Four enzymes of the gluconeogenic pathway in Sulfolobus solfataricus were purified and kinetically characterized. The enzymes were reconstituted in vitro to quantify the contribution of temperature instability of the pathway intermediates to carbon loss from the system. The reconstituted system, consisting of phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase and the fructose 1,6-bisphosphate aldolase/ phosphatase maintained a constant consumption rate of 3-phosphoglycerate and production of fructose 6-phosphate over a 1 hour period. Cofactors ATP and NADPH were regenerated via pyruvate kinase and glucose dehydrogenase. A mathematical model was constructed on the basis of the kinetics of the purified enzymes and the measured half-life times of the pathway intermediates. The model quantitatively predicted the systems uxes and metabolite concentrations. Relative enzyme concentrations were chosen such that half the carbon in the system was lost due to degradation of the thermolabile intermediates dihydroxyacetone phosphate, glyceraldehyde 3-phosphate and 1,3 bisphosphoglycerate, indicating that intermediate instability at high temperature can significantly affect pathway efficiency. This article is protected by copyright. All rights reserved.
SEEK ID: https://fairdomhub.org/publications/213
PubMed ID: 23865479
Projects: SulfoSys
Publication type: Not specified
Journal: FEBS J.
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Date Published: 20th Jul 2013
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Created: 1st Aug 2013 at 11:28
Last updated: 8th Dec 2022 at 17:26
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Silicon cell model for the central carbohydrate metabolism of the archaeon Sulfolobus solfataricus under temperature variation
Programme: SysMO
Public web page: http://sulfosys.com/
Organisms: Sulfolobus solfataricus
Kinetic characterisation of phosphoglycerate kinase
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Assay type: Enzymatic Assay
Technology type: Initial Rate Experiment
Investigation: Central Carbon Metabolism of Sulfolobus solfata...
Study: Model Gluconeogenesis
Organisms: Sulfolobus solfataricus
SOPs: Cloning and heterologous expression in E. coli, Preparation of cell extracts of recombinant E.coli, Protein purification from E. coli extracts
Data files: PGK Kinetic Data
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Mathematical model for PGK kinetics, saturation with ADP, ATP, 3PG and BPG.
Submitter: Jacky Snoep
Biological problem addressed: Enzymology
Investigation: Central Carbon Metabolism of Sulfolobus solfata...
Study: Model Gluconeogenesis
Organisms: Sulfolobus solfataricus
Models: PGK Kinetic Model
SOPs: No SOPs
Data files: PGK Kinetic Data, PGK Kinetics Simulation
Snapshots: No snapshots
Kinetic characterisation of glyceraldehyde 3-phosphate dehydrogenase
Submitter: Jacky Snoep
Assay type: Enzymatic Assay
Technology type: Initial Rate Experiment
Investigation: Central Carbon Metabolism of Sulfolobus solfata...
Study: Model Gluconeogenesis
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Snapshots: No snapshots
In vitro reconstitution of the PGK, GAPHD, TPI and FBPAase enzymes from S. solfataricus
Submitter: Jacky Snoep
Biological problem addressed: Metabolic Network
Investigation: Central Carbon Metabolism of Sulfolobus solfata...
Study: Model Gluconeogenesis
Organisms: Sulfolobus solfataricus
Models: FBPAase Kinetic Model, GAPDH Kinetic Model, Kinetic Model of Temperature Degradation of Glu..., Model of reconstituted gluconeogenesis system i..., PGK Kinetic Model, TPI Kinetic Model
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Biological problem addressed: Metabolic Network
Investigation: Central Carbon Metabolism of Sulfolobus solfata...
Organisms: Sulfolobus solfataricus
Models: Model of reconstituted gluconeogenesis system i...
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Mathematical model for GAPDH kinetics, saturation with BPG, NADPH, NADP, GAP and Pi
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Biological problem addressed: Enzymology
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Models: GAPDH Kinetic Model
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Data files: GAPDH Kinetic Data, GAPDH Kinetics Simulation
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Kinetic characterisation of triose phosphate isomerase
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Assay type: Enzymatic Assay
Technology type: Initial Rate Experiment
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Study: Model Gluconeogenesis
Organisms: Sulfolobus solfataricus
SOPs: Cloning and heterologous expression in E. coli, Preparation of cell extracts of recombinant E.coli, Protein purification from E. coli extracts
Data files: TPI Kinetic Data
Snapshots: No snapshots
Mathematical model for TPI kinetics, saturation with GAP and DHAP, and inhibition by 3PG and PEP
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Kinetic characterisation of fructose 1,6-bisphosphate aldolase phosphatase
Submitter: Jacky Snoep
Assay type: Enzymatic Assay
Technology type: Initial Rate Experiment
Investigation: Central Carbon Metabolism of Sulfolobus solfata...
Study: Model Gluconeogenesis
Organisms: Sulfolobus solfataricus
SOPs: Cloning and heterologous expression in E. coli, Preparation of cell extracts of recombinant E.coli, Protein purification from E. coli extracts
Data files: FBPAase Kinetic data
Snapshots: No snapshots
Mathematical model for FBPAase kinetics, saturation with DHAP and GAP
Submitter: Jacky Snoep
Biological problem addressed: Enzymology
Investigation: Central Carbon Metabolism of Sulfolobus solfata...
