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...