title: Absolute quantitation of glycolytic intermediates reveals thermodynamic shifts in Saccharomyces cerevisiae strains lacking PFK1 or ZWF1 genes.
abstract: Internal standard based absolute quantitation of glycolytic intermediates was performed to characterize the thermodynamic states of Saccharomyces cerevisiae metabolism. A mixture of (13)C-labeled glycolytic intermediates was prepared via extraction from S. cerevisiae cells cultivated using a synthetic medium containing [U-(13)C] glucose as the sole carbon source. The (13)C-labeled metabolite mixture was used as an internal standard for the analysis of S. cerevisiae cultivated in a medium containing natural glucose. The methodology was employed for the absolute quantitation of glycolytic intermediates of BY4742, pfk1Δ, and zwf1Δ strains of S. cerevisiae. Fructose-1,6-bisphosphate was the most abundant intermediate in the BY4742 strains in the log phase of growth. Estimation of the Gibbs free energy change (ΔG) from the absolute concentration revealed that several reactions, such as those catalyzed by ribose-5-phosphate keto-isomerase and phosphoglucose isomerase, were commonly at near-equilibrium in all three strains. A significant shift in thermodynamic state was also observed for the transketolase-transaldolase reaction, for which ΔG was -6.6 ± 0.5 kJ mol(-1) in the BY4742 strain and 5.4 ± 0.3 kJ mol(-1) in the zwf1Δ strain.
authors: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan. Electronic address: syunsuke_nishino@bio.eng.osaka-u.ac.jp.
authors: Shunsuke
authors: S
authors: Nishino
authors: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan. Electronic address: nobuyuki_okahashi@bio.eng.osaka-u.ac.jp.
authors: Nobuyuki
authors: N
authors: Okahashi
authors: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan. Electronic address: fmatsuda@ist.osaka-u.ac.jp.
authors: Fumio
authors: F
authors: Matsuda
authors: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan. Electronic address: shimizu@ist.osaka-u.ac.jp.
authors: Hiroshi
authors: H
authors: Shimizu
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000378:metabolism; D005635:Fructosediphosphates / Q000378:metabolism; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D005956:Glucose-6-Phosphate Isomerase / Q000378:metabolism; D006019:Glycolysis; D012015:Reference Standards; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D013816:Thermodynamics; D014153:Transaldolase / Q000378:metabolism; D014174:Transketolase / Q000378:metabolism
chemical_list: D005635:Fructosediphosphates; D014174:Transketolase; D014153:Transaldolase; D019747:Aldose-Ketose Isomerases; C022548:ribosephosphate isomerase; D005956:Glucose-6-Phosphate Isomerase; D005947:Glucose; C029063:fructose-1,6-diphosphate
keywords: Absolute quantitation; Central carbon metabolism; Gibbs free energy change; Liquid chromatography–mass spectrometry; Saccharomyces cerevisiae