Recent evidence indicates that in as diverse organisms as unicellular eukaryotes, higher plants and prokaryotes, anaerobic glycolysis relies on a pyrophosphate-dependent phosphofructokinase instead of the classical ATP-dependent enzyme. This difference in phosphoryl donor specificity does not necessarily reflect a primitive metabolism, as thought earlier, but could rather be the result of convergent evolution, fostered by the energetic advantage conferred to the cell when glycolysis is the sole source of ATP.

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Phosphofructokinase 2 was purified from spinach leaves by fractionation with poly(ethy1ene glycol) and by chromatography on blue Sepharose, anion exchanger Mono-Q and blue Trisacryl. A low-K, fructose-2,6-bisphosphatase copurified with phosphofructokinase 2 and its constitutive subunits could be easily identified by sodium dodecyl sulphate gel electrophoresis thanks to the formation of a [32P]phosphoenzyme intermediate upon short-time incubation in the presence of 1 pM fructose 2,6-[2-32P]bisphosphate. On anion-exchange chromatography, two peaks of phosphofructokinase 2/fructose-2,6-bisphosphatase were resolved. The first one, called L (light), represented about 10% of the phosphofructokinase 2 activity and was characterized by a phosphofructokinase 2/fructose-2,6-bisphosphatase activity ratio close to 1, by an M , of 132000 as measured by gel filtration, and by a series of subunits of M , comprised between 44000 and 70000. The second and major peak of phosphofructokinase 2, called H (heavy), had a phosphofructokinase 2/fructose-2,6-bisphosphatase ratio close to 8, an M , of 390000 and was made of 90000-M, subunits. The H form of phosphofructokinase 2 had a lower K,,, for fructose 6-phosphate than the L form and a higher Ki for a series of physiological inhibitors. By contrast, the kinetics of fructose-2,6-bisphosphatase was the same for the two forms of the enzyme. Upon incubation in the presence of papain or of a crude spinach leaf extract, the purified H form gave rise to products made of subunits of M, comprised between 70000 and 44000 but also of lower values which maintained their fructose-2,6-bisphosphatase activity. The H and L forms of phosphofructokinase 2/fructose-2,6-bisphosphatase were also detected in crude homogenates of castor bean endosperm and of Jerusalem artichoke tubers.Fructose 2,6-bisphosphate is a newly discovered regulator of metabolism present in most eukaryotic cells (reviewed in [l -31). It is synthesized from Fru6P and ATP by PFK 2 and is hydrolyzed back to Fru6P and Pi by FBPase 2. In liver and muscle, PFK 2 and FBPase 2 are part of a single bifunctional dimeric protein with an M , close to I10000 [2 -51. Liver and yeast PFK 2 are substrates for cyclic-AMP-dependent protein kinase which causes the inactivation of the enzyme in liver but its activation in yeast [3]. Some kinetic properties of PFK 2 and FBPase 2 present in a partially purified preparation of spinach leaves have also been reported [6-8a] but the enzymes have not been characterized at the molecular level.

Rice (Oryza sativa) seeds were imbibed for 3 days and the seedlings were further incubated for 8 days in the presence of either air or nitrogen. In aerobiosis, the specific activity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase and that of the ATP-dependent phosphofructokinase increased about fourfold. In anaerobiosis, the specific activity of ATP-dependent phosphofructokinase remained stable, whereas that of pyrophosphate:fructose 6-phosphate 1-phosphotransferase increased as much as in the presence of oxygen and there was also a fourfold increase in the concentration of fructose 2,6-bisphosphate, a potent stimulator of that enzyme. These data suggest a preferential involvement of pyrophosphate:fructose 6-phosphate 1-phosphotransferase rather than of ATP-dependent phosphofructokinase in glycolysis during anaerobiosis.

The pyrophosphate-dependent phosphofructokinase (PPi-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified. The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation. The protein was fragmented and a number of peptides were sequenced. Based on this information a PCR product was obtained from T. vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones. Southern analysis indicated the presence of five genes. One open reading frame (ORF) was completely sequenced and for two others the 5' half of the gene was determined. The sequences were highly similar. The complete ORF corresponded to a polypeptide of about 46 kDa. All the peptide sequences obtained were present in the derived sequences. The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half. The T. vaginalis enzyme was most similar to PPi-PFK of the mitochondriate heterolobosean, Naegleria fowleri. Most of the residues shown or assumed to be involved in substrate binding in other PPi-PFKs were conserved in the T. vaginalis enzyme. Direct comparison and phylogenetic reconstruction revealed a significant divergence among PPi-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists. The separation of these groups is supported with a high percentage of bootstrap proportions. The short T. vaginalis PFK shares a most recent common ancestor with the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the alpha- and beta-subunits of plant PPi-PFKs. The third group ("X") containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorferi. The fourth group ("Y") comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences. The well-studied PPi-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups. These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier. These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers.

