The monomer of pyruvate kinase, subtype M1, is both a kinase and a cytosolic thyroid hormone binding protein

Using a T7 expression system, the monomer of rat pituitary pyruvate kinase, subtype M1 (PKM1), was overexpressed in Escherichia coli and purified to homogeneity. The monomeric p58-M1 has intrinsic enzymatic activity with a Vmax of 79 +/- 20 units/mg and Km's for ADP and PEP of 1.43 +/- 0.76 and 0.14 +/- 0.07 mM, respectively. The monomer binds 3,3',5-triiodo-L-thyronine (T3) with Ka = 1.5 x 10(7) M-1. The order of analog specificity is L-T3 greater than L-thyroxine greater than D-T3 greater than 3'-isopropyl-3,5-diiodo-L-thyronine greater than or equal to 3',5',3-triiodo-L-thyronine. In contrast, tetrameric PKM1 lacks T3 binding activity. The kinase activity of p58-M1 is inhibited by T3 and its analogs in a concentration-dependent manner with the order of inhibitory activity similar to that of binding activity. This inhibition, however, is reversed by the addition of fructose 1,6-bisphosphate. p58-M1 is the second PK isoenzyme monomer to be identified as having thyroid hormone binding activity.     

Regulation of thyroid hormone binding to its cytosolic binding protein by L-alpha-alanine

The human cytosolic thyroid hormone binding protein (p58) was recently shown to be a monomer of pyruvate kinase, subtype PKM2, and have intrinsic pyruvate kinase activity. The present study evaluated the effect of L-alpha-alanine on the binding of 3,3',5-triiodo-L-thyronine (T3) and enzymatic activity of p58. Analysis of the competitive binding data indicated that alanine, at the physiological concentration, is a non-competitive inhibitor of T3 binding to p58. Furthermore, alanine was found to be a "mixed" inhibitor of the substrate phosphoenol pyruvate. However, binding of alanine to p58 did not block the association of p58 to form the tetrameric pyruvate kinase.     

In vivo regulation of monomer-tetramer conversion of pyruvate kinase subtype M2 by glucose is mediated via fructose 1,6-bisphosphate

The activity of pyruvate kinase, subtype M2 (PKM2), is known to be increased by fructose 1,6-bisphosphate (Fru-1,6-P2), one of the metabolites in the glycolytic pathway. Recently, we have shown that in vitro, Fru-1,6-P2 activated the association of monomer to form the tetrameric PKM2. To ascertain whether this mode of regulation also occurs in vivo, we prepared monomer-specific monoclonal antibody and quantified the monomer formation in situ in cultured cells by immunocytochemistry. The intracellular Fru-1,6-P2 was manipulated by the glucose concentration in the media. At the physiological concentration of glucose (4-6 mM), 30-35% of PK existed as a monomer. However, PKM2 was dissociated into monomer within minutes after cells were deprived of glucose. The maximal level of monomer was detected after 1 h at 37 degrees C. Monomer was rapidly (within minutes) converted to tetramer after addition of glucose. Furthermore, when cells cultured in 10 mM of glucose were treated with cytochalasin B, an inhibitor of the glucose transporter, a maximal level of monomer was detected within 20-30 min. Determination of Fru-1,6-P2 indicated that its intracellular concentration decreased concomitantly with the reduction in glucose concentration in the medium. These results indicate that monomer-tetramer inter-conversion is a major in vivo cellular regulatory mechanism in response to changes in the extracellular glucose concentration via Fru-1,6-P2.     

Amino-acids imitate the EDTA activation on the fructose-1,6-bisphosphatase of mantle tissue from the sea mussel (Mytilus galloprovincialis Lmk).

In the absence of AMP and Fru-2,6-P2, several amino-acids such as histidine, lysine, alanine, aspartic acid, and other molecules, as reduced glutathione or citrate, activate FBPase-1 from Mytilus galloprovincialis mantle. AMP decreases Vmax and Km for Fru-1,6-P2 both in the absence and in the presence of activators; but the addition of Fru-2,6-P2 decreases the affinity of the enzyme by its substrate. Na+ acts as a inhibitor reducing both Vmax and Km. The Km value is lower than the physiological level of Fru-1,6-P2, suggesting that the enzyme is operative but its activity is very reduced.     

