ATP-dependent Phosphofructokinase Research Articles

Molecular and Biochemical Characterization of the ADP-dependent Phosphofructokinase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively.

Purification and properties of the pyrophosphate-dependent phosphofructokinase from Dictyoglomus thermophilum Rt46 B.1.

The distribution of phosphofructokinase phosphoryl donor subtypes (ATP-, ADP-, and pyrophosphate) in the deeply rooted phylogenetic lineages of thermophiles is of interest with regard to the evolution of phosphofructokinase activity and of the Embden-Meyerhof pathway. In this article we present the first biochemical description of a thermostable pyrophosphate-dependent phosphofructokinase from the hyperthermophilic bacterium Dictyoglomus thermophilum. The enzyme was not allosterically controlled by traditional modulators of phosphofructokinases and has significant activity with tripolyphosphate and polyphosphate. Kinetic parameters of the enzyme suggest it plays primarily a glycolytic role. The enzyme required Mg2+ for optimal activity, was partially activated by some monovalent and divalent cations, and was strongly inhibited by Cu2+. The sequence of the 21 N-terminal residues suggests that the enzyme is most similar to the pyrophosphate-dependent phosphofructokinases from Amycolatopsis methanolica and the hyperthermophilic crenarchaeon Thermoproteus tenax, enzymes which have been suggested to represent an ancient lineage of phosphofructokinases (Siebers et al. 1998). The unexpected finding of a pyrophosphate-dependent phosphofructokinase in Dictyoglomus thermophilum, which is phylogenetically related to Thermotoga maritima, previously shown to possess an ATP-dependent phosphofructokinase activity, is discussed.

PPi-dependent phosphofructokinase from Thermoproteus tenax, an archaeal descendant of an ancient line in phosphofructokinase evolution.

Flux into the glycolytic pathway of most cells is controlled via allosteric regulation of the irreversible, committing step catalyzed by ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC 2.7.1.11), the key enzyme of glycolysis. In some organisms, the step is catalyzed by PPi-dependent PFK (PPi-PFK; EC 2.7.1.90), which uses PPi instead of ATP as the phosphoryl donor, conserving ATP and rendering the reaction reversible under physiological conditions. We have determined the enzymic properties of PPi-PFK from the anaerobic, hyperthermophilic archaeon Thermoproteus tenax, purified the enzyme to homogeneity, and sequenced the gene. The approximately 100-kDa PPi-PFK from T. tenax consists of 37-kDa subunits; is not regulated by classical effectors of ATP-PFKs such as ATP, ADP, fructose 2,6-bisphosphate, or metabolic intermediates; and shares 20 to 50% sequence identity with known PFK enzymes. Phylogenetic analyses of biochemically characterized PFKs grouped the enzymes into three monophyletic clusters: PFK group I represents only classical ATP-PFKs from Bacteria and Eucarya; PFK group II contains only PPi-PFKs from the genus Propionibacterium, plants, and amitochondriate protists; whereas group III consists of PFKs with either cosubstrate specificity, i.e., the PPi-dependent enzymes from T. tenax and Amycolatopsis methanolica and the ATP-PFK from Streptomyces coelicolor. Comparative analyses of the pattern of conserved active-site residues strongly suggest that the group III PFKs originally bound PPi as a cosubstrate.

The glycosomal ATP-dependent phosphofructokinase of Trypanosoma brucei must have evolved from an ancestral pyrophosphate-dependent enzyme.

Trypanosoma brucei contains an ATP-dependent phosphofructokinase (PFK), located in its glycosomes, which are peroxisome-like organelles sequestering the majority of its glycolytic enzymes. In this paper, we report the cloning and sequencing of the single-copy gene encoding this enzyme. Its amino-acid sequence is more similar to pyrophosphate (PPi)-dependent PFKs than to other ATP-dependent PFKs. A phylogenetic analysis suggests that the enzyme must have been derived from a PPi-dependent ancestral PFK, which changed its phospho-donor specificity during evolution. The enzyme is no longer capable of using PPi as phospho substrate, nor can it catalyze the reverse reaction as PPi-PFKs generally can. Moreover, the presence of a high pyrophosphatase activity in the cell renders it unlikely that PPi can function as free-energy source in present-day trypanosomes. It remains to be determined which mutations were responsible for the change in phospho-substrate specificity of the trypanosomatid PFK. As a result of its particular evolutionary history, the T. brucei PFK shows many structural differences, even at the active site, when compared with other ATP-dependent PFKs. These differences offer great potential for the structure-based design of trypanocidal drugs.

