Kinetic studies on the activation of pyrophosphate-dependent phosphofructokinase from mung bean by fructose 2,6-bisphosphate and related compounds.
Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was purified from the mung bean Phaseolus aureus. The enzyme is activated by fructose 2,6-bisphosphate at nanomolar concentrations. The enzyme exhibits Michaelis-Menten kinetics, and the reaction mechanism, deduced from initial velocity studies in the absence of inhibitors as well as product and dead-end inhibition studies, is rapid equilibrium random in the presence and absence of fructose 2,6-bisphosphate. In the direction of fructose 6-phosphate phosphorylation, saturating fructose 2,6-bisphosphate (1 microM) increases V congruent to 9-fold and increases V/KMgPPi and V/KF6P about 30-fold. In the reverse direction (phosphate phosphorylation), the same concentration of activator has little if any effect on V or the Km for inorganic phosphate (Pi) and Mg2+ but does increase V/KFBP about 42-fold. No changes were observed in any of the other rate constants. The binding affinity of fructose 2,6-bisphosphate to all enzyme forms is identical. The activator site of the mung bean PPi-PFK binds fructose 2,6-bisphosphate with a Kact of 30 nM with the 2,5-anhydro-D-glucitol 1,6-bisphosphate (the most effective analogue) 33-fold less tightly. Of the alkanediol bisphosphate series, 1,4-butanediol bisphosphate exhibited the tightest binding (Kact congruent to 3 microM). These and a series of other activating analogues are discussed in relation to the activator site.
D011756:Diphosphates / Q000494:pharmacology*; D004789:Enzyme Activation; D007887:Fabaceae; D005635:Fructosediphosphates / Q000494:pharmacology*; D006598:Hexosediphosphates / Q000494:pharmacology*; D007700:Kinetics; D008433:Mathematics; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D010946:Plants, Medicinal; D013329:Structure-Activity Relationship; D013403:Sugar Phosphates / Q000494:pharmacology*
D011756:Diphosphates; D005635:Fructosediphosphates; D006598:Hexosediphosphates; D013403:Sugar Phosphates; C027652:fructose 2,6-diphosphate; D010732:Phosphofructokinase-1