Regulation of Phosphofructokinase from Aspergillus Niger: Effect of Fructose 2,6-Bisphosphate on the Action of Citrate, Ammonium Ions and AMP

Abstract

The role of fructose 2,6-bisphosphate in the regulation of glycolysis in the citric acid accumulating fungus Aspergillus niger was investigated. Fructose 2,6-bisphosphate stimulated the activity of partially purified phosphofructokinase by increasing the affinity of the enzyme for fructose 6-phosphate and relieving inhibition by ATP. Fructose 2,6-bisphosphate acted synergistically with AMP, but not with NH,+ ions, which otherwise also activate phosphofructokinase. Fructose 2,6-bisphosphate also partially antagonized citrate inhibition of phosphofructokinase; complete deinhibition against high (5 mM) concentrations of citrate (as occur during citric acid accumulation), however, required the simultaneous presence of fructose 2,6bisphosphate (0.1 PM), AMP (0.1 mM) and NH,+ ions (20 mM).

Organic Acid Production by Filamentous Fungi

“…Subsequently, some of these genes have been over-expressed in an effort to increase flux through this critical pathway leading to citric acid production. A. niger 6-phosphofructokinase (PFK), like the PFK from other organisms, is very sensitive to activation by fructose-2,6-bisphosphate (half-maximal stimulation at less than 0.2 M) (Arts et al, 1987). Citrate is a strong inhibitor of PFK and the simultaneous presence of the activators AMP (0.1 mM), NH 4 ϩ ions (20 mM) and fructose-2,6-bisphosphate (0.2 M) are required to overcome inhibition by citrate (5 mM) (Arts et al, 1987).…”

“…A. niger 6-phosphofructokinase (PFK), like the PFK from other organisms, is very sensitive to activation by fructose-2,6-bisphosphate (half-maximal stimulation at less than 0.2 M) (Arts et al, 1987). Citrate is a strong inhibitor of PFK and the simultaneous presence of the activators AMP (0.1 mM), NH 4 ϩ ions (20 mM) and fructose-2,6-bisphosphate (0.2 M) are required to overcome inhibition by citrate (5 mM) (Arts et al, 1987). The apparent requirement for significant intracellular NH 4 ϩ concentrations to relieve this inhibition may be of practical interest in the control of commercial citrate processes where NH 3 introduction is carefully metered to provide sufficient nitrogen to maintain citric acid metabolism without promoting accumulation of biomass or increasing the pH of the fermentation.…”

“…Thus, the authors propose that glyceraldehyde-3-phosphate dehydrogenase would be a promising target for metabolic engineering. Over the last decade, a number of glycolytic enzymes of A. niger have been purified, characterized, and cloned (see Table 12.2) (Habison et al, 1983;Meixner-Monori et al, 1984;Arts et al, 1987;Panneman et al, 1996;Panneman et al, 1998;. Subsequently, some of these genes have been over-expressed in an effort to increase flux through this critical pathway leading to citric acid production.…”

Posttranslational Modification of 6-Phosphofructo-1-Kinase in Aspergillus niger

“…Because of the importance of 6-phosphofructo-1-kinase in regulating the primary metabolism of A. niger, its kinetic properties have been extensively studied (1,8,9,20,22).…”

The dimorphic yeast Yarrowia lipolytica possesses an atypical phosphofructokinase: characterization of the enzyme and its encoding gene

“…To our knowledge, no data are available on the effect of this metabolite on Pfks from other yeast species, except S. cerevisiae. It has been reported (Habison et al, 1983) that partially purified Pfk from Aspergillus niger is inhibited by phosphoenolpyruvate with a K i value of 0?15 mM, and also by citrate (K i 0?1-0?4 mM), the latter inhibition being released by Fru-2,6-P 2 activation (Arts et al, 1987). Inhibition by citrate is a common feature of different eukaryotic Pfks; however, in Y. lipolytica, at least during growth in YPD, the role of this inhibitory effect is uncertain because of the high K i value of 3?3 mM, and the undetectable intracellular concentration of citrate under these conditions.…”