Carbon Partitioning during Sucrose Accumulation in Sugarcane Internodal Tissue.

The temporal relationship between sucrose (Suc) accumulation and carbon partitioning was investigated in developing sugarcane internodes. Radiolabeling studies on tissue slices, which contained Suc concentrations ranging from 14 to 42% of the dry mass, indicated that maturation coincided with a redirection of carbon from water-insoluble matter, respiration, amino acids, organic acids, and phosphorylated intermediates into Suc. It is evident that a cycle of Suc synthesis and degradation exists in all of the internodes. The decreased allocation of carbon to respiration coincides with a decreased flux from the hexose pool. Both the glucose and fructose (Fru) concentrations significantly decrease during maturation. The phosphoenolpyruvate, Fru-6-phosphate (Fru-6-P), and Fru-2,6-bisphosphate (Fru-2, 6-P2) concentrations decrease between the young and older internodal tissue, whereas the inorganic phosphate concentration increases. The calculated mass-action ratios indicate that the ATP-dependent phosphofructokinase, pyruvate kinase, and Fru-1,6-bisphosphatase reactions are tightly regulated in all of the internodes, and no evidence was found that major changes in the regulation of any of these enzymes occur. The pyrophosphate-dependent phosphofructokinase reaction is in apparent equilibrium in all the internodes. Substrate availability might be one of the prime factors contributing to the observed decrease in respiration.

Use of 3-D computer modelling and kinetic studies to analyse grapefruit pyrophosphate-dependent phosphofructokinase

The glycolytic reaction of grapefruit PPi-dependent phosphofructokinase (PFP) depends on the presence of Fru-2,6-P 2 ( K a=6.7 nM). This molecule was further demonstrated in grapefruit juice sac cells. Citrate, α-ketoglutarate and isocitrate competitively inhibited the binding of Fru-2,6-P 2 to PFP. The affinity for Fru-6-P ( K m=159 μM) and PPi ( K m=33 μM) were not affected by the addition of these molecules. In the gluconeogenic reaction, the presence of Fru-2,6-P 2 did not affect the K m of Fru-1,6-P 2 (61 μM) in contrast to orange fruit PFP. These results led to the building of a computer model of PFP, based on the known structure of Bacillus stearothermophilus ATP-dependent phosphofructokinase (ATP-PFK). The results show that catalysis of Fru-6-P in the α chain is most unlikely, due to amino-acid substitutions and that Fru-2,6-P 2 can bind between the α and β subunits.

Entamoeba histolytica:Computer-Assisted Modeling of Phosphofructokinase for the Prediction of Broad-Spectrum Antiparasitic Agents

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) is the rate-limiting glycolytic enzyme found in the pathogenic protistsEntamoeba histolytica, Giardia lamblia, Toxoplasma gondii, Trichomonas vaginalis,andNaegleria fowleri.The enzyme differs significantly from ATP-dependent phosphofructokinases found in humans and as such represents an important drug target. Current therapy for infections caused by these pathogens is inadequate, especially for children, pregnant women, and the immune compromised. The development of more selective, safer agents is imperative, as parasitic infections are currently a significant health threat worldwide and will likely become increasingly common agents of disease in the future. For the purpose of designing drugs to treat parasitic infections, we have constructed a model of PPi-PFK fromE. histolyticabased on the three-dimensional structure of the ATP-dependent PFK fromBacillus stearothermophilus.The model was used with the computer program Dock 3.5 (University of California, San Francisco) to predict the binding of pyrophosphate and selected bisphosphonates to the enzyme. The predicted drug–enzyme interactions suggested that two of these compounds would be competitive inhibitors of pyrophosphate. These drugs were tested againstE. histolyticaand inhibited the growth of amebaein vitro.This class of compounds may have broad-spectrum antiparasitic activity and, in the future, may facilitate the treatment of serious parasitic infections.

