Abstract
The intracellular concentrations of the valine and leucine pathway
intermediates in a Corynebacterium glutamicum strain were measured
during a transient state. The data were obtained by performing a glucose
stimulus-response experiment with the use of a rapid sampling device and
advanced mass spectrometry. The glucose stimulus resulted in a 3-fold
increase in the intracellular pyruvate concentration within less than a
second, demonstrating the very fast interactions in metabolic networks.
The samples were taken at subsecond intervals for a time period of 25 s.
The time courses of the metabolite concentrations formed the
experimental basis of a mathematical model simulating the fluxes and
concentrations in the valine/leucine pathway. The implementation of a
model selection criterion based on the second law of thermodynamics is
demonstrated to be essential for the identification of realistic and
unique models. Large differences between the enzyme properties
determined in vitro and those determined in vivo by the model were
observed with the in vivo maximal rates being almost an order of
magnitude larger than the in vitro maximal rates. The transamination of
ketoisovalerate (KIV) to valine is carried out mainly by the
transaminase B enzyme, with the transaminase C enzyme playing a minor
role. The availability of the cofactors NADP and NADPH only has modest
influence on the flux through the valine pathway, while the influence of
NAD and NADH on the flux through the leucine pathway is negligible.
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