GLUCONEOGENESIS FROM PYRUVATE IN THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS - INVOLVEMENT OF REACTIONS OF THE EMBDEN-MEYERHOF PATHWAY

The hyperthermophilic archaeon Pyrococcus furiosus was grown on pyruvate as carbon and energy source. The enzymes involved in gluconeogenesis were investigated. The following findings indicate that glucose-6-phosphate formation from pyruvate involves phosphoenolpyruvate synthetase, enzymes of the Embden-Meyerhof pathway and fructose-1,6-bisphosphate phosphatase. Cell extracts of pyruvate-grown P. furiosus contained the following enzyme activities: phosphoenolpyruvate synthetase (0.025 U/mg, 50-degrees-C), enolase (0.9 U/mg, 80-degrees-C), phosphoglycerate mutase (0.13 U/mg, 55-degrees-C), phosphoglycerate kinase (0.01 U/mg, 50-degrees-C), glyceraldehyde-3-phosphate dehydrogenase reducing either NADP+ or NAD+ (NADP+: 0.019 U/mg, NAD+: 0.009 U/mg; 50-degrees-C), triosephosphate isomerase (1.4 U/mg, 50-degrees-C), fructose-1,6-bisphosphate aldolase (0.0045 U/mg, 55-degrees-C), fructose-1,6-bisphosphate phosphatase (0.026 U/mg, 75-degrees-C), and glucose-6-phosphate isomerase (0.22 U/mg, 50-degrees-C). Kinetic properties (V(max) values and apparent Km values) of the enzymes indicate that they operate in the direction of sugar synthesis. The specific enzyme activities of phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase (NADP+-reducing) and fructose-1,6-bisphosphate phosphatase in pyruvate-grown P. furiosus were by a factor of 3, 10 and 4, respectively, higher as compared to maltose-grown cells suggesting that these enzymes are induced under conditions of gluconeogenesis. Furthermore, cell extracts contained ferredoxin: NADP+ oxidoreductase (0.023 U/mg, 60-degrees-C); phosphoenolpyruvate carboxylase (0.018 U/mg, 50-degrees-C) acts as an anaplerotic enzyme. Thus, in P. furiosus sugar formation from pyruvate involves reactions of the Embden-Meyerhof pathway, whereas sugar degradation to pyruvate proceeds via a modified ''non-phosphorylated'' Entner-Doudoroff pathway.