Citrate Inhibition-Resistant Form of 6-Phosphofructo-1-Kinase fromAspergillus niger

Abstract

Two forms of Aspergillus niger 6-phosphofructo-1-kinase (PFK1) have been described recently, the 85-kDa native enzyme and 49-kDa shorter fragment that is formed from the former by posttranslational modification. So far, kinetic characteristics have never been determined on the enzyme purified to near homogeneity. For the first time, kinetic parameters were determined for individual enzymes with respect to citrate inhibition. The native 85-kDa enzyme was found to be moderately inhibited by citrate, with the K i value determined to be 1.5 mM, in the system with 5 mM Mg 2؉ ions, while increasing magnesium concentrations relieved the negative effect of citrate. An identical inhibition coefficient was determined also in the presence of ammonium ions, although ammonium acted as a strong activator of enzyme activity. On the other hand, the shorter fragment of PFK1 proved to be completely resistant to inhibition by citrate. Allosteric citrate binding sites were most probably lost after the truncation of the C-terminal part of the native protein, in which region some binding sites for inhibitor are known to be located. At near physiological conditions, characterized by low fructose-6-phosphate concentrations, a much higher efficiency of the shorter fragment was observed during an in vitro experiment. Since the enzyme became more susceptible to the positive control by specific ligands, while the negative control was lost after posttranslational modification, the shorter PFK1 fragment seems to be the enzyme most responsible for generating undisturbed metabolic flow through glycolysis in A. niger cells.Phosphofructokinase (EC 2.7.1.11) catalyzes the first essentially irreversible reaction of glycolysis, the phosphorylation of fructose-6-phosphate to form fructose-1,6-bisphosphate using MgATP as the phosphoryl donor and releasing MgADP as a by-product. The eukaryotic enzyme has complex regulatory properties that are mediated by the interaction of allosteric ligands with a number of distinct binding sites and presents one of the principal regulatory steps in glycolysis (4, 30). Eukaryotic phosphofructo-1-kinases (PFK1s) are more than twice the size of prokaryotic PFKs and are under regulatory control by a wider array of effectors than seen with the simpler bacterial enzymes. Sequencing data of amino acid residues indicated an evolutionary relationship between bacterial and eukaryotic PFKs that suggests duplication, tandem fusion, and divergence of catalytic and effector binding sites of a prokaryotic ancestor to yield in eukaryotes a total of six organic ligand binding sites (22). One of these allosteric ligands is citrate, which acts as a potent allosteric inhibitor of the eukaryotic PFK enzymes. Studies on citrate allosteric sites of rabbit muscle PFK concluded that they developed from the phosphoenolpyruvate (PEP)/ADP allosteric site of Escherichia coli PFK and are scattered both on N-terminal and C-terminal parts of the enzyme (11, 18). On the other hand, the strict conservation between active site residues in the N-termi...

Changes in primary metabolism leading to citric acid overflow in Aspergillus niger

“…Other Pfk1 stimulators, AMP and ammonium ions, increased the activity of the shorter fragment more intensely than the activity of the native protein, while citrate, a well known allosteric inhibitor of eukaryotic Pfk1 enzymes, showed moderate inhibition of the native enzyme, while no inhibition of the fragment could be observed by concentrations up to 10 mM (Mlakar and Legiša 2006).…”

“…Regulation of the central part of hexose metabolism takes place at several levels: at the transcriptional level, by regulating the activity of allosteric enzymes by specific effectors and as suggested recently (Mesojednik and Legiša 2005;Mlakar and Legiša 2006), even by post-translational modification.…”

“…Six organic allosteric ligands either increase or decrease substrate binding affinity and concomitantly determine overall enzyme activity (Poorman et al 1984). Pfk1 from A. niger cells was a subject of various studies (Habison et al 1983;Schreferl et al 1986;Arts et al 1987;Legiša and Benčina 1994;Mesojednik and Legiša 2005;Mlakar and Legiša 2006). The enzyme attracted the interest of investigators, due to its ability to maintain a high glycolytic flux in spite of elevated intracellular concentrations of citrate, a well-known inhibitor of Pfk1, which was reported to reach concentrations between 4 mM (Rö hr and Kubicek 1981) and 10 mM (Legiša and Kidrič 1989).…”

“…The results showed different kinetic characteristics of the native Pfk1 enzyme and the shorter fragment (from Mesojednik and Legiša 2005 with permission) The effectors were used at near physiological concentrations found in Aspergillus niger cells during citric acid accumulation: fructose 2,6-bisphosphate (8 lmol), NH 4 + (5 mmol), AMP (0.3 mmol), citrate (10 mmol), ATP (2 mmol). Estimation was made on the results published in papers: Mesojednik andLegiša 2005 andMlakar andLegiša 2006. Symbols: + slight positive effect, ++ strong positive effect, +++ very strong positive effect; -weak negative effect, --strong negative effect leads to the re-activation of 49 kDa fragment (Mesojednik and Legiša 2005).…”

Organic acid production in Aspergillus niger and other filamentous fungi

“…As an example, forcing increased flux through PFK might result in higher citrate secretion in silico, but in vivo, only overexpression of a truncated (due to post-translational modification (Mesojednik and Legiša, 2005;Mlakar and Legiša, 2006)) version of PFK, which was not subject to repression by citrate, has so far been successful (Capuder et al, 2009). Similarly, co-factor requirements of metalloproteins are not taken into account with GSMs, allthough even a GSM including all co-factor requirements of a given organism would not be able to predict the need for the organism to secrete citrate to scavenge iron.…”