Cooperative effect of fructose bisphosphate and glyceraldehyde-3-phosphate dehydrogenase on aldolase action

The combination of binding and kinetic approaches is suggested to study (i) the mechanism of substrate-modulated dynamic enzyme associations; (ii) the specificity of enzyme interactions. The effect of complex formation between aldolase and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) on aldolase catalysis was investigated under pseudo-first-order conditions. No change in kcat but a significant increase in KM of fructose 1,6-bisphosphate for aldolase was found when both enzymes were obtained from muscle. In contrast, kcat rather than KM changed if dehydrogenase was isolated from yeast. Next, the conversion of fructose 1-phosphate was not affected by interactions between enzyme couples isolated from muscle. The influence of fructose phosphates on the enzyme-complex formation was studied by means of covalently attached fluorescent probe. We found that the interaction ws not perturbed by the presence of fructose 1-phosphate; however, fructose 1,6-bisphosphate altered the dissociation constant of the enzyme complex. A molecular model for fructose 1,6-bisphosphate-modulated enzyme interaction has been evaluated which suggests that high levels of fructose bisphosphate would drive the formation of the 'channelling' complex between aldolase and glyceraldehyde-3-phosphate dehydrogenase.     

Degradation of endogenous fructose during catabolism of sucrose and mannitol in halophilic archaebacteria

Metabolism of fructose arising endogenously from sucrose or mannitol was studied in halophilic archaebacteria Haloarcula vallismortis and Haloferax mediterranei. Activities of the enzymes of Embden-Meyerhof-Parnas (EMP) pathway, Entner-Doudoroff (ED) pathway and Pentose Phosphate (PP) pathway were examined in extracts of cells grown on sucrose or mannitol and compared to those grown on fructose and glucose. Sucrase and NAD-specific mannitol dehydrogenase were induced only when sucrose or mannitol respectively were the growth substrates. Endogenously arising fructose was metabolised in a manner similar to that for exogenously supplied fructose i.e. a modified EMP pathway initiated by ketohexokinase. While the enzymes for modified EMP pathway viz. ketohexokinase, 1-phosphofructokinase and fructose 1,6-bisphosphate aldolase were present under all growth conditions, their levels were elevated in presence of fructose. Besides, though fructose 1,6-bisphosphatase, phosphohexoseisomerase and glucose 6-phosphate dehydrogenase were present, the absence of 6-phosphogluconate dehydratase precluded routing of fructose through ED pathway, or through PP pathway directly as 6-phosphogluconate dehydrogenase was lacking. Fructose 1,6-bisphosphatase plays the unusual role of a catabolic enzyme in supporting the non-oxidative part of PP pathway. However the presence of constitutive levels of glucose dehydrogenase and 2-keto 3-deoxy 6-phosphogluconate aldolase when glucose or sucrose were growth substrates suggested that glucose breakdown took place via the modified ED pathway.Abbreviations EMP Embden Meyerhof Parnas - ED Entner Doudoroff - PP pentose phosphate - KHK ketohexokinase - 1-PFK 1-phosphofructokinase - PEP-PTS phosphoenolpyruvate phosphotransferase - 6-PFK 6-phosphofructokinase - FBPase fructose 1,6-bisphosphatase - PHI phosphohexoseisomerase - G6P-DH glucose 6-phosphate dehydrogenase - 6PG-DH 6-phosphogluconate dehydrogenase - GAPDH glyceraldehyde 3-phosphate dehydrogenase - FIP fructose 1-phosphate - GSH reduced glutathione - 2-ME -mercaptoethanol - FBP fructose 1,6-bisphosphate - KDPG 2-keto 3-deoxy 6-phosphogluconate - F6P fructose 6-phosphatez     

Accumulation of fructose 1,6-bisphosphate in mutant cells of mucoid Pseudomonas aeruginosa as an evidence of phosphofructokinase activity

