Phosphofructokinase from Dirofilaria immitis: effect of fructose 2,6-bisphosphate and AMP on the non-phosphorylated and phosphorylated forms of the enzyme

Abstract The enzyme responsible for the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P 2 ), 6-phosphofructo-2-kinase, was shown to be present in the heart worm, Dirofilaria immitis The level of Fru-2,6-P 2 was determined to be 4±0.3 nmol(g wet weight) −1 in the tissues of the filariid. Fru-2,6-P 2 stimulated the activity of both the non-phosphorylated and phosphorylated forms of the D. immitis phosphofructokinase (PFK). The K act values for Fru-2,6-P 2 were 378 ± 18 nM and 65 ± 6 nM for the non-phosphorylated and phosphorylated forms, respectively, at 1 mM fructose 6-phosphate (Fru-6-P) and 1 mM ATP at pH 6.8 AMP also stimulated the activity of both forms of the enzyme with K act values of 230 ± 10 μM and 37.3 ± 6.1 μM for the non-phosphorylated and phosphorylated forms, respectively. In the absence of any effectors, the S 0.5 values for Fru-6-P were 17.4 mM and 11.0 mM for the non-phosphorylated and phosphorylated forms, respectively, of the D. immitis PFK at 1 mM ATP, pH 6.8. These S 0.5 values were lowered to 0.03 mM by the combined effects of saturating levels of Fru-2,6-P 2 and AMP. A physiological assay was developed based on the level of metabolites in the parasite that influence the activity of PFK. This assay contained the known effectors of the PFK at concentrations approximating those found in the parasite. Under these conditions the K Fru-6-P values were 153 μM and 60 μM for the non-phosphorylated and phosphorylated forms of the PFK, respectively. These results suggest that in addition to activation by Fru-2,6-P 2 and AMP, phosphorylation also plays a key role in the regulation of PFK activity, and a combination of these three effectors is required for the full expression of PFK activity under cellular conditions in the tissues of the parasite.