Cyclic AMP, a possible regulator of glycolysis in the ectomycorrhizal fungus Amanita muscaria.

The amounts of cyclic AMP (cAMP), fructose-2,6-bisphosphate (F26BP), trehalose and glycogen were determined in cell suspension cultures of the ectomycorrhiza-forming fungus Amanita muscaria (L. ex Fr.) Hooker. For the assay of cAMF a protocol was developed that enabled the detection of as little as 50 fmol of this secondary messenger by an enzyme-linked immuno assay (EIA). Values varied from < 1 and up to 5 pmol cAMP mg1 d. wt according to the age of the fungal culture. Typically, a transient increase in cAMP occurred after c. 4 d of culture of the fungus on glucose-containing medium. This increase (up to 100%) was followed by the start of the logarithmic growth phase, and by a more persistent increase in F26BP. In parallel, glucose in the medium started to decrease, whilst the amounts of fungal carbohydrates, especially the disaccharide trehalose, increased, From these data we assume that a high initial rate of glucose uptake caused an increase in the fungal pools of storage carbohydrates and, via activation of an adenylate cyclase, of cAMP. According to data reported for yeast cells this should enhance the formation of F26BP by phosphorylation of relevant enzymes. In animal and yeast cells an increase in the concentration of F26BP stimulates glycolysis by activation of the ATP-dependent phosphofructokinase (PFK). A. muscaria also possesses an F26BP activated PFK and, under conditions of symbiosis, host-derived carbohydrates are supplied mainly in the form of glucose. The implications of these findings to the regulation of carbohydrate metabolism of symbiotic plant root/fungus structures (ectomycorrhiza) are discussed.

Disruption of the phosphate-starvation response of oilseed rape suspension cells by the fungicide phosphonate.

The influence of the anti-fungal agent phosphonate (Phi) on the response of oilseed rape (Brassica napus L. cv. Jet Neuf) cell suspensions to inorganic phosphate (Pi) starvation was examined. Subculture of the cells for 7 d in the absence of Pi increased acid phosphate (APase; EC 3.1.3.2) and pyrophosphate (PPi)-dependent phosphofructokinase (PFP; EC 2.7.1.90) activities by 4.5- and 2.8-fold, respectively, and led to a 19-fold increase in Vmax and a 14-fold decrease in Km (Pi) and Pi uptake. Addition of 2 mM Pi to the nutrient media caused dramatic reductions in the growth and Pi content of the Pi-starved, but not Pi-sufficient cells, and largely abolished the Pi-starvation-dependent induction of PFP, APase, and the high-affinity plasmalemma Pi translocator. Immunoblotting indicated the cells contain three APase isoforms that are synthesized de novo following Pi stress, and that Pi treatment represses this process. Phosphonate treatment of Pi-starved cells significantly altered the relative extent of in-vivo 32P-labelling of polypeptides having M(rs) of 66, 55, 45 and 40 kDa. However, Phi had no effect on the total adenylate pool of Pi-starved cells which was about 32% lower than that of Pi-sufficient cells by day 7. Soluble protein levels, and activities of pyruvate kinase (EC 2.7.1.40) and ATP-dependent phosphofructokinase (EC 2.7.1.11) were unaffected by Pi starvation and/or Phi treatment. The effects of Phi on the growth, and APase and PFP activities of Pi-starved B. napus seedlings were similar to those observed in the suspension cells. The results re consistent with the hypothesis that a primary site of Phi action in higher plants is at the level of the signal transduction chain by which plants perceive and respond to Pi stress at the molecular level.

Identification of ATP-dependent phosphofructokinase as a regulatory step in the glycolytic pathway of the actinomycete Streptomyces coelicolor A3(2).

