On the mechanism of inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate.
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Role of fructose 2,6-bisphosphate in the regulation of glycolysis and gluconeogenesis in chicken liver.
Glucagon and dibutyryl cyclic AMP inhibited glucose utilization and lowered fructose 2,6-bisphosphate levels of hepatocytes prepared from fed chickens. Partially purified preparations of chicken liver 6-phosphofructo-1-kinase and fructose 1,6-bisphosphatase were activated and inhibited by fructose 2,6-bisphosphate, respectively. The sensitivities of these enzymes and the changes observed in fructose 2,6-bisphosphate levels are consistent with an important role for this allosteric effector in hormonal regulation of carbohydrate metabolism in chicken liver. In contrast, oleate inhibition of glucose utilization by chicken hepatocytes occurred without change in fructose, 2,6-bisphosphate levels. Likewise, pyruvate inhibition of lactate gluconeogenesis in chicken hepatocytes cannot be explained by changes in fructose 2,6-bisphosphate levels. Exogenous glucose caused a marked increase in fructose 2,6-bisphosphate content of hepatocytes from fasted but not fed birds. Both glucagon and lactate prevented this glucose effect. Fasted chicken hepatocytes responded to lower glucose concentrations than fasted rat hepatocytes, perhaps reflecting the species difference in hexokinase isozymes.
Animals↗
Effect of Mn2+ on fructose 2,6-bisphosphate inhibition of mouse liver, intestinal, and muscle fructose-1,6-bisphosphatases.
Fructose 2,6-bisphosphate inhibited all three fructose-1,6-bisphosphatases from the liver, intestine, and muscle of the mouse. The sensitivity of the liver enzyme to the inhibitor was significantly diminished when Mg2+ was replaced by Mn2+ as the activating cation. Inhibition of the liver enzyme by fructose 2,6-bisphosphate decreased as the concentration of the metal activator, Mn2+ or Mg2+, increased. The respective I50 values obtained by extrapolation of metal ion concentrations to zero were 40 microM with Mn2+ and 0.25 microM with Mg2+. The extent of desensitization to either fructose 2,6-bisphosphate or AMP inhibition by Mn2+ decreased in the order of the liver, intestine, and muscle enzyme. Only in the case of the liver enzyme was the substrate cooperativity induced by fructose 2,6-bisphosphate in the presence of Mg2+. In all three isoenzymes from the mouse, fructose 2,6-bisphosphate greatly potentiated the AMP inhibition of the enzyme in the presence of either Mg2+ or Mn2+. The liver enzyme with Mn2+ in addition to Mg2+ was still active in the presence of less than 1 microM fructose 2,6-bisphosphate, even though AMP was present at 100-200 microM.
Adenosine Monophosphate↗
Fructose 1,6-bisphosphatase in rat liver cytosol: interactions between the effects of K+, Zn2+, Mn2+, and fructose 2,6-bisphosphate as measured in a steady-state assay.
Fructose 1,6-bisphosphatase activity was determined in rat liver cytosols using glyceraldehyde 3-phosphate as primary substrate. Fructose 1,6-bisphosphate was formed in situ and steady-state concentrations ranging from 1 to 30 microM were observed depending on the activity of fructose 1,6-bisphosphatase and the concentration of added glyceraldehyde 3-phosphate. The system was free of contaminating low-molecular-weight compounds, divalent cations, and chelators. Under these conditions, fructose 1,6-bisphosphatase was inhibited by K+ (less than or equal to 200 mM). This inhibition was due to a reduction of V and was observed in presence of low (0.4 mM) and high (5 mM) concentrations of Mg2+. In presence of 0.4 mM Mg2+, 1 microM Zn2+ inhibited fructose 1,6-bisphosphatase by 50%; the same effect was obtained with 0.3 microM Zn2+ when the system was supplemented with 100 mM KCl. On the other hand, 0.2 microM Zn2+ enhanced the inhibitory effect of K+ and decreased the concentration of K+ yielding half-maximal inhibition from 175 to 100 mM when measured at 0.4 mM Mg2+. The effect of Zn2+ on the inhibition by K+ could be abolished by Mn2+ (less than 5 microM) or by 5 mM Mg2+. One hundred millimolar K+ enhanced the inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate and changed the type of inhibition from mainly competitive to a mixed-type inhibition (increase of Km, decrease of V). Mn2+ (less than 10 microM) reduced the effect of fructose 2,6-bisphosphate, especially in the presence of K+. It is proposed that K+ and Mn2+ may play a role in the regulation of gluconeogenesis.