Phosphoglucose isomerase negative mutant of mucoid Pseudomonas aeruginosa accumulated relatively higher concentration of fructose 1,6-bisphosphate (Fru-1,6-P₂) when mannitol induced cells were incubated with this sugar alcohol. Also the toluene-treated cells of fructose 1,6-bisphosphate aldolase negative mutant of this organism produced Fru-1,6-P₂ from fructose 6-phosphate in presence of ATP, but not from 6-phosphogluconate. The results together suggested the presence of an ATP-dependent fructose 6-phosphate kinase (EC 2.7.1.11) in mucoid P. aeruginosa.Abbreviations ALD Fru-1,6-P₂ aldolse - DHAP dihydroxyacetone phosphate - F6P fructose 6-phosphate - G6P glucose 6-phosphate - Gly3P glyceraldehyde 3-phosphate - KDPG 2-keto 3-deoxy 6-phosphogluconate - PFK fructose 6-phosphate kinase - PGI phosphoglucose isomerase - 6PG 6-phosphogluconate     

Glucose 6-Phosphate and Fructose 1,6-Bisphosphate Can Be Used as ATP-Regenerating Systems by Cerebellum Ca2+-Transport ATPase

Abstract : In this work, it is shown that the Ca²⁺-transport ATPase found in the microsomal fraction of the cerebellum can use both glucose 6-phosphate/hexokinase and fructose 1,6-bisphosphate/phosphofructokinase as ATP-regenerating systems. The vesicles derived from the cerebellum were able to accumulate Ca²⁺ in a medium containing ADP when either glucose 6-phosphate and hexokinase or fructose 1,6-bisphosphate and phosphofructokinase were added to the medium. There was no Ca²⁺ uptake if one of these components was omitted from the medium. The transport of Ca²⁺ was associated with the cleavage of sugar phosphate. The maximal amount of Ca²⁺ accumulated by the vesicles with the fructose 1,6-bisphosphate system was larger than that measured either with glucose 6-phosphate or with a low ATP concentration and phosphoenolpyruvate/pyruvate kinase. The Ca²⁺ uptake supported by glucose 6-phosphate was inhibited by glucose, but not by fructose 6-phosphate. In contrast, the Ca²⁺ uptake supported by fructose 1,6-bisphosphate was inhibited by fructose 6-phosphate, but not by glucose. Thapsigargin, a specific SERCA inhibitor, impaired the transport of Ca²⁺ sustained by either glucose 6-phosphate or fructose 1,6-bisphosphate. It is proposed that the use of glucose 6-phosphate and fructose 1,6-bisphosphate as an ATP-regenerating system by the cerebellum Ca²⁺-ATPase may represent a salvage route used at early stages of ischemia ; this could be used to energize the Ca²⁺ transport, avoiding the deleterious effects derived from the cellular acidosis promoted by lactic acid.     

A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Application to a microassay of fructose 2,6-bisphosphate

Pyrophosphate : fructose-6-phosphate phosphotransferase (PPi-PFK) has been purified 150-fold from potato tubers and the kinetic properties of the purified enzyme have been investigated both in the forward and the reverse direction. Saturation curves for fructose 6-phosphate and also for fructose 1,6-bisphosphate were sigmoidal whereas those for PPi and Pi were hyperbolic. In the presence of fructose 2,6-bisphosphate, the affinity for fructose 6-phosphate and for fructose 1,6-bisphosphate were greatly increased and the kinetics became Micha?lian. The effect of fructose 2,6-bisphosphate was increased by the presence of fructose 6-phosphate and decreased by the presence of Pi. Consequently, the Ka for fructose 2,6-bisphosphate was as low as 5 nM for the forward reaction and reached 150 nM for the reverse reaction. On the basis of these properties, a procedure allowing one to measure fructose 2,6-bisphosphate in amounts lower than a picomole, is described.     

The rate of substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate in skeletal muscle.

