and R235A mutations. The effects of consecutive Q215A, Y217F, and R235A mutations on ΔG⧧ for wild-type enzyme-catalyzed decarboxylation sum to 11.6 kcal/mol, but to only 7.6 kcal/mol when starting from S154A mutant. This shows that the S154A mutation results in a (11.6–7.6) = 4.0 kcal/mol decrease in transition state stabilization from interactions with Q215, Y217, and R235. Mutant cycles show that ca. 2 kcal/mol of this 4 kcal/mol effect is from the direct interaction between the S154 and Q215 side chains and that ca. 2 kcal/mol is from a tightening in the stabilizing interactions of the Y217 and R235 side chains. The sum of the effects of individual A154S, A215Q, F217Y and A235R substitutions at the quadruple mutant of ScOMPDC to give the corresponding triple mutants, 5.6 kcal/mol, is much smaller than 16.0 kcal/mol, the sum of the effects of the related four substitutions in wild-type ScOMPDC to give the respective single mutants. The small effect of substitutions at the quadruple mutant is consistent with a large entropic cost to holding the flexible loops of ScOMPDC in the active closed conformation

  • enzyme architecture deconstruction of the enzyme activating phosphodianion interactions of orotidine 5 monophosphate decarboxylase
    Journal of the American Chemical Society, 2014
    Co-Authors: Lawrence M Goldman, Tina L. Amyes, John A Gerlt, Bogdana Goryanova, John P Richard
    Abstract:

    The mechanism for activation of orotidine 5′-monophosphate decarboxylase (OMPDC) by interactions of side chains from Gln215 and Try217 at a gripper loop and R235, adjacent to this loop, with the phosphodianion of OMP was probed by determining the kinetic parameters Kcat and Km for all combinations of single, double, and triple Q215A, Y217F, and R235A mutations. The 12 kcal/mol intrinsic binding energy of the phosphodianion is shown to be equal to the sum of the binding energies of the side chains of R235 (6 kcal/mol), Q215 (2 kcal/mol), Y217 (2 kcal/mol), and hydrogen bonds to the G234 and R235 backbone amides (2 kcal/mol). Analysis of a triple mutant cube shows small (ca. 1 kcal/mol) interactions between phosphodianion gripper side chains, which are consistent with steric crowding of the side chains around the phosphodianion at wild-type OMPDC. These mutations result in the same change in the activation barrier to the OMPDC-catalyzed reactions of the whole substrate OMP and the substrate pieces (1-β-d-er...

  • 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
    2012
    Co-Authors: Wing-yin Tsang, Mc Kay B Wood, Tina L. Amyes, John A Gerlt, Freeman M. Wong, John P Richard
    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 1H 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 Kcat 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 pKa 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 pKa values for proton transfer from C-6 of uridine (pKCH = 28.8) and 5-fluorouridine (pKCH = 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 × 109-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 Kcat/Km 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 × 1010 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

  • mechanism of the orotidine 5 monophosphate decarboxylase catalyzed reaction importance of residues in the orotate binding site
    Biochemistry, 2011
    Co-Authors: Vanessa Iiams, E V Fedorov, Steven C Almo, A. A. Fedorov, B Desai, John A Gerlt
    Abstract:

    The reaction catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) is accompanied by exceptional values for rate enhancement (Kcat/knon = 7.1 × 1016) and catalytic proficiency [(Kcat/KM)/knon = 4.8 × 1022 M–1]. Although a stabilized vinyl carbanion/carbene intermediate is located on the reaction coordinate, the structural strategies by which the reduction in the activation energy barrier is realized remain incompletely understood. This laboratory recently reported that “substrate destabilization” by Asp 70 in the OMPDC from Methanothermobacter thermoautotrophicus (MtOMPDC) lowers the activation energy barrier by ∼5 kcal/mol (contributing ∼2.7 × 103 to the rate enhancement) [Chan, K. K., Wood, B. M., Fedorov, A. A., Fedorov, E. V., Imker, H. J., Amyes, T. L., Richard, J. P., Almo, S. C., and Gerlt, J. A. (2009) Biochemistry 48, 5518–5531]. We now report that substitutions of hydrophobic residues in a pocket proximal to the carboxylate group of the substrate (Ile 96, Leu 123, and Val 155) with neutr...

Archie C Reyes - One of the best experts on this subject based on the ideXlab platform.

