Kinetic Characterisation of Phosphofructokinase Purified from Setaria cervi: A Bovine Filarial Parasite

Abstract

Phosphofructokinase (PFK), a regulatory enzyme in glycolytic pathway, has been purified to electrophoretic homogeneity from adult female Setaria cervi and partially characterized. For this enzyme, the Lineweaver-Burk's double reciprocal plots of initial rates and D-fructose-6-phosphate (F-6-P) or Mg-ATP concentrations for varying values of cosubstrate concentration gave intersecting lines indicating that K(m) values for F-6-P (1.05 mM) and ATP (3 μM) were independent of each other. S. cervi PFK, when assayed at inhibitory concentration of ATP (>0.1 mM), exhibited sigmoidal behavior towards binding with F-6-P with a Hill coefficient (n) value equal to 1.8 and 1.7 at 1.0 and 0.33 mM ATP, respectively. D-fructose-1,6-diphosphate (FDP) competitively inhibited the filarial enzyme: K(i) and Hill coefficient values being 0.18 μM and 2.0, respectively. Phosphoenolpyruvate (PEP) also inhibited the enzyme competitively with the K(i) value equal to 0.8 mM. The Hill coefficient values (>1.5) for F-6-P (at inhibitory concentration of ATP) and FDP suggested its positive cooperative kinetics towards F-6-P and FDP, showing presence of more than one binding sites for these molecules in enzyme protein and allosteric nature of the filarial enzyme. The product inhibition studies gave us the only compatible mechanism of random addition process with a probable orientation of substrates and products on the enzyme surface.

Figure 1

Effect of F-6-P concentration on the rate of S. cervi PFK catalyzed reaction. ATP concentration was 50 (○) and 100 (•) μM and Mg²⁺ concentration was 3.3 mM. Enzyme concentration was 6.6 μg/mL. Other conditions were same as in standard enzyme assay. (b) Double reciprocal plot of the data of (a). ATP concentrations were 50 (○) and 100 (•) μM.

Figure 2

Effect of ATP concentration on the rate of PFK catalyzed reaction. F-6-P concentration was 1.6 (○) and 3.3 (•) mM and Mg²⁺ concentration was 3.3 mM. Enzyme concentration was 10 μg/mL. Other conditions were same as in standard enzyme assay. (b) Double reciprocal plot of the data of (a). F-6-P concentrations were 1.6 (Δ) and 3.3 (▲) mM.

Figure 3

Effect of F-6-P concentration on the rate of S. cervi PFK catalyzed reaction at different fixed and high concentration of ATP. ATP concentration was 0.1 (Δ), 0.33 (○), and 1.0 (•) mM and Mg²⁺ concentration was 3.3 mM. Enzyme concentration was 3.3 μg/mL. Other conditions were same as in standard enzyme assay. Inset. Hill plot of the data of (a) at 0.2–6.0 mM F-6-P concentrations. ATP concentrations were 0.1 (Δ), 0.33 (○), and 1.0 (•).

Figure 4

Effect of ATP concentration on the rate of PFK catalyzed reaction at fixed concentration of F-6-P and Mg²⁺ (3.3 mM each). Inset shows the plot obtained on assaying the enzyme at variable inhibitory (>0.1 mM) concentration of ATP at two fixed concentrations of F-6-P such as 1.6 (•) and 3.3 (○) mM. Mg²⁺ concentration was constant (3.3 mM). Enzyme concentration was 10 μg/mL. Other conditions were same as in standard enzyme assay.

Figure 5

Effect of Mg²⁺ and ATP concentrations on the rate of PFK-catalyzed reaction. For (○), Mg²⁺ ion concentration was equal to and varied together with that of ATP. For (•), Mg²⁺ ion concentration was equal to 3.33 mM. F-6-P concentration in each case was 3.33 mM. Enzyme concentration was 10 μg/mL. Other conditions were same as in standard enzyme assay.

Figure 6

Effect of Mg²⁺ concentration on the rate of PFK-catalyzed reaction. Concentrations of F-6-P and ATP were 3.33 and 0.1 mM, respectively. Enzyme concentration was 10 μg/mL. Other conditions were same as in standard enzyme assay. Inset shows the double reciprocal plot of the data.

Figure 7

(a) Effect of FDP on the rate of PFK catalyzed reaction. FDP concentrations were nil (○), 1.0 (Δ), and 2.0 (▲) μM. Mg²⁺ concentration was constant (3.3 mM). The concentration of ATP was 0.1 mM. Enzyme concentration was 10 μg/mL. Other conditions were same as in standard enzyme assay. (b) Hill plot of the data of the effect of FDP on the rate of PFK catalyzed reaction. The concentrations of F-6-P, ATP, and Mg²⁺ were 3.3, 0.1, and 3.3 mM, respectively. Enzyme concentration was 6.6 μg/mL. V _i and V ₀ are the rates of reaction in the presence and absence of FDP. Other conditions were same as in standard enzyme assay.

Figure 8

Effect of PEP on the rate of PFK-catalyzed reaction. PEP concentrations were nil (□), 1.0 (○), 2.0 (•), and 5.0 (Δ) mM. Mg²⁺ and ATP concentrations were 3.3 and 0.1 mM, respectively. Enzyme concentration was 10 μg/mL. Other conditions were same as in standard enzyme assay.