Animals↗
Purification of the phosphofructokinase regulatory factors.
In this paper, the existence and purification of two species of phosphofructokinase regulatory factor activity are reported. The purification procedure included liver homogenization and ultracentrifugation, a 93 degrees C heat step on the supernate, precipitation with ammonium sulfate, DEAE-cellulose column chromatography, and Sephadex G-75 (fine) chromatography. Two discrete regions of factor activity were eluted from the DEAE-cellulose column with a 0 to 0.5 M linear NaCl gradient. The lesser anionic fraction was not significantly retarded by DEAE-cellulose at pH 7.6, and was referred to as factor A. The more anionic form, factor B, eluted at about 0.2 M NaCl. The presence of two active fractions was confirmed by separation of factor activity (prior to DEAE-cellulose chromatography) into two discrete species by preparative isoelectric focusing on granulated gel. The isoelectric points were approximately 7.0 for factor B and 8.5 for factor A. Factor A and factor B exhibited quite different elution volumes, i.e., apparent molecular weights, when applied to a Sephadex G-75 column. Rechromatography on a Sephadex G-75 column was used for further purification and estimation of native molecular weight. The gel filtration method yielded a molecular weight of 13,800 +/- 1,800 for factor A. Factor A activity eluted as a symmetrical protein peak of constant specific activity, suggesting a homogeneous preparation. For factor B, the absorption at 280 nm and activity profile did not directly overlap. When the peak absorbance at 280 nm was considered, a molecular weight range of 39,000 +/- 4,000 was found, and on the basis of activity the molecular weight range was 36,000 +/- 4,000. After the final Sephadex G-75 chromatographic step, sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis of each SDS-treated factor preparation indicated that factor A, after visualization by silver staining, was homogeneous, with a subunit molecular weight of approximately 12,000. The factor B preparation consisted of two major polypeptides (11,000 and 18,000). The data appeared to support the conclusions that factor B was a dimer of the 18,000-Da subunit, and that the major contaminant was a tetramer of the 11,000-Da subunit.
Animals↗
Properties of the phosphofructokinase regulatory factors.
The work presented herein describes many of the physiological properties of the phosphofructokinase regulatory factors. Factor activity can be separated into two discrete fractions, which were designated factor A and factor B, based on their respective charges. A preparation containing both factor A and factor B did not protect the following key carbohydrate-metabolizing enzymes from thermal inactivation: glucokinase, glucose-6-phosphatase (solubilized or nonsolubilized forms), pyruvate kinase, glucose-6-P dehydrogenase, muscle-type phosphofructokinase, or the minor liver phosphofructokinase isozyme. Factor activity in this sample was found to be Pronase sensitive, irreversibly precipitated by trichloroacetic acid, reversibly precipitated by adjusting the sample to a pH of 3.0, and stable to heating at 98 degrees C for 20 min. Distribution studies indicated that factor activity was found only in the soluble cell fraction and not in the mitochondrial or nuclear fractions. Factor activity was retained by 12,000-14,000 molecular weight cut-off (MWCO) dialysis tubing, and not retained by 50,000 MWCO dialysis tubing. These studies indicate that fructose-2,6-P2, calmodulin, or insulin-generated mediator are not associated with factor activity. Although fructose-2,6-P2 did not, both factor preparations protect the major liver phosphofructokinase isozyme (liver PFK) from inactivation by lysosomal extracts. In the diabetic rat, the activities of both factors are greatly reduced but return to near normal levels after 48 h of insulin administration. These data suggest that factor B had little or no effect on the kinetic properties of liver PFK. However, factor A was a K-type activator with respect to fructose-6-P, increasing both the Km and Ki for ATP, and slightly increasing the Vm.
Amino Acids↗
Rat liver pyruvate kinase: influence of ligands on activity and fructose 1,6-bisphosphate binding.
