title: Intermediate instability at high temperature leads to low pathway efficiency for an in vitro reconstituted system of gluconeogenesis in Sulfolobus solfataricus.
abstract: 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.affiliation: Molecular Enzyme Technology and Biochemistry, Biofilm Centre, Faculty of Chemistry, University of Duisburg-Essen, Germany.
authors.forename: Theresa
authors.initials: T
authors.lastname: Kouril
authors.forename: Dominik
authors.initials: D
authors.lastname: Esser
authors.forename: Julia
authors.initials: J
authors.lastname: Kort
authors.forename: Hans V
authors.initials: HV
authors.lastname: Westerhoff
authors.forename: Bettina
authors.initials: B
authors.lastname: Siebers
authors.forename: Jacky L
authors.initials: JL
authors.lastname: Snoep
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D004099:Dihydroxyacetone Phosphate / Q000378:metabolism; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics / Q000378:metabolism*; D005636:Fructosephosphates / Q000096:biosynthesis; D005943:Gluconeogenesis / Q000235:genetics; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D005988:Glyceric Acids / Q000378:metabolism; D006207:Half-Life; D006358:Hot Temperature; D007700:Kinetics; D015233:Models, Statistical; D010735:Phosphoglycerate Kinase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D004163:Diphosphoglyceric Acids; D005636:Fructosephosphates; D005988:Glyceric Acids; D011994:Recombinant Proteins; D005986:Glyceraldehyde 3-Phosphate; C015891:glycerate 1,3-biphosphate; D004099:Dihydroxyacetone Phosphate; C027618:fructose-6-phosphate; C005156:3-phosphoglycerate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010735:Phosphoglycerate Kinase; D005634:Fructose-Bisphosphate Aldolase; D014305:Triose-Phosphate Isomerase
keywords: carbon loss; mathematical model; thermal instability; thermophile