Substrate cycling of fructose 6-phosphate through reactions catalysed by 6-phosphofructokinase and fructose-1,6-bisphosphatase was measured in skeletal muscles of the rat in vitro. The rate of this cycle was calculated from the steady-state values of the 3H/14C ratio in hexose monophosphates and fructose 1,6-bisphosphate after the metabolism of either [5-3H,6-14C]glucose or [3-3H,2-14C] glucose. Two techniques for the separation of hexose phosphates were studied; t.l.c. chromatography on poly(ethyleneimine)-cellulose sheets or ion-exchange chromatography coupled with enzymic conversion. These two methods gave almost identical results, suggesting that either technique could be used for determination of rates of fructose 6-phosphate/fructose 1,6-bisphosphate cycling. It was found that more than 50% of the 3H was retained in the fructose 1,6-bisphosphate; it is therefore probable that previous measurement of cycling rates, which have assumed complete loss of 3H, have underestimated the rate of this cycle. The effects of insulin, adrenaline and adrenergic agonists and antagonists on rates of fructose 6-phosphate/fructose 1,6-bisphosphate cycling were investigated. In the presence of insulin, adrenaline (1 microM) increased the cycling rate by about 10-fold in epitrochlearis muscle in vitro; the maximum rate under these conditions was about 2.5 mumol/h per g of tissue. The concentration of adrenaline that increased the cycling rate by 50% was about 50 nM. This effect of adrenaline appears to be mediated by the beta-adrenergic receptor, since the rate was increased by beta-adrenergic agonists and blocked by beta-adrenergic antagonists. From the knowledge of the precise rate of this cycle, the possible physiological importance of cycling is discussed.     

Assessment of a futile cycle involving reconversion of fructose 6-phosphate to fructose 1,6-bisphosphate during gluconeogenic growth of Escherichia coli.

In gluconeogenesis, fructose 6-phosphate is formed from fructose 1,6-bisphosphate, and if fructose 1,6-bisphosphate were reformed by the phosphofructokinase reaction there would be a "gluconeogenic futile cycle." We assessed the extent of this cycling in Escherichia coli growing on glycerol 3-phosphate, using a medium containing 32Pi. Fructose 1,6-bisphosphate coming from glycerol 3-phosphate should be unlabeled, but any coming from fructose 6-phosphate should contain label from the gamma-position of ATP. The amount of labeling of the 1-position of fructose 1,6-bisphosphate was only 2 to 10% of that of the gamma-position of ATP in a series of isogenic strains differing in phosphofructokinases (Pfk-1, Pfk-2, or Pfk-2). In control experiments with glucose 6-phosphate instead of glycerol 3-phosphate, the two positions were equally labeled. Thus, although the presence of Pfk-2 causes gluconeogenic impairment (Daldal et al., Eur. J. Biochem., 126:373-379, 1982), gluconeogenic futile cycling cannot be the reason.     

Glucose metabolism in the mucosa of the small intestine. Glycolysis in subcellular preparations from the cat and rat

1. Lactic acid formation in supernatant fractions of homogenates of cat or rat small-intestinal mucosa was measured under optimum conditions with glucose, fructose, glucose 6-phosphate, fructose 1,6-diphosphate or 3-phosphoglycerate as substrate. 2. Between 80 and 107% of the glycolytic activity of the homogenate was recovered in these particle-free preparations when glucose, fructose, glucose 6-phosphate or fructose 1,6-diphosphate was used as substrate. 3. Evidence was obtained that hexokinase and phosphofructokinase were the rate-limiting enzymes in the initial sequence of glycolytic reactions. The limitation of rate by hexokinase was much more pronounced in preparations from the cat than in those from the rat. 4. With subcellular preparations from cat or rat small intestine lactic acid was also formed from ribose 5-phosphate and at rates similar to those observed with glucose. 5. A higher rate of glycolysis was observed with glucose 6-phosphate as substrate with preparations from the proximal half of the small intestine of the rat as compared with the distal half. 6. Mucosal preparations from rats starved for 24-48hr. exhibited only about one-quarter of the glycolytic activity of those of fed control groups. The decreased rate of formation of lactic acid from either glucose or fructose was mainly due to a decrease in the activity of hexokinase(s). The activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and a number of other enzymes were not significantly decreased by starvation. 7. The results are discussed in relation to metabolic control of glycolysis in other mammalian tissues.     