The ATP-dependent phosphofructokinase (ATP-PFK) of Streptomyces coelicolor A3(2) was purified to homogeneity (1,600-fold) and characterized (110 kDa, with a single type of subunit of 40 kDa); it is allosterically inhibited by phosphoenolpyruvate. Cloning of the pfk gene of S. coelicolor A3(2) and analysis of the deduced amino acid sequence (343 amino acids; 36,667 Da) revealed high similarities to the PPi-PFK enzyme from Amycolatopsis methanolica (tetramer, nonallosteric; 70%) and to the allosteric ATP-PFK enzymes from other bacteria, e.g., Escherichia coli (tetramer; 37%) and Bacillus stearothermophilus (tetramer, 41%). Further structural and functional analysis of the two actinomycete PFK enzymes should elucidate the features of these proteins that determine substrate specificity (ATP versus PPi) and allosteric (in)sensitivity.

Evidence for an up-regulation of the host and a down-regulation of the fungal phosphofructokinase activity in ectomycorrhizas of Norway spruce and fly agaric.

For an understanding of metabolic interactions in ectomycorrhizal associations it is essential to distinguish enzyme activities of the symbionts. For the ATP-dependent phosphofructokinase (PFK) from ectomycorrhizas of fly agaric (Amanita muscaria (L. ex Fr.) Hooker) on Norway spruce (Picea abies (L.) Karst.) we were able to achieve a symbiont-specific differentiation by special assay conditions. Substrate concentrations, Mg2+ : ATP ratio and pH values which were optimum for the fly agaric PFK completely suppressed the PFK activity of spruce roots. On the other hand, under the optimum assay conditions for the spruce root PFK, the fungal PFK activity was reduced by more than 90%. The most pronounced difference between the enzymes of both organisms was the response towards fructose-2,6-bisphosphate (F26BP); whereas F26BP had no influence on the spruce PFK activity, the fly agaric PFK activity was strongly enhanced by very low levels of F26BP. The distinction of the partner-specific PFK activities illustrated that mycorrhiza formation exerted partner-specific effects. On the basis of host d. wt of the mycorrhizas, the host-specific PFK activity was more than doubled compared with that of the non-mycorrhizal short roots. By contrast, the fungal PFK activity, on a fungal d.wt basis, was reduced in the mycorrhizas to less than 1/4 of the activity of the free-living mycelium.

Effects of subsoiling on lateral roots, sucrose metabolizing enzymes, and soil ergosterol in two Jeffrey pine stands.

We determined the effects of subsoiling on woody lateral roots and enzyme activities involved in stem carbon metabolism of 90- to 100-year-old Jeffrey pine (Pinus jeffreyi Grev. and Balf.) growing on the eastern side of the California Sierra Nevada Range. Twelve 1.0-ha plots were established on each of two sites. Four site treatments-thinning and subsoiling entire plots, thinning and no subsoiling, thinning and subsoiling skid trails only, and no thinning or subsoiling (undisturbed control)-were replicated three times on each site. Root excavations and assays of stem cambium sucrose metabolizing enzymes were carried out during the summer and fall of 1994 and 1995. Subsoiled plots had more roots < 1 cm in diameter exhibiting dieback than undisturbed plots. No relationship was found for stem cambial sucrose synthase (SS) activity among treatments or sites; however, a strong seasonal pattern was observed. Activity of pyrophosphate-dependent phosphofructokinase (PPi-PFK) was seasonal, whereas ATP-dependent phosphofructokinase (ATP-PFK) activity was constant throughout the seasons. Neither of the PFK activities responded to treatment or site. Among plots, ergosterol, a surrogate for estimating live fungal biomass, was present in highest concentrations in soil and rhizosphere samples from subsoiled plots.

The Fungicide Phosphonate Disrupts the Phosphate-Starvation Response in Brassica nigra Seedlings.

