Studies on heart phosphofructokinase. Use of fructose 6-sulfate as an alternative substrate to study the mechanism of action and active site specificity.
Fructose 6-sulfate was synthesized by direct sulfurylation of fructose and was isolated by two selective steps: (a) conversion of the 6-sulfuryl ester to fructose 1-phosphate-6-sulfate with phosphofructokinase; (b) conversion of fructose 1-phosphate-6-sulfate to fructose 6-sulfate by fructose-1,6-diphosphatase. Utilizing crystalline sheep heart phosphofructokinase, kinetic studies with the alternative substrate were carried out at pH 8.2 which is optimal for nonallosteric kinetics. The data are consistent with an ordered addition of the two substrates with the first, MgATP, being at thermodynamic equilibrium. The Vmax and Km obtained with fructose 6-sulfate were 0.03- and 100-fold, respectively, that obtained with the natural substrate. The study suggests that the divalent phosphoryl moiety is intimately involved in the active site conformation. Identification of the product of the reaction, fructose 1-phosphate-6-sulfate, was confirmed through studies with aldolase, fructose-1,6-diphosphatase, and by 31P NMR. The utilization of fructose 6-sulfate as a substrate by yeast glucose-6-phosphate isomerase could not be demonstrated.
D000818:Animals; D001665:Binding Sites; D005636:Fructosephosphates; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D009206:Myocardium / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D012756:Sheep; D013329:Structure-Activity Relationship; D013464:Sulfuric Acids / Q000494:pharmacology
D005636:Fructosephosphates; D013464:Sulfuric Acids; D010732:Phosphofructokinase-1