Reversible ligand-induced dissociation of a tryptophan-shift mutant of phosphofructokinase from Bacillus stearothermophilus

The biophysical properties of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus (BsPFK) have been examined. The mutant, designated W179Y/Y164W, has kinetic and thermodynamic properties similar to the wild-type enzyme. A 2-fold decrease in kcat is observed, and the mutant displays a 3-fold smaller K(0.5) for the substrate, fructose-6-phosphate (Fru-6-P), as compared to the wild-type enzyme. The dissociation constant for the inhibitor, phospho(enol)pyruvate (PEP), increases 2-fold, and the coupling parameter, Q(ay), decreases 2-fold. This suggests that while the mutant displays a slightly decreased affinity for PEP, PEP is still an effective inhibitor once bound. The new position of the tryptophan in W179Y/Y164W is approximately 6 A from the Fru-6-P portion of the active site. A 25% decrease in fluorescence intensity is observed upon Fru-6-P binding, and an 80% decrease in fluorescence intensity is observed with PEP binding. In addition, the intrinsic fluorescence polarization increases from 0.327 +/- 0.001 to 0.353 +/- 0.001 upon Fru-6-P binding, but decreases to 0.290 +/- 0.001 when PEP binds. Most notably, the presence of PEP induces dissociation of the tetramer. Dissociation of the tetramer into dimers occurs along the active site interface and can be monitored by the loss in activity or the loss in tryptophan fluorescence that is observed when the enzyme is titrated with PEP. Activity can be protected or recovered by incubating the enzyme with Fru-6-P. Recovery of activity is enzyme concentration dependent, and the rate constant for association is 6.2 +/- 0.3 M(-1) x s(-1). Ultracentrifugation experiments revealed that in the absence of PEP the mutant enzyme exists in an equilibrium between the dimer and tetramer forms with a dissociation constant of 11.8 +/- 0.5 microM, while in the presence of PEP the enzyme exists in equilibrium between the dimer and monomer forms with a dissociation constant of 7.5 +/- 0.02 microM. A 3.1 A crystal structure of the mutant enzyme suggests that the amino acid substitutions have not dramatically altered the tertiary structure of the enzyme. While it is clear that wild-type BsPFK exists as a tetramer under these same conditions, these results suggest that quaternary structural changes probably play an important role in allosteric communication.     

Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase appears to be the only enzyme catalyzing the formation and hydrolysis of Fru-2,6-P2. The enzyme as we isolate it, contains a trace of tightly bound Fru-6-P. In this condition, it exhibited an ATPase activity comparable to its kinase activity. Inorganic phosphate stimulated all of its activities, by increasing the affinity for all substrates and increasing the Vmax of ATP and Fru-2,6-P2 hydrolysis. The enzyme catalyzed ADP/ATP and Fru-6-P/Fru-2,6-P2 exchanges at rates comparable to net reaction rates. It was phosphorylated by both [gamma-32P]ATP and [2-32P] Fru-2,6-P2, and the label from either donor was chased by either unlabeled donor, showing that the bound phosphate is hydrolyzed if not transferred to an acceptor ligand. The rate of labeling of the enzyme by [2-32P]Fru-2,6-P2 was 2 orders of magnitude greater than the maximal velocity of the bisphosphatase and therefore sufficiently fast to be a step in the hydrolysis. Both inorganic phosphate and Fru-6-P increased the rate and steady state of enzyme phosphorylation by ATP. Fru-2,6-P2 inhibited the ATPase and kinase reactions and Fru-6-P inhibited the Fru-2,6 bisphosphatase reaction while ATP and ADP had no effect. Removal of the trace of Fru-6-P by Glu-6-P isomerase and Glu-6-P dehydrogenase reduced enzyme phosphorylation by ATP to very low levels, greatly inhibited the ATPase, and rendered it insensitive to Pi, but did not affect ADP/ATP exchange. (alpha + beta)Methylfructofuranoside-6-P did not increase the rate or steady state labeling by ATP. These results suggest that labeling of the enzyme by ATP involved the production of [2-32P]Fru-2,6-P2 from the trace Fru-6-P. The 6-phosphofructo-2-kinase, fructose 2,6-bisphosphatase, and ATP/ADP exchange were all inhibited by diethylpyrocarbonate, suggesting the involvement of histidine residues in all three reactions. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a Fru-2,6 bisphosphatase site which is readily phosphorylated by Fru-2,6-P2.     