The development of Brassica nigra seedlings over 20 d of growth was disrupted by the fungicide phosphonate (Phi) in a manner inversely correlated with nutritional inorganic phosphate (Pi) levels. The growth of Pi-sufficient (1.25 mM Pi) seedlings was suppressed when 10, but not 5, mM Phi was added to the nutrient medium. In contrast, the fresh weights and root:shoot ratios of Pi-limited (0.15 mM) seedlings were significantly reduced at 1.5 mM Phi, and they progressively declined to about 40% of control values as medium Phi concentration was increased to 10 mM. Intracellular Pi levels generally decreased in Phi-treated seedlings, and Phi accumulated in leaves and roots to levels up to 6- and 16-fold that of Pi in Pi-sufficient and Pi-limited plants, respectively. Extractable activities of the Pi-starvation-inducible enzymes phosphoenolpyruvate phosphatase and inorganic pyrophosphate-dependent phosphofructokinase were unaltered in Pi-sufficient seedlings grown on 5 or 10 mM Phi. However, when Pi-limited seedlings were grown on 1.5 to 10 mM Phi (a) the induction of phosphoenolpyruvate phosphatase and inorganic pyrophosphate-dependent phosphofructokinase activities by Pi limitation was reduced by 40 to 90%, whereas (b) soluble protein concentrations and the activities of the ATP-dependent phosphofructokinase and pyruvate kinase were unaffacted. It is concluded that Phi specifically interrupts processes involved in regulation of the Pi-starvation response in B. nigra.

Cloning, sequencing and expression of the pyrophosphate-dependent phosphofructo-1-kinase from Naegleria fowleri

The cDNA for the PPi-dependent phosphofructo-1-kinase has been cloned and sequenced from a cDNA library prepared from the free-living amoeba Naegleria fowleri. The coding sequence of the cDNA consists of 1311 bases which translates into 437 amino acids with a molecular mass of 48095 Da. Comparison of the sequence with those of the previously described sequences of PPi-dependent phosphofructokinases from Propionibacterium freudenreichii and potato tuber revealed amino acid identities of 23 and 28% respectively and high conservation in those regions assumed to be part of the active site. The reading frame was cloned into an expression vector, which was transformed into Escherichia coli. Extracts of the transformed cells contained PPi-dependent phosphofructokinase activity that could be purified to homogeneity. The activity was lost on incubation with the chaotropic agent, KSCN, and recovered by subsequent incubation with AMP. These properties are consistent with those described by Mertens, De Jonckheere and Van Schaftingen [Biochem. J. (1993) 292, 797-803] for the enzyme prepared from Naegleria and support the idea that the cloned cDNA coded for the complete native enzyme. No nucleotide-binding motif or evidence for a nucleotide-binding site characteristic of the ATP-dependent phosphofructokinases could be found within the primary structure.

Structure of the genes encoding the α- and β-subunits of castor pyrophosphate-dependent phosphofructokinase

Full-length genomic clones encoding the α- and β-subunits of the pyrophosphate-dependent phosphofructokinase (PFP) from the castor plant have been isolated and sequenced. The gene (PFPα) encoding PFPα is approx. 5.8 kb in length and contains 19 exons, which collectively encode a protein of 617 amino acids (aa) having a deduced Mr of 67 360. PFPβ is approx. 4.6-kb long and contains 16 exons. Together, these exons encode a protein (PFPβ), of 552 aa with a deduced Mr of 60 114. The intron-exon splice junctions in both genes contain the consensus sequences typical for plants. An alignment of intron placement in castor PFPα and PFPβ with introns in the 5' portion of the gene encoding the ATP-dependent phosphofructokinase (PFK) from rabbit muscle, indicates that only one intron occupies the same position in all three genes. Furthermore, within castor PFPα and PFPβ, only two introns are identically placed. Within the promoter regions of castor PFPα and PFPβ, there are short sequences having high homology to each other (up to 65%). The results demonstrate, for the first time, that there is little homology between PFP and PFK, nor are PFPα and PFPβ closely related. This lack of homology suggests PFP did not evolve from PFK, but rather, that PFP and PFK have probably evolved from a common ancestral gene.