The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P2 per enzyme tetramer. The Hill coefficient (nH), and the concentration of Fru-1,6-P2 giving half-maximal binding [FBP]1/2, are influenced by several factors. In 150 mM Tris-HCl, 70 mM KCl, 11 mM MgSO4 at pH 7.4, [FBP]1/2 is 2.6 microM and nH is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P2 by decreasing [FBP]1/2. ADP and ATP alone had little influence on Fru-1,6-P2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P2 needed for maximum binding. Phosphorylation also decreased the nH for Fru-1,6-P2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn2+ for Mg2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 mM. As a consequence, Mn2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibits at saturating concentrations of the substrate or in the presence of Fru-1,6-P2. Both Mg2+ and Mn2+ diminish binding of low concentrations of Fru-1,6-P2; however, the concentrations of the metal ions needed to influence Fru-1,6-P2 binding exceed those needed to support catalytic activity.
Alanine↗
Activation of phosphofructokinase from rat tissues by 6-phosphogluconate and fructose 2,6-bisphosphate.
6-Phosphogluconate activates phosphofructokinase from liver, adipose tissue, kidney, and skeletal muscle by decreasing the apparent S0.5 for fructose 6-phosphate without affecting the maximum velocity. The response of phosphofructokinase to 6-phosphogluconate is hyperbolic, with apparent activation constants similar to concentrations of 6-phosphogluconate in tissues. Phosphofructokinase from these tissues is also activated by fructose 2,6-bisphosphate, but the apparent activation constants are much less than the concentrations of fructose 2,6-bisphosphate in tissues. Under most conditions, the effects of 6-phosphogluconate and fructose 2,6-bisphosphate are additive. However, with low concentrations of fructose 6-phosphate there is synergism between the effectors. Whereas fructose 2,6-bisphosphate overcomes the inhibition of phosphofructokinase by high concentrations of ATP, 6-phosphogluconate does not. Thus, the effectors probably act at different sites on the enzyme. The relative effect of 6-phosphogluconate is much greater on phosphofructokinase from the lipogenic tissues, adipose, and liver, than it is on the enzyme from kidney or skeletal muscle. Thus, the influence of 6-phosphogluconate on phosphofructokinase, which could coordinate the disposition of glucose 6-phosphate between the oxidative branch of the hexosemonophosphate pathway and glycolysis, may be important for lipogenesis.
Adenine Nucleotides↗
Purification and properties of rat brain pyruvate kinase.
Rat brain pyruvate kinase was purified to near homogeneity by a three-step process involving ammonium sulfate precipitation and phosphocellulose and Blue-Sepharose CL-6B column chromatography. The enzyme migrated on polyacrylamide gel along with a commercial sample of rabbit muscle pyruvate kinase. The enzyme showed a hyperbolic relationship with phosphoenolpyruvate and ADP, with apparent Km's of 0.18 and 0.42 X 10(-3) M, respectively. The enzyme was inhibited by ATP, the effect being more pronounced at unsaturating concentrations of phosphoenolpyruvate. L-Phenylalanine was found to be a strong inhibitor of the enzyme, with the Ki for inhibitor being 0.11 mM. The inhibition by phenylalanine was more pronounced at pH 7.4 than at pH 7.0, and appeared to be competitive with phosphoenolpyruvate. L-Alanine and fructose 1,6-bisphosphate prevented the inhibition of the enzyme by phenylalanine. Ca2+ was found to be a strong inhibitor of the enzyme, and the inhibition was more marked at saturating phosphoenolpyruvate concentrations. The kinetic properties of the purified brain pyruvate kinase suggest that the enzyme may be distinct from the muscle or liver enzymes.
Adenosine Monophosphate↗
Inhibition of fructose bisphosphatase and stimulation of phosphofructokinase by a stable isosteric phosphonate analog of fructose 2,6-bisphosphate.
We describe the synthesis of a mixture of D-manno- and D-gluco-2,5-anhydro-1-deoxy-1-phosphonohexitol 6-phosphate via a Horner-Emmons reaction of 2,3,5-tri-O-benzyl-beta-D-arabinofuranose followed by phosphorylation of the equivalent 6-position and subsequent deprotection. This mixture inhibits fructose-1,6-bisphosphatase; the concentration required for half-maximal effect in the presence of 25 microM AMP is approximately 6 microM. The mixture of analogs also stimulates 6-phosphofructo-1-kinase from rabbit liver; the concentration required to reach one-half Vmax was found to be ca. 25 microM at 0.25 mM fructose 6-phosphate and 50 microM AMP. These analogs have replaced the labile anomeric phosphate of fructose 2,6-bisphosphate with a stable methylenephosphonate, and could be of great interest due to their appropriate physiological effects and their chemical stability.
