โ Scribed by Claudia T. Evans
No coin nor oath required. For personal study only.
This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and highโresolution ^13^C NMR. The oxidation of [1,2,3โ^13^C]propionate to glutamate via the TCA cycle was measured in wildโtype (WT) and a citrate synthase mutant (CS^โ^) strain of Escherichia coli transformed with allosteric E. coli citrate synthase (ECCS) or nonโallosteric pig citrate synthase (PCS). The ^13^C fractional enrichment in glutamate Cโ2, Cโ3, and Cโ4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total Cโ4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10โfold less total enzyme activity than PCS but only 50% less total labeling in glutamate Cโ4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure(TCACALC) and yielded an acetate/succinylโCoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the ^13^C spectra of cells presented with [3โ^13^C] propionate or [2โ^13^C]propionate. The results are consistent with the presence of an allosteric citrate synthase in ECCS and a nonโallosteric 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.
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