Fructose-1,6-bisphosphatase from rat liver. A comparison of the kinetics of the unphosphorylated enzyme and the enzyme phosphorylated by cyclic AMP-dependent protein kinase.
A purification procedure for rat hepatic fructose-1,6-bisphosphatase, described earlier, has been improved, resulting in an enzyme preparation with a neutral pH optimum and with both phosphorylatable serine residues present. The subunit Mr was 40,000. Phosphorylation in vitro with cyclic AMP-dependent protein kinase resulted in the incorporation of 1.4 mol of phosphate/mol of subunit and led to an almost 2-fold decrease in apparent Km for fructose-1,6-bisphosphate. In contrast to yeast fructose-1,6-bisphosphatase, fructose-2,6-bisphosphate had no effect on the rate of phosphorylation or dephosphorylation of the intact enzyme. The effects of the composition of the assay medium, with regard to buffering substance and Mg2+ concentration, on the apparent Km values of phosphorylated and unphosphorylated enzyme were investigated. The kinetics of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase were studied with special reference to the inhibitory effects of adenine nucleotides and fructose-2,6-bisphosphate. Unphosphorylated fructose-1,6-bisphosphatase was more susceptible to inhibition by both AMP and fructose 2,6-bisphosphate than phosphorylated enzyme, at high and low substrate concentrations. Both ATP and ADP had a similar effect on the two enzyme forms, ADP being the more potent inhibitor. Finally, the combined effect of several inhibitors at physiological concentrations was studied. Under conditions resembling the gluconeogenic state, phosphorylated fructose-1,6-bisphosphatase was found to have twice the activity of the unphosphorylated enzyme.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology*; D004789:Enzyme Activation; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D010750:Phosphoproteins; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D006597:Fructose-Bisphosphatase; D008274:Magnesium