This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high-resolution 13C NMR. The oxidation of [1,2,3-13C]propionate to glutamate via the TCA cycle was measured in wild-type (WT) and a citrate synthase mutant (CS-) strain of Escherichia coli transformed with allosteric E. coli citrate synthase (ECCS) or non-allosteric pig citrate synthase (PCS). The 13C fractional enrichment in glutamate C-2, C-3, and C-4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total C-4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10-fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C-4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure (TCACALC) and yielded an acetate/succinyl-CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the 13C spectra of cells presented with [3-13C]propionate or [2-13C]propionate. The results are consistent with the presence of an allosteric citrate synthase in ECCS and a non-allosteric citrate synthase in PCS. The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels.