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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
Author(s) -
Kouril Theresa,
Esser Dominik,
Kort Julia,
Westerhoff Hans V.,
Siebers Bettina,
Snoep Jacky L.
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12438
Subject(s) - sulfolobus solfataricus , dihydroxyacetone phosphate , biochemistry , triosephosphate isomerase , glyceraldehyde , dehydrogenase , chemistry , fructose , aldolase a , enzyme , phosphoglycerate mutase , phosphatase , biology , glycolysis , archaea , gene
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.