Differential effects of proteolysis and protein modification on the activities of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase catalyzes exchange reactions between ADP and ATP and between fructose-6-P and fructose-2,6-P2 at histidyl residues. Limited proteolysis of the enzyme with thermolysin yielded an enzyme core with a subunit molecular weight of 35,000-38,000. This enzyme core had no kinase activity and a 2-fold activated bisphosphatase activity whose sensitivity to the product inhibitor fructose-6-P was unchanged. The thermolysin-treated enzyme also did not catalyze the fructose-6-P/fructose-2,6-P2 exchange reaction but did catalyze the ADP/ATP exchange. These results suggest that 1) the enzyme's reactions may be catalyzed at two active sites, 2) there are at least two fructose-6-P binding sites, 3) the fructose-6-P/fructose-2,6-P2 exchange is catalyzed only at the kinase site, and 4) inactivation of the exchange and kinase reactions by thermolysin digestion is due to the loss of the fructose-6-P binding site of the kinase. Also consistent with these conclusions was the finding that oxidation of the enzyme with ascorbate/Fe3+ or H2O2 resulted in complete loss of the kinase activity as well as the fructose-6-P/fructose-2,6-P2 exchange but did not affect the bisphosphatase activity or the ADP/ATP exchange. Dithiothreitol could completely reactivate the ascorbate/Fe3+-inactivated enzyme, suggesting that oxidation occurred at a sulfhydryl group(s) essential for fructose-6-P binding in the kinase reaction. In addition, the kinase and fructose-6-P/fructose-2,6-P2 exchange reactions were more sensitive to inactivation by diethylpyrocarbonate than was the bisphosphatase. The different responses of the kinase and bisphosphatase reactions to the action of these various protein-modifying agents and to thermolysin digestion support the existence of a separate site for each reaction and an essential role for sulfhydryl groups at the sugar-phosphate-binding site(s) of the kinase.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D013820:Thermolysin / Q000494:pharmacology*
D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D013820:Thermolysin