Purification and regulatory properties of pyruvate kinase from Veillonella parvula.

The nonglycolytic, anaerobic organism Veillonella parvula M4 has been shown to contain an active pyruvate kinase. The enzyme was purified 126-fold and was shown by disc-gel electrophoresis to contain only two faint contaminating bands. The purified enzyme had a pH optimum of 7.0 in the forward direction and exhibited sigmoidal kinetics at varying concentrations o-f phosphoenol pyruvate (PEP), adenosine 5'-monophosphate (AMP), and Mg-2+ ions with S0.5 values of 1.5, 2.0, and 2.4 mM, respectively. Substrate inhibition was observed above 4 m PEP. Hill plots gave slope values (n) of 4.4 (PEP), 2.8 (adenosine 5'-diphosphate), and 2.0 (Mg-2+), indicating a high degree of cooperativity. The enzyme was inhibited non-competitively by adenosine 5'-triphosphate (Ki = 3.4 mM), and this inhibition was only slightly affected by increasing concentration of Mg-2+ ions to 30 mM. Competitive inhibition was observed with 3-phosphoglycerate, malate, and 2,3-diphosphoglycerate but only at higher inhibitor concentrations. The enzyme was activated by glucose-6-phosphate (P), fructose-6-P, fructose-1,6-diphosphate (P2), dihydroxyacetone-P, and AMP; the Hill coefficients were 2.2, 1.8, 1.5, 2.1, and 2.0, respectively. The presence of each these metabolites caused substrate velocity curves to change from sigmoidal to hyperbolic curves, and each was accompanied by an increase in the maximum activity, e.g., AMP greater than fructose-1,6-P2 greater than dihydroxyacetone-P greater than glucose-6-P greater than fructose-6-P. The activation constants for fructose-1,6-P2, AMP, and glucose-6-P were 0.3, 1.1, and 5.3 mM, respectively. The effect of 5 mM fructose-1,6-P2 was significantly different from the other compounds in that this metabolite was inhibitory between 1.2 and 3 mM PEP. Above this concentration, fructose-1,6-P2 activated the enzyme and abolished substrate inhibition by PEP. The enzyme was not affected by glucose, glyceraldehyde-3-P, 2-phosphoglycerate, lactate, malate, fumerate, succinate, and cyclic AMP. The results suggest that the pyruvate kinase from V. parvula M4 plays a central role in the control of gluconeogenesis in this organism by regulating the concentration of PEP.     

Altered glucose 1,6-bisphosphate and fructose 2,6-biphosphate levels in low-frequency stimulated rabbit fast-twitch muscle

Glucose 1,6-bisphosphate (Glc-1,6-P2) and fructose 2,6-bisphosphate (Fru-2,6-P2) concentrations display pronounced increases in rabbit fast-twitch muscle during chronic low-frequency stimulation. These increases are first seen after stimulation periods exceeding 3 h and reach maxima after 12-24 h of stimulation (approximately 3-fold for Glc-1,6-P2 and 5-fold for Fru-2,6-P2). Both metabolites regress to normal values after stimulation periods longer than 4 days. The fact that their increases coincide with the replenishment of glycogen after its initial depletion, could point to a role of Glc-1,6-P2 and Fru-2,6-P2 in glycogen metabolism.     

