citrate inhibition-resistant form of 6-phosphofructo-1-kinase from Aspergillus niger.
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 Ki value determined to be 1.5 mM, in the system with 5 mM Mg2+ 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.
Mlakar
Tina
T
National Institute of Chemistry, Department of Biotechnology, Hajdrihova 19, SI-1001 Ljubljana, Slovenia.
Legisa
Matic
M
D000595:Amino Acid Sequence; D000818:Animals; D001234:Aspergillus niger / Q000187:drug effects / Q000201:enzymology*; D019343:Citric Acid / Q000494:pharmacology*; D004789:Enzyme Activation; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D011817:Rabbits; D016415:Sequence Alignment
D000644:Quaternary Ammonium Compounds; D019343:Citric Acid; D010732:Phosphofructokinase-1; D008274:Magnesium