6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase from rat liver.
Rat liver 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was phosphorylated by [gamma-32P]ATP plus fructose-6-P or [2-32P]fructose-2,6-P2. The radioactivity co-migrated with homogeneous 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase during sodium dodecyl sulfate-disc gel electrophoresis and the phosphoenzyme was acid-labile and base-stable. Hydrolysis of the phosphate group from phosphoenzyme prepared with either donor and the hydrolysis of their tryptic phosphopeptides depended on pH similarly. The pH dependence suggested that phosphate was linked to the N3 of a histidine residue. Co-electrophoresis and co-chromatography of alkaline hydrolysates of the labeled phosphoenzyme prepared from either substrate allowed the definitive identification of 3-phosphohistidine in the degradation products. Modification of the enzyme with diethylpyrocarbonate inactivated both the phosphotransferase and phosphohydrolase activities and suppressed the phosphorylation of the enzyme by ATP and fructose-6-P or by fructose-2,6-P2. Trypsin digestion of the phosphoenzyme formed upon incubation with ATP or fructose-2,6-P2 yielded an identical phosphopeptide after high pressure liquid chromatography. All the above data are consistent with this enzyme catalyzing both phosphohydrolase and phosphotransferase reactions with the mediation of phosphohistidine in the reaction scheme(s).
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004355:Drug Stability; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D005636:Fructosephosphates / Q000378:metabolism; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D014357:Trypsin
D005636:Fructosephosphates; D010750:Phosphoproteins; D006639:Histidine; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D006597:Fructose-Bisphosphatase; D014357:Trypsin; D004047:Diethyl Pyrocarbonate