  • the organization of active site side chains of glycerol 3 phosphate dehydrogenase promotes efficient enzyme catalysis and rescue of variant enzymes
    Biochemistry, 2020
    Co-Authors: Judith R Cristobal, Archie C Reyes, John P Richard
    Abstract:

    A comparison of the values of Kcat/Km for reduction of dihydroxyacetone phosphate (DHAP) by NADH catalyzed by wild type and K120A/R269A variant glycerol-3-phosphate dehydrogenase from human liver (hlGPDH) shows that the transition state for enzyme-catalyzed hydride transfer is stabilized by 12.0 kcal/mol by interactions with the cationic K120 and R269 side chains. The transition state for the K120A/R269A variant-catalyzed reduction of DHAP is stabilized by 1.0 and 3.8 kcal/mol for reactions in the presence of 1.0 M EtNH3+ and guanidinium cation (Gua+), respectively, and by 7.5 kcal/mol for reactions in the presence of a mixture of each cation at 1.0 M, so that the transition state stabilization by the ternary E·EtNH3+·Gua+ complex is 2.8 kcal/mol greater than the sum of stabilization by the respective binary complexes. This shows that there is cooperativity between the paired activators in transition state stabilization. The effective molarities (EMs) of ∼50 M determined for the K120A and R269A side chains are ≪106 M, the EM for entropically controlled reactions. The unusually efficient rescue of the activity of hlGPDH-catalyzed reactions by the HPi/Gua+ pair and by the Gua+/EtNH3+ activator pair is due to stabilizing interactions between the protein and the activator pieces that organize the K120 and R269 side chains at the active site. This "preorganization" of side chains promotes effective catalysis by hlGPDH and many other enzymes. The role of the highly conserved network of side chains, which include Q295, R269, N270, N205, T264, K204, D260, and K120, in catalysis is discussed.

  • enzyme architecture breaking down the catalytic cage that activates orotidine 5 monophosphate decarboxylase for catalysis
    Journal of the American Chemical Society, 2018
    Co-Authors: Archie C Reyes, Tina L. Amyes, John A Gerlt, David C. Plache, Astrid P. Koudelka, John P Richard
    Abstract:

    We report the results of a study of the catalytic role of a network of four interacting amino acid side chains at yeast orotidine 5′-monophosphate decarboxylase (ScOMPDC), by the stepwise replacement of all four side chains. The H-bond, which links the −CH2OH side chain of S154 from the pyrimidine umbrella loop of ScOMPDC to the amide side chain of Q215 in the phosphodianion gripper loop, creates a protein cage for the substrate OMP. The role of this interaction in optimizing transition state stabilization from the dianion gripper side chains Q215, Y217, and R235 was probed by determining the kinetic parameter Kcat/Km for 16 enzyme variants, which include all combinations of single, double, triple, and quadruple S154A, Q215A, Y217F, and R235A mutations. The effects of consecutive Q215A, Y217F, and R235A mutations on ΔG⧧ for wild-type enzyme-catalyzed decarboxylation sum to 11.6 kcal/mol, but to only 7.6 kcal/mol when starting from S154A mutant. This shows that the S154A mutation results in a (11.6–7.6) = ...

  • primary deuterium kinetic isotope effects a probe for the origin of the rate acceleration for hydride transfer catalyzed by glycerol 3 phosphate dehydrogenase
    Biochemistry, 2018
    Co-Authors: Archie C Reyes, Tina L. Amyes, John P Richard
    Abstract:

    Abstract. The following primary deuterium kinetic isotope effects (1o DKIEs) on Kcat/Km are reported for wildtype and mutant glycerol-3-phosphate dehydrogenase (GPDH)-catalyzed reduction of DHAP by NADL (L = H, D) over an 11.5 kcal/mol range of activation barriers: wildtype GPDH, ΔΔG‡ = 0 kcal/mol, 1o DKIE = 1.5; N270A, 5.6 kcal/mole, 3.1; R269A, 9.1 kcal/mol, 2.8; R269A + 1.0 M guanidine, 2.4 kcal/mol, 2.7; R269A/N270A, 11.5 kcal/mol, 2.4. Similar 1o DKIEs were observed on Kcat. The narrow range of 1o DKIEs (2.4 – 3.1) observed for a 9.1 kcal/mol change in reaction driving force provides strong evidence that these are intrinsic 1o DKIE on rate determining hydride transfer. Evidence is presented that the intrinsic DKIE on wildtype GPDH-catalyzed reduction of DHAP lies in this range. A similar range of 1o DKIE (2.4 – 2.9) on (Kcat/KGA, M -1 s -1 ) was reported for dianion activated hydride transfer from NADL to glycolaldehyde (GA) [J. Am. Chem. Soc. 2016, 138, 14526-14529]. These 1o DKIEs are much smaller ...

