The effective molarity of the substrate phosphoryl group in the transition state for yeast OMP decarboxylase

Sievers, Annette; Wolfenden, Richard

doi:10.1016/j.bioorg.2004.08.005

Cited by 31 publications

(51 citation statements)

References 20 publications

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“…37 This sequence is the same as that observed in the published crystal structure of wild-type yeast OMPDC, 26 except for the C155S mutation, which was introduced to enhance the protein stability. 38 The S2H, C155S, A160S, and N267D mutations do not cause a significant change in the kinetic parameters for wild-type OMPDC.…”

Section: Methodsmentioning

confidence: 87%

Enzyme Architecture: Deconstruction of the Enzyme-Activating Phosphodianion Interactions of Orotidine 5′-Monophosphate Decarboxylase

et al. 2014

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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-erythrofuranosyl)orotic acid (EO) and phosphite dianion. This shows that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The 12 kcal/mol intrinsic phosphodianion binding energy of OMP is divided between the 8 kcal/mol of binding energy, which is utilized to drive a thermodynamically unfavorable conformational change of the free enzyme, resulting in an increase in (kcat)obs for OMPDC-catalyzed decarboxylation of OMP, and the 4 kcal/mol of binding energy, which is utilized to stabilize the Michaelis complex, resulting in a decrease in (Km)obs.

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Section: Methodsmentioning

confidence: 87%

Enzyme Architecture: Deconstruction of the Enzyme-Activating Phosphodianion Interactions of Orotidine 5′-Monophosphate Decarboxylase

et al. 2014

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“…These interactions are important for catalysis: 1) k cat /K m for OMP exceeds that for orotidine by a factor of ~10 11 for the OMPDC from Saccharomyces cerevisiae (ScOMPDC) (10); 2) k cat /K m for OMP exceeds that for 1-(β-D-erythrofuranosyl)orotic acid (EO; 5′-truncated OMP analog) by factors of 5.2 × 10 8 and 3.6 × 10 8 for ScOMPDC and MtOMPDC, respectively (11, 12); and 3 ) phosphite dianion ( H P i ) activates decarboxylation of EO by factors of 5.6 × 10 5 M −1 and 2.9 × 10 5 M −1 for ScOMPDC and MtOMPDC, respectively (11, 12). The “intrinsic binding energy” [IBE; (13)] of the 5′-phosphate/HP i 1) increases the affinity for the substrate (e.g., OMP vs. orotidine/EO); and 2) enables decarboxylation by juxtaposition of the substrate with the active site hydrogen-bonded networks (substrate destabilization and intermediate stabilization).…”

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confidence: 99%

Conformational Changes in Orotidine 5′-Monophosphate Decarboxylase: “Remote” Residues That Stabilize the Active Conformation

et al. 2010

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The structural factors responsible for the extraordinary rate enhancement (~1017) of the reaction catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) have not been defined. Catalysis requires a conformational change that closes an active site loop and “clamps” the orotate base proximal to hydrogen-bonded networks that destabilize the substrate and stabilize the intermediate. In the OMPDC from Methanobacter thermoautotrophicus, a “remote” structurally conserved cluster of hydrophobic residues that includes Val 182 in the active site loop is assembled in the closed, catalytically active conformation. Substitution of these residues with Ala decreases kcat/Km with a minimal effect on kcat, providing evidence that the cluster stabilizes the closed conformation. The intrinsic binding energies of the 5′-phosphate group of OMP for the mutant enzymes are similar to that for the wild type, supporting this conclusion.

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“…S4) is decarboxylated by yeast ODCase with a ~10 11 –fold lower k cat / K m value than OMP. 64 Mutations of the phosphate binding arginine (R203) result in 10 3 –10 4 –fold lower k cat / K m values. 65 These drastic effects on catalytic efficiency confirm the importance of the phosphate part of OMP for the catalytic reaction.…”

Section: Resultsmentioning

confidence: 99%

Substrate Distortion Contributes to the Catalysis of Orotidine 5′-Monophosphate Decarboxylase

et al. 2013

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Orotidine 5'-monophosphate decarboxylase (ODCase) accelerates the decarboxylation of orotidine 5'-monophosphate (OMP) to uridine 5'-monophosphate (UMP) by 17 orders of magnitude. Eight new crystal structures with ligand analogues combined with computational analyses of the enzyme’s short-lived intermediates and the intrinsic electronic energies to distort the substrate and other ligands improve our understanding of the still controversially discussed reaction mechanism. In their respective complexes, 6-methyl-UMP displays significant distortion of its methyl substituent bond, 6-amino-UMP shows the competition between the K72 and C6 substituents for a position close to D70, and the methyl- and ethyl-ester of OMP both induce rotation of the carboxylate group substituent out of the plane of the pyrimidine ring. MD and QM/MM computations of the enzyme-substrate (ES) complex also show the bond between the carboxylate group and the pyrimidine ring to be distorted with the distortion contributing a 10–15% decrease of the ΔΔG‡ value. These results are consistent with ODCase using both substrate distortion as well as transition state stabilization, primarily exerted by K72, in its catalysis of the OMP decarboxylation reaction.

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The effective molarity of the substrate phosphoryl group in the transition state for yeast OMP decarboxylase

Cited by 31 publications

References 20 publications

Enzyme Architecture: Deconstruction of the Enzyme-Activating Phosphodianion Interactions of Orotidine 5′-Monophosphate Decarboxylase

Enzyme Architecture: Deconstruction of the Enzyme-Activating Phosphodianion Interactions of Orotidine 5′-Monophosphate Decarboxylase

Conformational Changes in Orotidine 5′-Monophosphate Decarboxylase: “Remote” Residues That Stabilize the Active Conformation

Substrate Distortion Contributes to the Catalysis of Orotidine 5′-Monophosphate Decarboxylase

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