Effects of starvation and exercise on concentrations of citrate, hexose phosphates and glycogen in skeletal muscle and heart. Evidence for selective operation of the glucose-fatty acid cycle.

Concentrations of citrate, hexose phosphates and glycogen were measured in skeletal muscle and heart under conditions in which plasma non-esterified fatty acids and ketone bodies were physiologically increased. The aim was to determine under what conditions the glucose-fatty acid cycle might operative in skeletal muscle in vivo. In keeping with the findings of others, starvation increased the concentrations of glycogen, citrate and the fructose 6-phosphate/fructose 1,6-bisphosphate ratio in heart, indicating that the cycle was operative. In contrast, it decreased glycogen and had no effect on the concentration of citrate or the fructose 6-phosphate/fructose 1,6-bisphosphate ratio in the soleus, a slow-twitch red muscle in which the glucose-fatty acid cycle has been demonstrated in vitro. In fed rats, exercise of moderate intensity caused glycogen depletion in the soleus and red portion of gastrocnemius muscle, but not in heart. In starved rats the same exercise had no effect on the already diminished glycogen contents in skeletal muscle, but it decreased cardiac glycogen by 25-30%. After exercise, citrate and the fructose 6-phosphate/fructose 1,6-bisphosphate ratio were increased in the soleus of the starved rat. Significant changes were not observed in fed rats. The data suggest that in the resting state the glucose-fatty acid cycle operates in the heart, but not in the soleus muscle, of a starved rat. In contrast, the metabolite profile in the soleus was consistent with activation of the glucose-fatty acid cycle in the starved rat during the recovery period after exercise. Whether the cycle operates during exercise itself is unclear.     

Glycolysis at the climacteric of bananas.

This work was carried out to investigate the relative roles of phosphofructokinase and pyrophosphate-fructose-6-phosphate 1-phosphotransferase during the increased glycolysis at the climacteric in ripening bananas (Musa cavendishii Lamb ex Paxton). Fruit were ripened in the dark in a continuous stream of air in the absence of ethylene. CO2 production, the contents of glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate and PPi; and the maximum catalytic activities of pyrophosphate-fructose-6-phosphate 1-phosphotransferase, 6-phosphofructokinase, pyruvate kinase and phosphoenolpyruvate carboxylase were measured over a 12-day period that included the climacteric. Cytosolic fructose-1,6- bisphosphatase could not be detected in extracts of climacteric fruit. The peak of CO2 production was preceded by a threefold rise in phosphofructokinase, and accompanied by falls in fructose 6-phosphate and glucose 6-phosphate, and a rise in fructose 1,6-bisphosphate. No change in pyrophosphate-fructose-6-phosphate 1-phosphotransferase or pyrophosphate was found. It is argued that phosphofructokinase is primarily responsible for the increased entry of fructose 6-phosphate into glycolysis at the climacteric.     

Ribose 1,5-bisphosphate is a putative regulator of fructose 6-phosphate/fructose 1,6-bisphosphate cycle in liver

6-Phosphofructo-1-kinase and fructose-1,6-bisphosphatase are rate-limiting enzymes for glycolysis and gluconeogenesis respectively, in the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver. The effect of ribose 1,5-bisphosphate on the enzymes was investigated. Ribose 1,5-bisphosphate synergistically relieved the ATP inhibition and increased the affinity of liver 6-phosphofructo-1-kinase for fructose 6-phosphate in the presence of AMP. Ribose 1,5-bisphosphate synergistically inhibited fructose-1,6-bisphosphatase in the presence of AMP. The activating effect on 6-phosphofructo-1-kinase and the inhibitory effect on fructose-1,6-bisphosphatase suggest ribose 1,5-bisphosphate is a potent regulator of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver.     