  • enzyme architecture erection of active orotidine 5 monophosphate decarboxylase by substrate induced conformational changes
    Journal of the American Chemical Society, 2017
    Co-Authors: Archie C Reyes, Tina L. Amyes, John P Richard
    Abstract:

    Orotidine 5′-monophosphate decarboxylase (OMPDC) catalyzes the decarboxylation of 5-fluoroorotate (FO) with Kcat/Km = 1.4 × 10–7 M–1 s–1. Combining this and related kinetic parameters shows that the 31 kcal/mol stabilization of the transition state for decarboxylation of OMP provided by OMPDC represents the sum of 11.8 and 10.6 kcal/mol stabilization by the substrate phosphodianion and the ribosyl ring, respectively, and an 8.6 kcal/mol stabilization from the orotate ring. The transition state for OMPDC-catalyzed decarboxylation of FO is stabilized by 5.2, 7.2, and 9.0 kcal/mol, respectively, by 1.0 M phosphite dianion, d-glycerol 3-phosphate and d-erythritol 4-phosphate. The stabilization is due to the utilization of binding interactions of the substrate fragments to drive an enzyme conformational change, which locks the orotate ring of the whole substrate, or the substrate pieces in a caged complex. We propose that enzyme-activation is a possible, and perhaps probable, consequence of any substrate-induc...

  • enzyme architecture self assembly of enzyme and substrate pieces of glycerol 3 phosphate dehydrogenase into a robust catalyst of hydride transfer
    Journal of the American Chemical Society, 2016
    Co-Authors: Archie C Reyes, Tina L. Amyes, John P Richard
    Abstract:

    The stabilization of the transition state for hlGPDH-catalyzed reduction of DHAP due to the action of the phosphodianion of DHAP and the cationic side chain of R269 is between 12.4 and 17 kcal/mol. The R269A mutation of glycerol-3-phosphate dehydrogenase (hlGPDH) results in a 9.1 kcal/mol destabilization of the transition state for enzyme-catalyzed reduction of dihydroxyacetone phosphate (DHAP) by NADH, and there is a 6.7 kcal/mol stabilization of this transition state by 1.0 M guanidine cation (Gua+) [J. Am. Chem. Soc. 2015, 137, 5312–5315]. The R269A mutant shows no detectable activity toward reduction of glycolaldehyde (GA), or activation of this reaction by 30 mM HPO32–. We report the unprecedented self-assembly of R269A hlGPDH, dianions (X2– = FPO32–, HPO32–, or SO42–), Gua+ and GA into a functioning catalyst of the reduction of GA, and fourth-order reaction rate constants Kcat/KGAKXKGua. The linear logarithmic correlation (slope = 1.0) between values of Kcat/KGAKX for dianion activation of wildtype ...

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  • mechanistic studies of formate oxidase from aspergillus oryzae a novel member of the glucose methanol choline oxidoreductase enzyme superfamily that oxidizes carbon acids
    Archives of Biochemistry and Biophysics, 2018
    Co-Authors: John M Robbins, Andreas S Bommarius, Giovanni Gadda
    Abstract:

    Abstract Formate oxidase (FOX) from Aspergillus oryzae is the only GMC member that oxidizes a carbon acid rather than alcohols; thus, its catalytic mechanism may be different from that of other GMC members. We have used pH, solvent viscosity, and deuterium kinetic isotope effects, to investigate the catalytic mechanism of FOX. The enzyme followed a Bi-Bi sequential steady-state kinetic mechanism. The Kcat value was pH-independent between pH 2.8 and 6.8, suggesting a lack of ionizable groups in kinetic step(s) that limit the overall turnover of the enzyme. The Kcat/Kformate value decreased from a value of 10,000 M−1s−1 at low pH with a pKa value of 4.4, consistent with the requirement of a protonated group for substrate binding. An inverse viscosity dependence on the Kcat/Kformate value indicated an isomerization of the Michaelis complex. The Kcat/Koxygen value was 340,000 M−1s−1 and pH independent up to pH 6.0. The DKcat and D(Kcat/Kformate) values were 2.5 and 1.9, respectively, indicating that substrate CH bond cleavage is rate-limiting for FOX catalysis. Analytical ultracentrifugation indicated a concentration dependence of the oligomeric state of FOX. The appkred,H value was ∼75% that of Kcat,H, indicating that the anaerobic reduction of FOX was dependent on the oligomeric state of FOX.