Adenosine Monophosphate↗
Phosphofructokinase from Fasciola hepatica: activation by phosphorylation and other regulatory properties distinct from the mammalian enzyme.
Phosphofructokinase from the liver fluke, Fasciola hepatica, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase isolated from this organism. Phosphorylated fluke phosphofructokinase had a sevenfold lower apparent Km for its substrate, Fru-6-P, and an eightfold higher 0.5 Vopt for ATP, the enzyme's primary inhibitor, than native phosphofructokinase. Activation of fluke phosphofructokinase following phorphorylation by a mammalian protein kinase catalytic subunit was previously reported (E. S. Kamemoto and T. E. Mansour (1986) J. Biol. Chem. 261, 4346-4351). The catalytic subunit of protein kinase isolated from the liver fluke phosphorylated sites on fluke phosphofructokinase similar to those phosphorylated by the mammalian enzyme. Maximal phosphate incorporation was 0.3 mol P/mol of protomer. The native enzyme was found to contain 1.3 mol P/mol of protomer. In contrast to fluke phosphofructokinase, activity of the mammalian heart enzyme was slightly decreased following phosphorylation. The dependence of allosteric interaction on an acidic pH observed with the mammalian phosphofructokinase was not observed with the fluke enzyme. Unlike mammalian phosphofructokinase, allosteric kinetics of the fluke enzyme was observed at alkaline pH (8.0). Fluke phosphofructokinase was found to be relatively insensitive to inhibition by citrate, a known potent inhibitor of the mammalian enzyme. Fru-2,6-P2, a potent modifier of phosphofructokinase from a variety of sources, was found to activate both native and phosphorylated fluke phosphofructokinase. The most potent activators of fluke phosphofructokinase were found to be Fru-2,6-P2, AMP, and phosphorylation. The endogenous level of Fru-2,6-P2 in the flukes was determined to be 29 +/- 1.3 nmol/g wet wt, a level that may well modulate enzyme activity. Fru-6-P,2-kinase, the enzyme responsible for synthesis of Fru-2,6-P2, was found to be present in the flukes. Our results suggest physiological roles for phosphorylation and Fru-2,6-P2 in regulation of fluke phosphofructokinase.
Adenosine Triphosphate↗
Inhibition of hexokinase activity by a fructose 2,6-bisphosphate-dependent cytosolic protein from liver.
Mammalian and yeast hexokinases were found to be reversibly inhibited by fructose 2,6-bisphosphate, an effect requiring the presence of a cytosolic protein factor. Experimental evidence suggests that this factor (inhibitor) is a regulatory protein, the interactions of which with hexokinases are modulated by fructose 2,6-bisphosphate. The Vmax of hexokinase D was decreased, and no changes on other kinetic parameters were observed. The inhibitor was present in fresh liver cytosol filtered through Sephadex G-25 and was partially isolated by negative absorption on DEAE-cellulose followed by ammonium sulfate fractionation. The inhibitor was also present in brain and kidney, but not in muscle. A molecular mass of 200,000 was determined by gel filtration. The inhibition was dependent on the concentrations of both the inhibitory protein and fructose 2,6-bisphosphate. No delay in fructose 2,6-bisphosphate inhibition was observed. Several other hexose phosphates were tested and were not effective. In the presence of amounts of inhibitor sufficient to produce complete inhibition of hexokinase D, the concentration of fructose 2,6-bisphosphate required to produce 50% inhibition was about 0.5 microM. The inhibitor was unstable and was stabilized by the presence of fructose 2,6-bisphosphate.
Animals↗
Phosphofructokinase is responsible for the fructose 2,6-bisphosphate inhibition of hexokinase in tissue extracts.