Regulation of Hexose-Phosphate Cycle Determines Glucose and Fructose Accumulation in Cherimoya ( Annona cherimola Mill.) during Ripening

Summary Cherimoya ( Annona cherimola Mill. cv. Fino de Jete ) is a climacteric fruit. Starch stores are converted mainly to glucose and fructose in equimolar concentrations during ripening. There is little accumulation of sucrose, which is consistent with the induction of invertase 2 days after harvesting. Activities of enzymes involved in the hexose-phosphate cycle have been determined during ripening. ATP-dependent phosphofructokinase shows a slight increase in maximum velocity independent of the presence of fructose 2,6-bisphosphate. Pyrophosphate-dependent phosphofructokinase and fructose 1,6-bisphosphatase, on the other hand, are activated and inhibited by fructose 2,6-bisphosphate, respectively, at the onset of ripening. Activities of these enzymes, as well as concentrations of fructose 2,6-bisphosphate, fructose 1,6bisphosphate and fructose 6-phosphate, suggest that glycolysis and gluconeogenesis are differentially activated and hexose-phosphate cycling is important in the regulation of carbon flux from starch during ripening.

Source of apparent ADP-dependent phosphofructokinase activity in plant extracts

This work investigates the ability of ADP to act as a phosphoryl donor for the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate in plants. Apparent ADP-dependent phosphofructokinase activity was detected in extracts of arum spadix, carrot tap root, spinach leaf, potato tuber and banana fruit, and in phosphofructokinase purified to near homogeneity from rabbit muscle. This apparent activity was an artifact resulting from adenylate kinase contamination of the triose phosphate isomerase/glycerol 3-phosphate dehydrogenase mixture used in the spectrophotometric assay of phosphofructokinase. This contaminant results in the conversion of ADP to AMP and ATP, the latter providing the substrate for conventional ATP-dependent phosphofructokinase. The following observations support this proposal. (i) The apparent ADP-dependent activity was observed only after incubations support this and ADP before starting the assay. (ii) ATP was formed during this preincubation. (iii) Contaminating adenylate kinase activity was sufficient to support the observed rate of ADP-dependent phosphorylation of fructose 6-phosphate. (iv) No ADP-dependent phosphofructokinase activity was detected using a discontinuous assay. Similar contamination may contribute to the apparent ADP-dependent phosphofructokinase activity reported by others (K.K. Surendranathan and P.M. Nair, Plant Science 59 (1989) 45–51).

Transgenic potato plants with strongly decreased expression of pyrophosphate:fructose-6-phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in their tubers

