We propose, first, a practical method for studying the isotopic transformation of glutamate or any other metabolite isotopomers in the citric acid and the glyoxylate cycles; second, two mathematical models, one for evaluating the flux through the citric acid cycle and the other for evaluating the flux through the latter coupled to the glyoxylate cycle in yeast. These models are based on the analysis of 13C-NMR spectra of glutamate obtained from Saccharomyces cerevisiae, NCYC strain, fed with 100% enriched [2-13C]acetate. The population of each glutamate isotopomer, the change in intensity of each multiplet component or the enrichment of any glutamate carbon is expressed by a specific analytical equation from which the flux in the citric acid and the glyoxylate cycles can be deduced.
The aerobic metabolism of 100% [2-13C]acetate in acetate-grown S. cerevisiae cells was studied as a function of time using 13C-NMR, 1H-NMR and biochemical techniques. The C1 and C6 doublet and singlet of labeled trehalose increase continuously with time indicating that there is no isotopic transformation between trehalose isotopomers even though the corresponding formation rates are different. By contrast, the glutamate C4 singlet increases then decreases with time. The C4 doublet, which is lower than the singlet for t < 60 min, increases continuously and becomes higher than the singlet for t 90 min. A similar observation was made for the C2 resonance singlet and doublet. In addition, the glutamate C2 multiplet consists of only seven instead of nine peaks as in random labeling. These results agree well with our models and demonstrate that, in the presence of acetate, anaplerotic carbon sources involved in the synthesis of acetyl-CoA are negligible in yeast.
The flux in the citric acid cycle was deduced from a plot of the C4 area versus incubation time, while the flux within the glyoxylate cycle was determined from the relative intensity of the glutamate C4 doublet and singlet. The fluxes in the citric acid and the glyoxylate cycles were found to be comparable. The proportion of glutamate in isotopic exchange with the citric acid cycle is about 2.5% min -1 in yeast.