Regulation of mitochondrial NAD-malic enzyme involved in C4 pathway photosynthesis.

Mitochondrial NAD-malic enzyme isolated from bundle sheath cells of different C₄ species was activated 5- to 15-fold by fructose 1,6-phosphate. With 2.5 mm malate, fructose 1,6-phosphate was optimally active between 30 and 100 μm and activation was similar to that previously reported for CoA and acetyl-CoA. 3-Phosphoglycerate and isocitrate were less effective activators and other metabolites including fructose 6-phosphate and glyceraldehyde 3-phosphate were without effect. Depending on the source of the enzyme, the response to increasing fructose 1,6-phosphate was either sigmoidal, with activation resulting from an increase in the affinity of the enzyme for malate, or hyperbolic, in which case the activator increased maximum velocity. Bicarbonate inhibited NAD-malic enzyme activity, acting competitively with respect to both malate and the activators fructose 1,6-phosphate and CoA. The enzyme was also inhibited in a similar competitive manner by higher concentrations of chloride and nitrate ions. Decarboxylation of C₄ acids by isolated mitochondria was inhibited by bicarbonate and nitrate ions, and, as with isolated NAD-malic enzyme, inhibition was competitive with respect to malate. The rate of C₄ acid decarboxylation by freshly prepared mitochondria was not increased by adding fructose 1,6-phosphate or CoA. However, decarboxylating activity declined after incubating mitochondria at 30 °C without C₄ acids, and this loss of activity was largely prevented by fructose 1,6-phosphate. Mitochondria were found to decarboxylate oxaloacetate as rapidly as aspartate.     

The Formation of Glucose 1,6-Diphosphate from Fructose 1,6-Diphosphate by Yeast Phosphoglucomutase

It was found that fructose 1,6-diphosphate, the main intermediate of glycolysis, was able to act as a coenzyme of yeast phosphoglucomutase reaction. The mechanism of the coenzymatic activity of fructose 1,6-diphosphate was studied. It was indicated in the fructose 1,6-diphosphate dependent reaction that glucose 1,6-diphosphate was formed by the phosphate-transfer of fructose 1,6-diphosphate to glucose 1-phosphate in the first step, and in the second step the conversion of glucose 1-phosphate to glucose 6-phosphate, the original mutase reaction, occurred in the presence of glucose 1,6-diphosphate. The kinetic constants in the reaction of the first step were determined from the time courses of the fructose 1,6-diphosphate dependent reaction.     

Irreversible transitions in the 6-phosphofructokinase/fructose 1,6-bisphosphatase cycle.

The dynamics of the fructose 6-phosphate fructose-1,6-bisphosphate cycle operating in an open and homogeneous system reconstituted from purified enzymes was extensively studied. In addition to 6-phosphofructokinase and fructose-1,6-bisphosphatase, pyruvate kinase, adenylate kinae and glucose-6-phosphate isomerase were involved. In that multi-enzyme system, the main source of non-linearity is the reciprocal effect of AMP on the activities of 6-phosphofructokinase and fructose-1,6-bisphosphatase. Depending upon the experimental parameter values, stable attractors, various types of multiple states and sustained oscillations were shown to occur. In the present report we show that irreversible transitions are also likely to occur for realistic operating conditions. Two parameters of the system, that is the adenylate energy charge of the influx and the fructose-1,6-bisphosphatase maximal activity, are potential candidates to provoke such irreversible transitions from one steady state to the other: (a) when varying the maximal activity of fructose-1,6-bisphosphatase, the system can jump irreversibly from a low to a high stable steady state, and (b) when the adenylate energy charge of the influx is the changing parameter, irreversible transitions occur from a high stable steady state to a stable oscillatory state (limit cycle motion). This behavior can be predicted by constructing the loci of limit points and Hopf bifurcation points.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Pyrophosphate: fructose 6-phosphate phosphotransferase in germinating castor bean seedlings