Study: Model Gluconeogenesis
Organisms: Sulfolobus solfataricus
Models: FBPAase Kinetic Model
SOPs: No SOPs
Data files: FBPAase Kinetic data, FBPAase Kinetics Simulation
Snapshots: No snapshots
Temperature degradation of BPG, GAP and DHAP
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Assay type: Metabolite Concentration
Technology type: Technology Type
Investigation: Central Carbon Metabolism of Sulfolobus solfata...
Study: Model Gluconeogenesis
Organisms: Sulfolobus solfataricus
SOPs: No SOPs
Data files: Temperature Degradation of Gluconeogenic Interm...
Snapshots: No snapshots
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Biological problem addressed: Metabolism
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Submitter: Jacky Snoep
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Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis
Assays: TPI Modelling
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Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis
Simulation results of experimental data of the reconstituted gluconeogenic system
Creators: Jacky Snoep, Theresa Kouril
Submitter: Jacky Snoep
Simulation results of PGK experimental data for ADP, ATP, 3PG and BPG saturation.
Creator: Jacky Snoep
Submitter: Jacky Snoep
Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis
Assays: PGK Modelling
Simulation results of FBPAase of experimental data for DHAP and GAP saturation
Creator: Jacky Snoep
Submitter: Jacky Snoep
Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis
Assays: FBPAase Modelling
Simulation results of GAPDH experimental data for BPG, NADPH, NADP, GAP, and Pi
Creators: Jacky Snoep, Theresa Kouril
Submitter: Jacky Snoep
Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis
Assays: GAPDH Modelling
Kinetic characterisation of FBPAase. Expermental data for enzyme reaction rates with increasing concentrations of DHAP and GAP.
Creators: Jacky Snoep, Theresa Kouril
Submitter: Jacky Snoep
Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis
Assays: FBPAase, FBPAase Modelling
Kinetic characterisation of TPI. Experimental data for enzyme reaction rates with increasing concentrations of DHAP and GAP, and inhibition with 3PG and PEP.
Creators: Jacky Snoep, Theresa Kouril
Submitter: Jacky Snoep
Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis
Assays: TPI, TPI Modelling
Kinetic characterisation of GAPDH. Expermental data for enzyme reaction rates with increasing concentrations of BPG, NADPH, NADP, GAP and Pi.
Creators: Jacky Snoep, Theresa Kouril
Submitter: Jacky Snoep
Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis
Assays: GAPDH, GAPDH Modelling
Experimental data for determination of half-life of gluconeogenic intermediates (BPG, GAP and DHAP).
Creators: Jacky Snoep, Theresa Kouril
Submitter: Jacky Snoep
Kinetic characterisation of PGK. Expermental data for enzyme reaction rates with increasing concentrations of ATP, ADP, BPG and 3PG.
Creators: Jacky Snoep, Theresa Kouril
Submitter: Jacky Snoep
Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis
Assays: PGK, PGK Modelling
Model of reconstituted gluconeogenesis system in S. solfataricus based on the individual kinetic models for PGK, GAPDH, TPI, FBPAase.
Creator: Jacky Snoep
Submitter: Jacky Snoep
Model type: Ordinary differential equations (ODE)
Model format: SBML
Environment: JWS Online
Exponential decay model of gluconeogenic intermediates
Creator: Jacky Snoep
Submitter: Jacky Snoep
Model type: Ordinary differential equations (ODE)
Model format: Mathematica
Environment: Not specified
Organism: Sulfolobus solfataricus
Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis
Assays: Modelling Metabolite Degradation at High Temper..., Reconstituted Gluconeogenesis System
Mathematical model for TPI kinetics, GAP and DHAP saturation, and inhibition with 3PG and PEP.
Creator: Jacky Snoep
Submitter: Jacky Snoep
Model type: Ordinary differential equations (ODE)
Model format: Mathematica
Environment: Not specified
Organism: Sulfolobus solfataricus
Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis
Mathematical model for GAPDH kinetics, BPG, NADPH, NADP, GAP and Pi saturation.
Creator: Jacky Snoep
Submitter: Jacky Snoep
Model type: Ordinary differential equations (ODE)
Model format: Mathematica
Environment: Not specified
Organism: Sulfolobus solfataricus
Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis
Assays: GAPDH Modelling, Reconstituted Gluconeogenesis System
Mathematical model for FBPAase kinetics, saturation with DHAP and GAP
Creator: Jacky Snoep
Submitter: Jacky Snoep
Model type: Ordinary differential equations (ODE)
Model format: Mathematica
Environment: Not specified
Organism: Sulfolobus solfataricus
Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis
Assays: FBPAase Modelling, Reconstituted Gluconeogenesis System
Mathematical model for PGK kinetics, ADP, ATP, 3PG and BPG saturation.
Creator: Jacky Snoep
Submitter: Jacky Snoep
Model type: Ordinary differential equations (ODE)
Model format: Mathematica
Environment: Not specified
Organism: Sulfolobus solfataricus
Investigations: Central Carbon Metabolism of Sulfolobus solfata...
Studies: Model Gluconeogenesis