Activation of mammalian phosphofructokinases by ribose 1,5-bisphosphate

Ribose 1,5-bisphosphate (Rib-1,5-P2), a newly discovered activator of rat brain phosphofructokinase, forms rapidly during the initiation of glycolytic flux and disappears within 20 s (Ogushi, S., Lawson, J.W. R., Dobson, G.P., Veech, R.L., and Uyeda, K. (1990) J. Biol. Chem. 265, 10943-10949). Activation of various mammalian phosphofructokinases and plant pyrophosphate-dependent phosphofructokinases by Rib-1,5-P2 was investigated. The order of decreasing potency for activation of rabbit muscle phosphofructokinase was: fructose (Fru) 2,6-P2, Rib-1,5-P2, Fru-1,6-P2, Glc-1,6-P2, phosphoribosylpyrophosphate, ribulose-1,5-P2, sedoheptulose-1,7-P2, and myoinositol-1,4-P2. The K0.5 values for activation by Rib-1,5-P2 of rat brain, rat liver, and rabbit muscle phosphofructokinases and potato and mung bean pyrophosphate-dependent phosphofructokinases were 64 nM, 230 nM, 82 nM, 710 nM, and 80 microM, respectively. The corresponding K0.5 values for Fru-2,6-P2 were 9, 8.6, 10, 7, and 65 nM, respectively. Rib-1,5-P2 was a competitive inhibitor of Fru-2,6-P2, binding to the muscle enzyme with Ki of 26 microM. Citrate increased the K0.5 for Rib-1,5-P2 without affecting the maximum activation, and AMP lowered the K0.5 for Rib-1,5-P2 without affecting the maximum activation. These effects of citrate and AMP were similar to those observed with Fru-2,6-P2 and different from those with Fru-1,6-P2. Rib-1,5-P2 is the second most potent activator of phosphofructokinase thus far discovered. The Rib-1,5-P2-activated conformation of the enzyme seems to be similar to that induced by Fru-2,6-P2, but different from that induced by Fru-1,6-P2.     

The inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate is enhanced by EDTA and diminished by zinc(II)

The sensitivity of the Mg(II)-dependent activity of rabbit liver fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) to inhibition by fructose 2,6-bisphosphate (Fru-2,6-P2) was enhanced by EDTA and diminished to negligible levels by 0.5-2 microM Zn(II) added as another FBPase inhibitor. Fru-2,6-P2 was more efficient in the presence of the synergistic effector AMP: still, the Fru-2,6-P2 concentration inhibiting 50% changed from 3 microM (with EDTA) to higher than 50 microM (with Zn(II]. On the other hand, the Zn(II)-dependent FBPase activity was inhibited by Fru-2,6-P2 to a much lesser extent than the Mg(II)-dependent activity.     

Effect of buffer solutions on activation of Shamouti orange pyrophosphate-dependent phosphofructokinase by fructose 2,6-bisphosphate

Shamouti phosphofructokinase (PFP) activation depends on the presence of fructose 2,6-bisphosphate (Fru-2,6-P2) in the glycolytic reaction. The effect of activation by Fru-2,6-P2 differs considerably, however, according to the buffer (pH 8.0) in which the reaction is performed: Ka = 2.77 +/- 0.3 nM in Hepes-NaOH and 7.75 +/- 1.49 nM in Tris-HCl. The presence of chloride ions (39 mM) in the Tris-HCl buffer inhibits PFP. Indeed, when using a Hepes-NaOH buffer and then adding 39 mM NaCl, Ka = 8.12 +/- 0.52 nM. The Ki for chloride ions is approximately 21.7 mM. In the gluconeogenic reaction, Shamouti PFP generally showed a high endogenous activity. Addition of Fru-2,6-P2 did not modify the velocity and the Vmax of the enzyme; however, its presence increased the affinity of the enzyme for Fru-1,6-P2 from 200 +/- 15.6 microM in absence of Fru-2,6-P2 to 89 +/- 10.3 microM in its presence (10 microM). In the presence of chloride (39 mM), the affinity for the substrate decreased with K(m) = 150 +/- 14 microM. The calculated Ki for chloride ions equals 56.9 mM. In both the glycolytic and the gluconeogenic reactions, Vmax is not affected; therefore, the inhibition mode of chloride is competitive.     

Regulation by thyroid hormone of the synthesis of a cytosolic thyroid hormone binding protein during liver regeneration.

To understand the regulation by thyroid hormone, 3,3',5-triiodo-L-thyronine (T3), of the synthesis of a cytosolic thyroid hormone binding protein (p58-M2) during liver regeneration, the synthesis of p58-M2 was evaluated. The synthesis of p58-M2 was measured by metabolic labeling of primary cultures derived from the regenerating liver of euthyroid, hypo- or hyperthyroid rats. During regeneration, the increase in the liver/body weight ratio is approximately 25% higher in hyper- than in hypothyroid rats. However, T3 has no effect on the rate of overall liver regeneration observed in four days. In mature liver, T3 increased the synthesis of p58-M2 by approximately 2.5-fold. During regeneration, however, the change in the synthesis of p58-M2 varied with the thyroid status. In euthyroid rats, the synthesis of p58-M2 continued to increase up to 2-fold during liver regeneration. In hyperthyroid rats, after an initial increase by 1.5-fold on day 1, the synthesis of p58-M2 subsequently declined during regeneration. In hypothyroid rats, the synthesis of p58-M2 remained virtually unchanged during regeneration. These results indicate that T3 regulates the synthesis of p58-M2 in mature and regenerating liver.     