Mammalian and yeast hexokinases were reported to be reversibly inhibited by fructose 2,6-bisphosphate in the presence of cytosolic proteins (H. Niemeyer, C. Cerpa, and E. Rabajille (1987) Arch. Biochem. Biophys. 257, 17-26). Reinvestigation of this finding using a radioassay with [14C]glucose as substrate showed no effect of fructose 2,6-bisphosphate on hexokinase activity of rat liver cytosols. Detailed reexamination of the spectrophotometric assay resulted in the observation that the fructose 2,6-bisphosphate-dependent inhibition was a function of the cytosolic phosphoglucose isomerase and phosphofructokinase activities compared to the amount of glucose-6-phosphate dehydrogenase used as auxiliary enzyme. The diminution or loss of the fructose 2,6-bisphosphate-dependent inhibition produced in aged cytosols was restored by addition of crystalline muscle phosphofructokinase, as well as by decreasing the amount of glucose-6-phosphate dehydrogenase in the assay. When phosphoglucose isomerase, phosphofructokinase, and hexokinase activities were separated by DEAE-chromatography of liver cytosol, no fructose 2,6-bisphosphate-dependent inhibition of hexokinase was found in any single fraction of the chromatogram. However, combination of fractions containing both phosphoglucose isomerase and phosphofructokinase displayed the fructose 2,6-bisphosphate-dependent inhibition on either endogenous hexokinase or added yeast hexokinase. From these results we conclude that the activation of phosphofructokinase elicited by fructose 2,6-bisphosphate is responsible for the hexokinase inhibition observed in the coupled spectrophotometric assay.
Animals↗
Purification and characterization of fructose-1,6-bisphosphatase from bovine brain.
Fructose-1,6-bisphosphatase from bovine brain tissue has been purified to near homogeneity. This enzyme is similar to other mammalian fructose-1,6-bisphosphatases in many respects, and its properties are distinctly different from those reported for the enzyme from rat brain [A. L. Majumder and F. Eisenberg (1977) Proc. Natl. Acad. Sci. USA 74, 3222-3225; S. Chattoraj and A. L. Majumder (1986) Biochem. Biophys. Res. Commun. 139, 571-580]. The bovine enzyme (sp act 4, pH ratio (7.5/9.6) = 3.6) has a pH optimum of 7.5. The Km is 2 microM. Divalent metal ion is required for activity, and Vmax is obtained at either 4 mM Mg2+ or 0.3 mM Mn2+. Fructose 2,6-bisphosphate is a competitive inhibitor (Ki = 0.07 microM), and AMP a noncompetitive inhibitor (kis = 24 microM, Kii = 10 microM) of bovine brain fructose-1,6-bisphosphatase. The enzyme activity is enhanced by small amounts of EDTA relative to metal, and AMP inhibits fructose-1,6-bisphosphatase in either the presence or absence of the metal chelator; however, AMP is more effective in the absence of EDTA.
Adenosine Monophosphate↗
Impaired reductive activation of stromal bisphosphatases in tomato leaves following low-temperature exposure at high light.
Photosynthesis in domestic tomato (Lycopersicon esculentum L.) is highly sensitive to low temperature, particularly when accompanied by high light. Since previous studies have established that the inhibited plants retain photosynthetic electron transfer and ATP formation competence, we sought to identify specific steps in the photosynthetic carbon reduction pathway that could account for the lost photosynthetic capacity. Measurements of steady-state photosynthetic metabolite pool sizes showed an accumulation of fructose 1,6-bisphosphate and sedohepulose 1,7-bisphosphate following chilling in the light. Measurements of in vivo turnover rates of the metabolite pools accompanied by direct determinations of enzymatic activity showed that the capacity of the stromal bisphosphatases was substantially reduced following chilling in the light and was the cause of the bisphosphate accumulation. The time course of the loss of phosphatase activity closely mimicked that of the inhibition of net photosynthesis, further indicating that impaired phosphatase function is the underlying cause of the sensitivity of photosynthesis in tomato to light and chilling. Fructose 1,6-bisphosphatase extracted from inhibited tomato plants could be fully activated in the presence of dithiothreitol, indicating that chilling in the light disrupts the normal, thioredoxin-dependent, activation pathway of the stromal bisphosphatases. This disruption could involve a change in the redox potential of the functional disulfide on the phosphatases.
Cold Temperature↗
Regulation of 6-phosphofructo-1-kinase activity in ras-transformed rat-1 fibroblasts.
