title: The sugar phosphate specificity of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
abstract: The sugar phosphate specificity of the active site of 6-phosphofructo-2-kinase and of the inhibitory site of fructose-2,6-bisphosphatase was investigated. The Michaelis constants and relative Vmax values of the sugar phosphates for the 6-phosphofructo-2-kinase were: D-fructose 6-phosphate, Km = 0.035 mM, Vmax = 1; L-sorbose 6-phosphate, Km = 0.175 mM, Vmax = 1.1; D-tagatose 6-phosphate, Km = 15 mM, Vmax = 0.15; and D-psicose 6-phosphate, Km = 7.4 mM, Vmax = 0.42. The enzyme did not catalyze the phosphorylation of 1-O-methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, 2,5-anhydro-D-mannitol 6-phosphate, D-ribose 5-phosphate, or D-arabinose 5-phosphate. These results indicate that the hydroxyl group at C-3 of the tetrahydrofuran ring must be cis to the beta-anomeric hydroxyl group and that the hydroxyl group at C-4 must be trans. The presence of a hydroxymethyl group at C-2 is required; however, the orientation of the phosphonoxymethyl group at C-5 has little effect on activity. Of all the sugar monophosphates tested, only 2,5-anhydro-D-mannitol 6-phosphate was an effective inhibitor of the kinase with a Ki = 95 microM. The sugar phosphate specificity for the inhibition of the fructose-2,6-bisphosphatase was similar to the substrate specificity for the kinase. The apparent I0.5 values for inhibition were: D-fructose 6-phosphate, 0.01 mM; L-sorbose 6-phosphate, 0.05 mM; D-psicose 6-phosphate, 1 mM; D-tagatose 6-phosphate, greater than 2 mM; 2,5-anhydro-D-mannitol 6-phosphate, 0.5 mM. 1-O-Methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, and D-arabinose 5-phosphate did not inhibit. Treatment of the enzyme with iodoacetamide decreased sugar phosphate affinity in the kinase reaction but had no effect on the sensitivity of fructose-2,6-bisphosphatase to sugar phosphate inhibition. The results suggest a high degree of homology between two separate sugar phosphate binding sites for the bifunctional enzyme.
authors.forename: S J
authors.initials: SJ
authors.lastname: Pilkis
authors.forename: J
authors.initials: J
authors.lastname: Pilkis
authors.forename: M R
authors.initials: MR
authors.lastname: el-Maghrabi
authors.forename: T H
authors.initials: TH
authors.lastname: Claus
mesh_terms: D000818:Animals; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism / Q000494:pharmacology
chemical_list: D013403:Sugar Phosphates; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D007460:Iodoacetamide