A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure

We have engineered a variant of the lactate dehydrogenase enzyme from Bacillus stearothermophilus in which arginine-173 at the proposed regulatory site has been replaced by glutamine. Like the wild-type enzyme, this mutant undergoes a reversible, protein-concentration-dependent subunit assembly, from dimer to tetramer. However, the mutant tetramer is much more stable (by a factor of 400) than the wild type and is destabilized rather than stabilized by binding the allosteric regulator, fructose 1,6-biphosphate (Fru-1,6-P2). The mutation has not significantly changed the catalytic properties of the dimer (Kd NADH, Km pyruvate, Ki oxamate and kcat), but has weakened the binding of Fru-1,6-P2 to both the dimeric and tetrameric forms of the enzyme and has almost abolished any stimulatory effect. We conclude that the Arg-173 residue in the wild-type enzyme is directly involved in the binding of Fru-1,6-P2, is important for allosteric communication with the active site, and, in part, regulates the state of quaternary structure through a charge-repulsion mechanism.     

The pH dependence of the allosteric response of human liver pyruvate kinase to fructose-1,6-bisphosphate, ATP, and alanine

The allosteric regulation of human liver pyruvate kinase (hL-PYK) by fructose-1,6-bisphosphate (Fru-1,6-BP; activator), ATP (inhibitor) and alanine (Ala; inhibitor) was monitored over a pH range from 6.5 to 8.0 at 37 °C. As a function of increasing pH, hL-PYK’s affinity for the substrate phosphoenolpyruvate (PEP), and for Fru-1,6-BP decreases, while affinities for ATP and alanine slightly increases. At pH 6.5, Fru-1,6-BP and ATP elicit only small allosteric impacts on PEP affinity. As pH increases, Fru-1,6-BP and ATP elicit greater allosteric responses, but the response to alanine is relatively constant. Since the magnitudes of the allosteric coupling for ATP and for alanine inhibition are different and the pH dependences of these magnitudes are not similar, these inhibitors likely elicit their responses using different molecular mechanisms. In addition, our results fail to support a general correlation between pH dependent changes in effector affinity and pH dependent changes in the corresponding allosteric response.     

Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases

The specificities of cAMP-dependent and cGMP-dependent protein kinases were studied using synthetic peptides corresponding to the phosphorylation site in 6-phosphofructo-2-kinase/Fru-2,6-P2ase (Murray, K.J., El-Maghrabi, M.R., Kountz, P.D., Lukas, T.J., Soderling, T.R., and Pilkis, S.J. (1984) J. Biol. Chem. 259, 7673-7681) as substrates. The peptide Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase on predominantly the first of its 2 seryl residues. The Km (4 microM) and Vmax (14 mumol/min/mg) values were comparable to those for the phosphorylation of this site within native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. An analog peptide containing only two arginines was phosphorylated with poorer kinetic constants than was the parent peptide. These results suggest that the amino acid sequence at its site of phosphorylation is a major determinant that makes 6-phosphofructo-2-kinase/Fru-2,6-P2ase an excellent substrate for cAMP-dependent protein kinase. Although 6-phosphofructo-2-kinase/Fru-2,6-P2ase was not phosphorylated by cGMP-dependent protein kinase, the synthetic peptide corresponding to the cAMP-dependent phosphorylation site was a relatively good substrate (Km = 33 microM, Vmax = 1 mumol/min/mg). Thus, structures other than the primary sequence at the phosphorylation site must be responsible for the inability of cGMP-dependent protein kinase to phosphorylate native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. Peptides containing either a -Ser-Ser- or -Thr-Ser- moiety were all phosphorylated by cGMP-dependent kinase to 1.0 mol of phosphate/mol of peptide, but the phosphate was distributed between the two hydroxyamino acids. Substitution of a proline in place of the glycine between the three arginines and these phosphorylatable amino acids caused the protein kinase selectively to phosphorylate the threonyl or first seryl residue and also enhanced the Vmax values by 4-6-fold. These results are consistent with a role for proline in allowing an adjacent threonyl residue to be readily phosphorylated by cGMP-dependent protein kinase.     

