The control of glycolytic flux in the yeast Saccharomyces cerevisiae was studied by using permeabilized cells. Cells were harvested from chemostat cultures and, after removal of the cell wall, nystatin was used to permeabilize the spheroplasts. By this method it is possible to study the performance and regulation of a complete and functional metabolic pathway and not only a single enzymatic step. The results showed that ATP has a strong negative effect on glycolytic activity affecting several of the glycolytic enzymes. However, the main targets for ATP inhibition was phosphofructokinase and pyruvate kinase. Phospofructokinase was inhibited by ATP concentrations starting at about 1-2 mM, while pyruvate kinase required ATP levels above 2.5 mM before any inhibition was visible. These ATP concentrations were in the same range as measured for nitrogen- and glucose-limited cells cultivated in chemostat cultures. Other potential candidates as enzymes susceptible to ATP inhibition included hexokinase and enolase. The ATP:ADP ratio, as well as trehalose-6-phosphate levels, did not seem to influence the glycolytic activity.
Copyright 2000 John Wiley & Sons, Ltd.