The distribution of enzymes interconverting fructose 6-phosphate and fructose 1,6-bisphosphate has been studied in a range of tissues from castor bean seedlings. In each tissue the activity of PP_i:fructose 6-phosphate phosphotransferase was greater than phosphofructokinase and substantial compared with fructose 1,6-bisphosphatase. PP_i:fructose 6-phosphate phosphotransferase in endosperm is apparently confined to the cytoplasm. The role of this latter enzyme in vivo is discussed.     

Reversal of glycolysis in yeast.

When a buffered, aerobic suspension of ethanol-grown cells of Saccharomyces cerevisiae is treated with ethanol, a rapid flux of metabolism is observed from endogenous phosphoenolpyruvate to hexose monophosphates. Intracellular concentrations of phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate record a monotonic drop, while those of triose phosphates and fructose 1,6-diphosphate fall after an early rise; fructose 6-phosphate, mannose 6-phosphate, and glucose 6-phosphate levels rise to a plateau. Prior growth on glucose extinguishes fructose 1,6-diphosphatase activity and completely arrests the rise of the hexose monophosphates. By using mutants blocked at a number of glycolytic steps it has been concluded that the metabolic flow takes place along the Embden-Meyerhof pathway in the reverse direction bypassing pyruvate kinase and fructose 6-phosphate kinase. Ethanol acts as a trigger by supplying NADH at the glyceraldehyde 3-phosphate dehydrogenase step. The rate of the reversal in the span phosphoenolpyruvate to fructose 1,6-diphosphate approaches 40 μ mol of 3-carbon units per minute per gram of wet cells. The in vivo activity of fructose 1,6-diphosphatase is nearly a quarter of this rate.     

Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase

Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the bridge position of pyrophosphate to a nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. The maximum rates of isotope exchange at equilibrium for the [14C]fructose 1,6-bisphosphate in equilibrium fructose 6-phosphate, [32P]Pi in equilibrium MgPPi, and Mg[32P]PPi in equilibrium fructose 1,6-bisphosphate exchange reactions increasing all four possible substrate-product pairs in constant ratio are identical, consistent with a rapid equilibrium mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate (F6P)/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate (FBP) pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi, in agreement with initial velocity studies [Bertagnolli, B.L., & Cook, P.F. (1984) Biochemistry 23, 4101]. Neither back-exchange by [32P]Pi nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction.     

Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.     

Kinetic properties of pyrophosphate:fructose-6-phosphate phosphotransferase from germinating castor bean endosperm

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was purified over 500-cold from endosperm of germinating castor bean (Ricinus commiunis L. var. Hale). The kinetic properties of the purified enzyme were studied. PFP was specific for pyrophosphate and had a requirement for a divalent metal ion. The pH optimum for activity was 7.3 to 7.7. The enzyme had similar activities in the forward and reverse directions and exhibited hyperbolic kinetics with all substrates. Kinetic constants were determined in the presence of fructose 2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for fructose 6-phosphate, fructose 1,6-bisphosphate, and pyrophosphate up to 10-fold. Half-maximum activation of PFP by fructose 2,6-bisphosphate was obtained at 10 nanomolar. The affinity of PFP for this activator was reduced by decreasing the concentration of fructose 6-phosphate or increasing that of phosphate. Phosphate inhibited PFP when the reaction was measured in the reverse direction, i.e. fructose 6-phosphate production. In the presence of fructose 2,6-bisphosphate, phosphate was a mixed inhibitor with respect to both fructose 6-phosphate and pyrophosphate when the reaction was measured in the forward direction, i.e. fructose 1,6-bisphosphate production. The possible roles of fructose 2,6-bisphosphate, fructose 6-phosphate, and phosphate in the control of PFP are discussed.