  • solvent isotope and viscosity effects on the steady state kinetics of the flavoprotein nitroalkane oxidase
    FEBS Letters, 2013
    Co-Authors: Giovanni Gadda, Paul F. Fitzpatrick
    Abstract:

    The flavoprotein nitroalkane oxidase catalyzes the oxidative denitrification of a broad range of primary and secondary nitroalkanes to yield the respective aldehydes or ketones, hydrogen peroxide and nitrite. With nitroethane as substrate the D2O(Kcat/KM) value is 0.6 and the D2OKcat value is 2.4. The Kcat proton inventory is consistent with a single exchangeable proton in flight, while the Kcat/KM is consistent with either a single proton in flight in the transition state or a medium effect. Increasing the solvent viscosity did not affect the Kcat or Kcat/KM value significantly, establishing that nitroethane binding is at equilibrium and that product release does not limit Kcat.

  • contribution of flavin covalent linkage with histidine 99 to the reaction catalyzed by choline oxidase
    Journal of Biological Chemistry, 2009
    Co-Authors: Osbourne Quaye, Sharonda Cowins, Giovanni Gadda
    Abstract:

    The FAD-dependent choline oxidase has a flavin cofactor covalently attached to the protein via histidine 99 through an 8α-N(3)-histidyl linkage. The enzyme catalyzes the four-electron oxidation of choline to glycine betaine, forming betaine aldehyde as an enzyme-bound intermediate. The variant form of choline oxidase in which the histidine residue has been replaced with asparagine was used to investigate the contribution of the 8α-N(3)-histidyl linkage of FAD to the protein toward the reaction catalyzed by the enzyme. Decreases of 10-fold and 30-fold in the Kcat/Km and Kcat values were observed as compared with wild-type choline oxidase at pH 10 and 25 °C, with no significant effect on Kcat/KO using choline as substrate. Both the Kcat/Km and Kcat values increased with increasing pH to limiting values at high pH consistent with the participation of an unprotonated group in the reductive half-reaction and the overall turnover of the enzyme. The pH independence of both D(Kcat/Km) and DKcat, with average values of 9.2 ± 3.3 and 7.4 ± 0.5, respectively, is consistent with absence of external forward and reverse commitments to catalysis, and the chemical step of CH bond cleavage being rate-limiting for both the reductive half-reaction and the overall enzyme turnover. The temperature dependence of the Dkred values suggests disruption of the preorganization in the asparagine variant enzyme. Altogether, the data presented in this study are consistent with the FAD-histidyl covalent linkage being important for the optimal positioning of the hydride ion donor and acceptor in the tunneling reaction catalyzed by choline oxidase.

  • Oxygen- and temperature-dependent kinetic isotope effects in choline oxidase: correlating reversible hydride transfer with environmentally enhanced tunneling.
    Journal of the American Chemical Society, 2005
    Co-Authors: Giovanni Gadda
    Abstract:

    Choline oxidase catalyzes the flavin-linked oxidation of choline to glycine betaine, with betaine aldehyde as intermediate and oxygen as electron acceptor. Here, the effects of oxygen concentration and temperature on the kinetic isotope effects with deuterated choline have been investigated. The D(Kcat/Km) and DKcat values with 1,2-[2H4]-choline were pH-independent at saturating oxygen concentrations, whereas they decreased at high pH to limiting values that depended on oxygen concentration at ≤0.97 mM oxygen. The Kcat/Km and Kcat pH profiles had similar patterns reaching plateaus at high pH. Both the limiting Kcat/Km at high pH and the pKa values were perturbed to lower values with choline and ≤0.25 mM oxygen. These data suggest that oxygen availability modulates whether the reduced enzyme-betaine aldehyde complex partitions forward to catalysis rather then reverting to the oxidized enzyme-choline alkoxide species. At saturating oxygen concentrations, the D(Kcat/Km) was 10.6 ± 0.6 and temperature indepen...