Limited proteolysis and photoaffinity labeling with 8-azido-ATP of fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase.
Limited proteolysis and photoaffinity labeling of fructose-6-P,2-kinase and fructose-2,6-bisphosphatase were studied. Proteolysis by trypsin proceeds in two stages in which the first cleavage yields a product, Mr about 53,000, which has lost 90% of fructose-6-P,2-kinase, but retains nearly 80% of fructose-2,6-bisphosphatase. Further digestion of this product yields a second cleavage product, Mr about 50,000, which is completely devoid of the kinase and most of the phosphatase activities. These results indicate that fructose-6-P,2-kinase resides only in the original ("native") enzyme (Mr = 55,000), but fructose-2,6-bisphosphatase activity is present in both the native enzyme and the cleavage product(s). All three activities of fructose-6-P,2-kinase including the forward, the reverse, and ATP-ADP exchange activities are lost to the same degree by the mild proteolysis. Ki of fructose-6-P for fructose-2,6-bisphosphatase is not altered by the proteolysis. Partial protection against the proteolysis is provided by ATP, fructose-6-P, and fructose-2,6-P2. When the tryptic digestion of fructose-6-P,2-kinase:fructose-2,6-bisphosphatase was performed before and after phosphorylation of the enzyme by cAMP-dependent protein kinase, both the first and the second cleavage products contained the phosphorylation site. 8-Azido-ATP serves as a substrate for fructose-6-P,2-kinase with a Km of about 1 mM. Exposure of the enzyme-8-azido-ATP complex results in covalent incorporation (0.7 mol/mol of subunit) and 90% inactivation of fructose-6-P,2-kinase without loss of fructose 2,6-bisphosphatase. When the native and the first cleavage product of tryptic digestion were photoaffinity labeled with [alpha-32P]8-azido-ATP, the radiolabel occurred only in the native enzyme. These results provide evidence in support of, although not conclusive, the idea that the active sites of this bifunctional enzyme are different and located in two distinct sites.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D025481:Phosphofructokinase-2; D010770:Phosphotransferases / Q000378:metabolism*; D051381:Rats; D014357:Trypsin
D000345:Affinity Labels; D001386:Azides; D010446:Peptide Fragments; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D006597:Fructose-Bisphosphatase; D014357:Trypsin