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PGK and FBPAase titrations

Experimental analysis of pathway fluxes with varying PGK and FBPAase concentrations in the incubation assay.

SEEK ID: https://demo.fairdomhub.org/assays/30

Experimental Assay

Jacky Snoep

Projects: SulfoSys

Investigation: Central Carbon Metabolism of Sulfolobus solfataricus

Study: Pathway optimisation

Assay type: Extracellular Metabolite Concentration

Technology type: Progressive Curve Experiment

Organisms: Sulfolobus solfataricus : P2 (wild-type / wild-type)

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Created: 3rd Feb 2013 at 11:18

Last updated: 26th Oct 2017 at 11:57

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Related items

SulfoSys

Silicon cell model for the central carbohydrate metabolism of the archaeon Sulfolobus solfataricus under temperature variation

Programme: SysMO

Public web page: http://sulfosys.com/

Central Carbon Metabolism of Sulfolobus solfataricus
SulfoSys

An investigation in the CCM of S. solfataricus with a focus on the unique temperature adaptations and regulation; using a combined modelling and experimental approach.

Snapshots: Snapshot 1

Pathway optimisation
SulfoSys

By varying the input or output flux via PGK and FBPAase manipulation respectively the pathway efficiency can be optimised.

Person responsible: Jacky Snoep

Snapshots: No snapshots

P2

Contributor: Not available

Provider Name: Not specified

Provider's strain ID: Not specified

Organism: Sulfolobus solfataricus

Genotypes: wild-type

Phenotypes: wild-type

Comment: Not specified

Model simulation and experimental data for PGK and FBPAase titrations
SulfoSys

Combined plot fo the model simulations and the experimental data for the PGK and FBPAase titrations.

Creator: Jacky Snoep

Contributor: Jacky Snoep

Relationship type: Not specified

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PGK titration of reconstituted system
SulfoSys

Increasing the amount of PGK in the incubation assay and measuring the 3PG and F6P fluxes.

Creator: Jacky Snoep

Contributor: Jacky Snoep

Relationship type: Not specified

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FBPAase titration of reconstituted system
SulfoSys

Increasing the amount of FBPAase in the reconstituted system and measuring the 3PG and F6P fluxes.

Creator: Jacky Snoep

Contributor: Jacky Snoep

Relationship type: Not specified

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Intermediate instability at high temperature leads to low pathway efficiency for an in vitro reconstituted system of gluconeogenesis in Sulfolobus solfataricus
SulfoSys

Abstract (Expand)

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-h 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 system fluxes 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.

Authors: T. Kouril, D. Esser, J. Kort, H. V. Westerhoff, B. Siebers, Jacky Snoep

Date Published: 22nd Aug 2013

Journal: FEBS J

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