Branch point control by the phosphorylation state of isocitrate dehydrogenase. A quantitative examination of fluxes during a regulatory transition.

To understand how enzymatic pathways respond to changing external conditions, the fluxes through the tricarboxylic acid cycle and ancillary reactions were determined under three different growth conditions in Escherichia coli. The velocities through the major steps in each pathway were measured (a) for growth on acetate alone, (b) for growth on acetate plus glucose, and (c) during the transition caused by addition of glucose to cells growing on acetate. During the transition, the carbon flow through the Krebs cycle decreased by a factor of 5 despite an increase in the growth rate of the culture. Under these conditions, the dephosphorylation of isocitrate dehydrogenase caused a 4-fold increase in its activity. This, together with the decreased rate of substrate production and the kinetic parameters of the branch point enzymes, led to a cessation of the flux through the glyoxylate shunt. The decreased rate of acetyl-CoA turnover, not an inhibition of acetate transport, caused a slower rate of acetate uptake in the presence of glucose. The modulation of protein phosphorylation and metabolite levels is one of the regulatory mechanisms which enables the bacterium to make dramatic shifts between metabolic pathways within a fraction of a doubling time.