| Metabolism is crucial for live cells. Great effort has been made to map out its network structure. However, understanding of quantitative aspects of metabolism, including the reaction rates (i.e., fluxes) and their regulation, is still lacking.;E.coli provides a tractable system for quantitative analysis of metabolism, due to a number of simplifying factors including absence of extensive compartmentalization. Using a liquid chromatography tandem mass spectrometry (LC-MS/MS) based high through-put assay, quantitative analyses of E. coli metabolism were carried out on three levels: experimental quantitation of metabolite concentrations and fluxes involved in nitrogen metabolism, quantitative investigation of global metabolic changes triggered by nutrient starvation, and examination of the integrated regulation of central nitrogen metabolism.;These analyses showed that the rate of amino acid and nucleotide production in exponentially growing E. coli agree well with those predicted to allow optimal biomass production. However, ammonia is assimilated mainly through an ATP-consuming pathway rather than the non ATP-consuming pathway as was previously predicted. Removal of either carbon or nitrogen source from cultural media triggered profound and extensive metabolite concentration changes, but apparent homeostasis can sometimes be maintained for certain groups of metabolites by reducing production and consumption simultaneously. Metabolic changes induced by nutrient starvation are largely conserved between E. coli and yeast, suggesting metabolic similarity across organisms possibly extends from the network topology to regulatory elements as well. Milder modulations of nitrogen availability to E. coli (e.g., changing media ammonia concentration) induced metabolic changes that were less profound and largely localized to metabolites involved in the nitrogen assimilation reactions. In contrast to the view that saturated enzymes are insensitive to substrate concentration, competition for the active sites of saturated enzymes was found to be a key determinant of enzyme fluxes in live cells. Combined with covalent modification reactions controlling glutamine synthetase activity, such active-site competition was sufficient to explain and predict the complex dynamic response patterns of central nitrogen metabolites.;Beside specific lessons learned about E. coli metabolism, useful tools for experimental quantitation of fluxes are developed; widely applicable methodology for quantitative investigation of metabolic regulation is established in this study. |
