The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13 C labeling experiments with [U-13 C 6 ]glucose in glucoseor ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.The central carbon pathways fulfill anabolic and catabolic functions by providing cofactors and building blocks for macromolecular synthesis as well as energy. While some singlegene knockout mutations in central metabolism preclude growth on glucose, a majority of such variations can potentially be compensated for either by isoenzymes (39) or by a rerouting of carbon fluxes through alternative pathways (7,15,20,42). Usually, the phenotypes of knockout mutants is characterized by quantitative physiological analysis, and conclusions on intracellular metabolism are then based on qualitative interpretation of these results. To reveal cause and effect relationships, however, it is important to gain deeper insight into the complex metabolic responses at the level of intracellular metabolite concentrations and fluxes. These intracellular carbon fluxes, or in vivo reaction rates, are per se nonmeasurable quantities that cannot usually be inferred directly from in vitro enzyme activities because not all in vivo effector concentrations are known. Hence, the intracellular reaction rates are commonly estimated by methods of metabolic flux analysis, which provide a holistic perspective on metabolism (52, 54).The most common approach is based on flux balancing analysis within a stoichiometric model of cellular metabolism (26,41, 50, 52). In this approach, uptake and secretion rates and biosynthetic requirements are balanced in a stoichiometric model, assuming quasi-steady-state material balances on the intermediates. In many cases, however, limited extracellular data and stoichiometric constraints lead to underdetermined systems, so that addition...