Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: IV. Enzymes and fluxes of pyruvate metabolism.

The activities of pyruvate kinase (PK), pyruvate: formate-lyase (PFL), pyruvate dehydrogenase (PDH), and citrate synthase (CS) involved in the anaerobic glycerol conversion by Klebsiella pneumoniae were studied in continuous culture under conditions of steady states and sustained oscillations. Both the in vitro and in vivo activities of PK, PFL, and PDH are strongly affected by the substrate concentration and its uptake rate, as is the in vitro activity of CS. The flux from phosphoenolpyruvate to pyruvate is found to be mainly regulated on a genetic level by the synthesis rate of PK, particularly at low substrate concentration and low growth rate. In contrast, the conversion of pyruvate to acetyl-CoA is mainly regulated on a metabolic level by the in vivo activities of PFL and PDH. The ratio of in vitro to in vivo activities is in the range of 1 to 1.5 for PK, 5 to 17 for PFL and 5 to 80 for PDH under the experimental conditions. The regulation of in vivo activity and synthesis of these enzymes is sensitive to fluctuations of culture conditions, leading to oscillations of both the in vitro and in vivo activities. In particular, PFL is strongly affected during oscillations; its average in vitro activity is only about half of its corresponding steady-state value under similar environmental conditions. The average in vitro activities of PDH and PK under oscillations are close to their corresponding steady-state values. In contrast to all other enzymes measured for the glycerol metabolism by K. pneumoniae PFL and PDH are more effectively in vivo utilized under oscillations than under steady state, underlining the peculiar role of pyruvate metabolism in the dynamic responses of the culture.