Potato (Solanum tuberosum L.) plants were transformed with “antisense” constructs to the genes encoding the α-and β-subunits of pyrophosphate: fructose-6-phosphate phosphotransferase (PEP), their expression being driven by the constitutive CaMV 35S promotor. (i) In several independent transformant lines, PFP expression was decreased by 70–90% in growing tubers and by 88–99% in stored tubers. (ii) The plants did not show any visual phenotype, reduction of growth or decrease in total tuber yield. However, the tubers contained 20–40% less starch than the wild type. Sucrose levels were slightly increased in growing tubers, but not at other stages. The rates of accumulation of sucrose and free hexoses when tubers were stored at 4° C and the final amount accumulated were the same in antisense and wild-type tubers. (iii) Metabolites were investigated at four different stages in tuber life history; growing (sink) tubers, mature tubers, cold-sweetening tubers and sprouting (source) tubers. At all stages, compared to the wild type, antisense tubers contained slightly more hexose-phosphates, two- to threefold less glycerate-3-phosphate and phosphoenolpyruvate and up to four-to fivefold more fructose-2,6-bisphosphate. (iv) There was no accumulation or depletion of inorganic pyrophosphate (PPi), or of UDP-glucose relative to the hexose-phosphates. (v) The pyruvate content was unaltered or only marginally decreased, and the ATP/ADP ratio did not change. (vi) Labelling experiments on intact tubers did not reveal any significant decrease in the unidirectional rate of metabolism of [U-14C]sucrose to starch, organic acids or amino acids. Stored tubers with an extreme (90%) reduction of PFP showed a 25% decrease in the metabolism of [U14-C] sucrose. (vii) Metabolism (cycling) of [U-14C]glucose to surcrose increased 15-fold in discs from growing antisense tubers, compared with growing wild-type tubers. Resynthesis of sucrose was increased by 10–20% when discs from antisense and wild-type tubers stored at 4° C (cold sweetening) were compared. The conversion of [U-14C]glucose to starch was decreased by about 30% and 50%, respectively. (viii) The randomisation of [1-13C]glucose in the glucosyl and fructosyl moieties of sucrose was decreased from 13.8 and 15.7% in the wild type to 3.6 and 3.9% in an antisense transformant. Simultaneously, randomisation in glucosyl residues isolated from starch was reduced from 14.4 to 4.1%. (ix) These results provide evidence that PFP catalyses a readily reversible reaction in tubers, which is responsible for the recycling of label from triose-phosphates to hexose-phosphates, but with the net reaction in the glycolytic direction. The results do not support the notion that PFP is involved in regulating the cytosolic PPi concentration. They also demonstrate that PFP does not control the rate of glycolysis, and that tubers contain exessive capacity to phosphorylate fructose-6-phosphate. The decreased concentration of phosphoenolpyruvate and glycerate-3-phosphate compensates for the decrease of PFP protein by stimulating ATP-dependent phosphofructokinase, and by stimulating fructose-6-phosphate,2-kinase to increase the fructose-2,6-bisphosphate concentration and activate the residual PFP. The decreased starch accumulation is explained as an indirect effect, caused by the increased rate of resynthesis (cycling) of sucrose in the antisense tubers.

Cloning, sequencing, and expression in Escherichia coli of the gene coding for phosphofructokinase in Lactobacillus bulgaricus

A fragment of 1,185 bp containing the gene coding for phosphofructokinase (ATP:D-fructose-6-phosphate-1-phosphotransferase; EC 2.7.1.11) in Lactobacillus bulgaricus has been cloned, sequenced, and expressed in Escherichia coli. The amino acid sequence of this enzyme was homologous to those of the ATP-dependent phosphofructokinases from E. coli, Thermus thermophilus, Spiroplasma citri, and Bacillus stearothermophilus, suggesting that these enzymes have closely related structures despite their different regulatory properties. The recombinant protein had the same structural and functional properties as did the original enzyme. The 3' end of the 1,185-bp fragment showed the presence of an open reading frame corresponding to the N-terminal amino acid sequence of the pyruvate kinase from L. bulgaricus. This gene organization, the same as that in S. citri (C. Chevalier, C. Saillard, and J. M. Bové, J. Bacteriol. 172:2693-2703, 1990) and B. stearothermophilus (D. Walker, W. N. Chia, and H. Muirhead, J. Mol. Biol. 228:265-276, 1992; H. Sakai and T. Ohta, Eur. J. Biochem. 311:851-859, 1993) but different from that in E. coli (H. W. Hellinga and P. R. Evans, Eur. J. Biochem. 149:363-373, 1985), indicated that the same transcription unit apparently contained the genes for phosphofructokinase and pyruvate kinase, the two key enzymes of glycolysis. The possibility that these genes could be transcribed at the same time suggested that in L. bulgaricus, the coordinated regulation of phosphofructokinase and pyruvate kinase occurs at the levels of both biosynthesis and enzymatic activity.

Pyrophosphate-dependent phosphofructo-1-kinase complements fructose 1,6-bisphosphatase but not phosphofructokinase deficiency in Escherichia coli.

The gene from Propionibacterium freudenreichii for PPi-dependent phosphofructo-1-kinase, an enzyme that is found in some bacteria, in a number of anaerobic protists, and in plants, complements the absence of fructose 1,6-bisphosphatase in Escherichia coli but does not complement the deficiency of the ATP-dependent phosphofructokinase.