PPi-dependent phosphofructokinase (PPi-PFK) was detected in extracts of the amoeba Naegleria fowleri, with a specific activity of about 15-30 nmol/min per mg of protein, which was increased about 2-fold by 0.5 mM AMP. PPi-PFK was inactivated upon gel filtration and could be re-activated by incubation at 30 degrees C in the presence of AMP. N. fowleri PPi-PFK was purified more than 1100-fold to near homogeneity with a yield of about 25%. The pure enzyme had a specific activity of 65 mumol/min per mg of protein, and SDS/PAGE analysis showed a single band, of 51 kDa. Size-exclusion chromatography revealed the existence of two forms: a large one (approximately 180 kDa), presumably a tetramer, which was active, and a smaller one (approximately 45 kDa), presumably the monomer, which was inactive, but could be re-activated and converted into the large form by incubation at 30 degrees C in the presence of 0.5 mM AMP. Reactivation was also observed at 30 degrees C in the absence of AMP, particularly at higher enzyme concentration or in the presence of poly(ethylene glycol). Inactivation of the tetrameric enzyme was promoted by 0.25 M potassium thiocyanate. The enzyme displayed Km values of 10 and 15 microM for fructose 6-phosphate and PPi, respectively, in the forward reaction, and of 35 and 590 microM for fructose 1,6-bisphosphate and Pi in the backward reaction. The activity was dependent on the presence of Mg2+. AMP increased Vmax. about 2-fold without changing the affinity for the substrates; its half-maximal effect was observed at 2 microM.

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Preclimacteric bananas fruits were treated for 12 h with ethylene to induce the climacteric rise in respiration. One day after the end of the hormonal treatment, the two activities of the bifunctional enzyme, phosphofructokinase 2/fructose-2,6-bisphosphatase started to increase to reach fourfold their initial value 6 days later. By contrast, the activities of the pyrophosphate-dependent and of the ATP-dependent 6-phosphofructo-1 -kinases remained constant during the whole experimental period, the first one being fourfold greater than the second. The concentrations of fructose 2,6-bisphosphate and of fructose 1,6-bisphosphate increased in parallel during 4 days and then slowly decreased, the second one being always about 100-fold greater than the first. The change in fructose 2,6-bisphosphate concentration can be partly explained by the rise of the bifunctional enzyme, but also by an early increase in the concentration of fructose 6-phosphate, the substrate of all phosphofructokinases, and also by the decrease in the concentration of glycerate 3-phosphate, a potent inhibitor of phosphofructokinase 2. The burst in fructose 2,6-bisphosphate and the activity of the pyrophosphate-dependent phosphofructokinase, which is in banana the only enzyme known to be sensitive to fructose 2,6-bisphosphate, can explain the well-known increase in fructose 1,6-bisphosphate which occurs during ripening.In ripening fruits, the presence of ethylene has been associated with a large increase in respiratory metabolism, particularly in so-called climacteric fruits in which low concentrations of exogenous ethylene induce autocatalytic ethylene production and accelerate the ripening process [1, 21. During this ethylene-induced burst in respiration, glycolysis is activated as indicated by the 10 -20-fold increase in Fru(l,6)Pz concentration [3-51. The mechanism which allows the increase of the glycolytic flux is not clear; it was therefore of interest to investigate its relationship to Fru(2,6)Pz, a new regulator of glycolysis discovered 6 years ago [6]. Fru(2,6)P2 is a regulatory molecule which promotes glycolysis and restricts gluconeogenesis in very different types of eukaryotic cells (reviewed in [7]). In higher plants, Fru(2,6)Pz activates PP1-PFK and inhibits cytoplasmic FBPase 1 [7]. A dramatic increase in its concentration has been observed in various situations in which a resumption of metabolic activity occurs [8-101.Climacteric fruits, such as banana, are good models to reevaluate the link between ethylene and glycolysis. The aim of the present work was to determine whether Fru(2,6)Pz