Fructose 2,6-bisphosphate (F-2,6-P2) stimulated glycolysis in cell-free extracts of both normal and ras-transfected rat-1 fibroblasts. The extract of the transformed cell glycolyzed more rapidly in both the absence and the presence of F-2,6-P2 than the extract of the parent fibroblast. Addition of mitochondrial ATPase (F1) or inorganic phosphate (Pi) further stimulated lactate production in both cell lines. F-2,6-P2 stimulated the 6-phosphofructo-1-kinase (PFK-1) activity in extracts of normal and transfected cells. The activity in extracts of transformed cells tested with a fructose 6-phosphate regenerating system was considerably higher than in the extract of normal cells. Stimulation of PFK-1 activity by cAMP of both cell lines was not as pronounced as that by F-2,6-P2. In the absence of F-2,6-P2 the PFK-1 activity was strongly inhibited in the transformed cell by ATP concentrations higher than 1 mM, whereas in the normal cell only a marginal inhibition was noted even at 2 or 3 mM ATP. F-2,6-P2 reversed the inhibition of PFK-1 by ATP. Nicotinamide adenine dinucleotide (NAD) at 100 microM (in the presence of 2 mM ATP and 1 microM F-2,6-P2) stimulated PFK-1 activity only in the transformed cell, whereas nicotinamide adenine dinucleotide phosphate (NADP) inhibited PFK-1 activity (in the presence or absence of 1 microM F-2,6-P2) in extracts of both cell lines. No previous observations of stimulation or inhibition by NAD or NADP on PFK-1 activity appear to have been reported. A threefold increase in the intracellular concentration of F-2,6-P2 was observed after transfection of rat-1 fibroblast by the ras oncogene. We conclude from these data that the PFK-1 activity of ras-transfected rat-1 fibroblasts shows a greater response to certain stimulating and inhibitory regulating factors than that of the parent cell.
Animals↗
Phosphofructokinase from mollusc muscle is activated by phosphorylation.
Phosphofructokinase was purified from muscle tissue of two different molluscs, edible snails, Helix pomatia (gastropoda), and mussels, Mytilus edulis (bivalvia). Under denaturing conditions, both enzymes had a molecular mass of 82 kDa. In the presence of ATP-Mg2+, the enzymes were rapidly phosphorylated in vitro by the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase purified from snail muscle and also by the C subunit of protein kinase from bovine heart. The extent of phosphorylation was 0.6 and 0.5 phosphate residues per subunit for the snail and the mussel phosphofructokinase, respectively. Phosphorylation of both phosphofructokinases effected a decrease in ATP inhibition at neutral or slightly acidic pH values and increased the affinity for fructose 6-phosphate. The resulting activation in the presence of suboptimum fructose 6-phosphate concentrations was more distinct for the snail enzyme. In addition, phosphorylated phosphofructokinase from mussels exhibited a marked increase in Vmax when activated by either 5'-AMP or fructose 2,6-bisphosphate.
Adenosine Monophosphate↗
Redox active sulfhydryls are required for fructose 2,6-bisphosphate activation of plant pyrophosphate fructose-6-phosphate 1-phosphotransferase.
The classical, alpha/beta-subunit form (Q2) of green tomato pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90), a cytosolic enzyme functional in carbohydrate metabolism, was rapidly inactivated on incubation with the oxidant 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Analysis of the DTNB-treated sample by a fluorescence procedure revealed that inactivation was accompanied by oxidation of sulfhydryl groups, primarily on the alpha-subunit. Phosphate metabolites--fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, Pi, and PPi--protected against DTNB inactivation to varying degrees. The Km values for fructose 6-phosphate and PPi were not changed by DTNB treatment, but the capability for activation by fructose 2,6-bisphosphate was severely diminished. The oxidative inactivation of PFP was reversed by dithiothreitol, but not by monothiols (reduced glutathione or beta-mercaptoethanol). Reactivation was accompanied by restoration of the ability to undergo activation by fructose 2,6-bisphosphate. The findings suggest that sulfhydryl groups are essential for the activation of PFP by fructose 2,6-bisphosphate and raise the possibility that a reversible change in their redox status can take place under certain conditions. Evidence that this is the case was obtained with a preparation from wheat flour which, in the absence of an added oxidant, required reduction by a dithiol for activation by fructose 2,6-bisphosphate (dithiothreitol and reduced thioredoxin h).
Dithionitrobenzoic Acid↗