Carbanions from decarboxylation of orotate analogs: Stability and mechanistic implications

Yeoh, Fong Ying; Cuasito, Roxanne R.; Capule, Christina C.; Wong, Freeman M.; Wu, Weiming

doi:10.1016/j.bioorg.2007.02.001

Cited by 16 publications

(17 citation statements)

References 17 publications

(39 reference statements)

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“…The rate of exchange of H6 of the UMP product with solvent deuterium can be used to estimate its pK a within the active site. In solution, the pK a of 1,3-dimethyluracil is estimated as 34 ± 2 (18) and 30 (19); in the active sites of both ScOMPDC and MtOMPDC, the pK a of UMP is ≤ 22 (17). The difference establishes that the active site stabilizes the vinyl carbanion by ≥ 14 kcal/mol (producing a rate acceleration ≥ 10 10 ).…”

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confidence: 99%

Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization^,

et al. 2009

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The reaction catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) involves a stabilized anionic intermediate, although the structural basis for the rate acceleration (k cat /k non , 7.1 × 10 16 ) and proficiency [(k cat /K M )/k non , 4.8 × 10 22 M −1 ] is uncertain. That the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC) catalyze the exchange of H6 of the UMP product with solvent deuterium allows an estimate of a lower limit on the rate acceleration associated with stabilization of the intermediate and . We investigated that hypothesis by structural and functional characterization of the D70N and D70G mutants of MtOMPDC and the D91N mutant of ScOMPDC. The substitutions for Asp 70 in MtOMPDC significantly decrease the value of k cat for decarboxylation of FOMP (a more reactive substrate analog) but have little effect on the value of k ex for exchange of H6 of FUMP with solvent deuterium; the structures of wild type MtOMPDC and its mutants are superimposable when complexed with 6-azaUMP. In contrast, the D91N mutant of ScOMPDC does not catalyze exchange of H6 of FUMP; the structures of wild type ScOMPDC and its D91N mutant are not superimposable when complexed with 6-azaUMP, with differences in both the conformation of the active site loop and the orientation of the ligand vis á vis the active site residues. We propose that the differential effects of substitutions for Asp 70 of MtOMPDC on decarboxylation and exchange provide additional evidence for a carbanionic intermediate as well as the involvement of Asp 70 in substrate destabilization. † This research was supported by NIH GM039754 (to J.P.R.) and GM065155 (to J.A.G.). Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). The X-ray coordinates and structure factors for the following structures have been deposited in the Protein Data Bank with the indicated accession codes: wild type MtOMPDC in the absence of ligands (PDB accession code 3G18), wild type MtOMPDC complexed with 6-azaUMP (3G1A), wild type MtOMPDC complexed with UMP (3G1D), wild type MtOMPDC complexed with H 2 OMP (3D1F), wild type MtOMPDC complexed with H 2 UMP (3D1H), the D70N mutant of MtOMPDC in the absence of ligands (3G1Y), the D70N mutant of MtOMPDC complexed with 6-azaUMP (3G24), the D70N mutant of MtOMPDC complexed with UMP (3G22), the D70G mutant of MtOMPDC in the absence of ligands (3G1S), the D70G mutant of MtOMPDC complexed with UMP (3G1X), the D70G mutant of MtOMPDC complexed with FOMP (3G1V), wild type ScOMPDC in the absence of ligands (3GDK), wild type ScOMPDC complexed with 6-azaUMP (3GDL), the D91N mutant of ScOMPDC in the absence of ligands (3GDR), and the D91N mutant of ScOMPDC complexed with 6-azaUMP (3GDT).

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confidence: 99%

Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization^,

et al. 2009

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“…The ability of molecules 4 – 6 to quench the color (as shown in Table 4) allows the bracketing of their acidity in the form of p K values. 14 The deprotonation of the methyl group in pyridone 5 may also be an issue here, but this has no effect on the obsevation that uracil 4 is more acidic than pyridones 5 and 6 .…”

Section: Resultsmentioning

confidence: 94%

“…7,8,13,14 The gas phase study was carried out in a quadrupole ion trap mass spectrometer where the carbanions were generated via collision activated dissociation (CAD) of precursor carboxylate anions derived from acids 1 – 3 as the result of electrospray ionization. 7,8 The carbanions were allowed to react with a series of neutral carbon acids with varying strength as seen in Table 1.…”

Section: Resultsmentioning

confidence: 99%

“…In one set of experiments, carbanions 7 – 9 were generated through the decarboxylation of their corresponding carboxylates at 230 °C in DMSO and were allowed to react with weak carbon acids of varying strength as shown in Table 3. 14 The weak acids in Table 3 were chosen for the range of p K a values, and most importantly, for the fact that their conjugate bases (carbanions) are colored. 21 These weak acid thus act as indicators in this experiment.…”

Section: Resultsmentioning

confidence: 99%

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Stabilities of uracil and pyridone-based carbanions: a systematic study in the gas phase and solution and implications for the mechanism of orotidine-5′-monophosphate decarboxylase

et al. 2013

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The stabilities of the C6-centered carbanions derived from 1,3-dimethyluracil, N-methyl-2-pyridone, and N-methyl-4-pyridone were systematically investigated in the gas phase and in DMSO and water solutions. The stabilities of the carbanions in the gas phase and DMSO were directly measured through their reactions with carbon acids with known proton affinity or pKa values. The stabilities of the carbanions in DMSO were also probed through their kinetic isotope effects of protonation over deuteriation using acids with different acidity. The stabilities of the carbanions in water were determined through the rates of hydrogen-deuterium exchange reactions of the corresponding conjugate acids. The carbanions derived from the two pyridones were found to have the same stability, whereas the carbanion derived from 1,3-dimethyluracil was more stable. The order of the stability of the carbanions showed no correlation with the decarboxylation rates of their corresponding carboxylic acids. The implications of the results for the mechanism of orotidine-5′-monophosphate decarboxylase (ODCase) are discussed.

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“…67–69 ) and 29 (ref. 70 ) in water and in DMSO, respectively, whereas the p K a in yeast ODCase is estimated as ≤ 22 (ref. 38 ).…”

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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Carbanions from decarboxylation of orotate analogs: Stability and mechanistic implications

Cited by 16 publications

References 17 publications

Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization^,

Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization^,

Stabilities of uracil and pyridone-based carbanions: a systematic study in the gas phase and solution and implications for the mechanism of orotidine-5′-monophosphate decarboxylase

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

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Carbanions from decarboxylation of orotate analogs: Stability and mechanistic implications

Cited by 16 publications

References 17 publications

Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization,

Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization,

Stabilities of uracil and pyridone-based carbanions: a systematic study in the gas phase and solution and implications for the mechanism of orotidine-5′-monophosphate decarboxylase

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

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Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization^,

Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization^,