In the current study we report the properties of P

In the current study, we report the properties of P. anserina mutants carrying mutations in a gene encoding the bi-functional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). This enzyme catalyzes two opposite reactions (Pilkis et al., 1995, Rider et al., 2004): synthesis of Fru-2,6-P2 (fructose 2,6-biphosphate) from Fru-6-P (fructose-6-phosphate) and ATP via its phosphofructose-2-kinase activity (PFK-2), and degradation of Fru-2,6-P2 to Fru-6-P and inorganic phosphate via its fructose-2,6-bisphosphatase activity (FBPase-2). A mutant carrying a single substitution in a conserved motif of the phosphatase domain was first isolated as a suppressor of a respiratory-deficient mutant. Null mutations of the kinase and phosphatase domains were then constructed. Their analysis showed that only mutations affecting the phosphatase domain are able to suppress the respiratory mutant phenotype and that this suppression is correlated with a strong expression of the AOX due to the activation of the RSEs transcription factors. Moreover we show that the key SynaptoRedTM C2 of gluconeogenesis are also strongly overexpressed. The phenotype of the phosphatase mutants was intriguing since two other genes predicted to each also encode a PFK-2/FBPase-2 are present in all the tested Ascomycetes, one of them being probably mono-functional, lacking FBPase-2 activity.
Materials and methods
Results
Discussion
Acknowledgements
Mammalian muscle fructose 1,6-bisphosphatase (FBPase) [EC 3.1.3.11] is a regulatory enzyme of glyconeogenesis. The enzyme is strongly inhibited by AMP and the determined for AMP of muscle FBPase is about 100 times lower than the concentration of AMP in myocyte, which indicates that muscle FBPase should be almost completely inhibited in vivo , , . On the other hand, the synthesis of glycogen from carbohydrate precursors in skeletal muscle, in which FBPase is indispensable, has also been observed , , . Recently we have shown that interaction of muscle FBPase with muscle aldolase in vitro results in the formation of heterologous complex, and that FBPase associated with aldolase is entirely insensitive to AMP inhibition , . Through an immunocytochemical study we have found that human muscle FBPase is located on both sides of the Z-line, where it supposedly interacts with α-actinin . Assuming that in vivo aldolase binds to FBPase, the presence of aldolase on both sides of the Z-line may also be expected.
Introduction Fructose 1,6-bisphosphatase (FBPase) [EC 3.1.3.11] catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate (Benkovic and deMaine, 1982, Tejwani, 1983). Two isozymes of FBPase, liver and muscle, have been found in vertebrate tissues (Tejwani, 1983, Al-Robaiy and Eschrich, 1999, Rakus and Dzugaj, 2000). The expression of two FBPase isozymes in mammals is the effect of the existence of two different genes that were created by the duplication of one FBPase gene in mammalian ancestors (Tillmann et al., 2002). Kinetic properties of both isozymes are virtually identical: both require magnesium, manganese, zinc or cobalt in order to be active; both are activated by potassium and ammonium ions and inhibited competitively by fructose 2,6-bisphosphate and allosterically by AMP (Pilkis et al., 1981, Benkovic and deMaine, 1982, Tejwani, 1983, Zhang et al., 1996, Rakus and Dzugaj, 2000). The liver and muscle isozymes have been recognized as respective regulatory enzymes of gluconeogenesis and glyconeogenesis – the synthesis of glycogen from noncarbohydrates. The basic difference between them concerns their sensitivity toward the AMP inhibition. A determined I0.5 value for the muscle isozyme is in the range of 0.05–0.1μM, which is 50–100 times lower than the corresponding value for the liver isozyme (Rakus and Dzugaj, 2000, Rakus et al., 2003b). The sensitivity of muscle isozyme toward AMP raises a question of how this enzyme can be active in the presence of a physiological concentration of AMP, nearly 100 times higher than I0.5 of FBPase (Skalecki et al., 1995). Quite recently we have found that the muscle aldolase [EC 4.1.2.13] interacts with muscle FBPase in vitro, which results in the formation of a heterologous complex, in which FBPase is insensitive to AMP inhibition (Rakus et al., 2003b). Aldolase catalyzes a reversible reaction of synthesis of fructose 1,6-bisphosphate from 3-phosphoglyceraldehyde and phosphodihydroxyacetone. In the gluconeogenesis, as well as in the glyconeogenesis, aldolase supplies substrate to FBPase (Littlechild and Watson, 1993).