Changes in the state of subunit association of lactate dehydrogenase from Bacillus stearothermophilus

Time-resolved measurements of the fluorescence anisotropy of an extrinsic dye-group attached to lactate dehydrogenase from B. stearothermophilus revealed that the rotational correlation time of the enzyme at low concentrations is 55 ns, while at high enzyme concentrations or in the presence of fructose 1,6-bisphosphate (Fru-1,6-P2) the correlation time increases to 95 ns. These correlation times are consistent with a change in Mr from 85 000 +/- 12 000 (dimer) to 150 000 +/- 22 000 (tetramer) and show that the tetrameric state can be induced either by raising the protein concentration or by the addition of the ligand. We have confirmed this change in molecular weight by gel-filtration experiments. In the ligand-induced tetramer, two Fru-1,6-P2 molecules are bound.     

MgATP-dependent activation by phosphoenolpyruvate of the E187A mutant of Escherichia coli phosphofructokinase

Using enzymatic assays and steady-state fluorescence emission, we performed a linkage analysis of the three-ligand interaction of fructose 6-phosphate (Fru-6-P), phosphoenolpyruvate (PEP), and MgATP on E187A mutant Escherichia coli phosphofructokinase (PFK). PEP allosterically inhibits Fru-6-P binding to E. coli PFK. The magnitude of antagonism is 90-fold in the absence and 60-fold in the presence of a saturating concentration of MgATP [Johnson, J. J., and Reinhart, G. D. (1997) Biochemistry 36, 12814-12822]. Substituting an alanine for the glutamate at position 187, located in the allosteric site (i.e., mutant E187A), activates Fru-6-P binding and inhibits the maximal rate of enzyme turnover [Lau, F. T.-K., and Fersht, A. R. (1987) Nature 326, 811-812]. The allosteric action of PEP appears to depend on the presence of the cosubstrate MgATP. In the presence of a saturating concentration of MgATP, PEP enhances the binding of Fru-6-P to the enzyme by a modest 2-fold. Decreasing the concentration of MgATP mitigates the extent of activation. At MgATP concentrations approaching 25 microM, PEP becomes insensitive to the binding of Fru-6-P. At MgATP concentrations < 25 microM, PEP "crosses over" and becomes antagonistic toward substrate binding. The present study examines the role of Glu 187 at the allosteric site in the binding of Fru-6-P and offers a more complex explanation of the mechanism than that described by traditional allosteric mechanistic models.     

Covalent control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: insights into autoregulation of a bifunctional enzyme.

The hepatic bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P2ase), E.C. 2.7-1-105/E.C. 3-1-3-46, is one member of a family of unique bifunctional proteins that catalyze the synthesis and degradation of the regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P2). Fru-2,6-P2 is a potent activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, and provides a switching mechanism between these two opposing pathways of hepatic carbohydrate metabolism. The activities of the hepatic 6PF-2-K/Fru-2,6-P2ase isoform are reciprocally regulated by a cyclic AMP-dependent protein kinase (cAPK)-catalyzed phosphorylation at a single NH2-terminal residue, Ser-32. Phosphorylation at Ser-32 inhibits the kinase and activates the bisphosphatase, in part through an electrostatic mechanism. Substitution of Asp for Ser-32 mimics the effects of cAPK-catalyzed phosphorylation. In the dephosphorylated homodimer, the NH2- and COOH-terminal tail regions also have an interaction with their respective active sites on the same subunit to produce an autoregulatory inhibition of the bisphosphatase and activation of the kinase. In support of this hypothesis, deletion of either the NH2- or COOH-terminal tail region, or both regions, leads to a disruption of these interactions with a maximal activation of the bisphosphatase. Inhibition of the kinase is observed with the NH2-truncated forms, in which there is also a diminution of cAPK phosphorylation to decrease the Km for Fru-6-P. Phosphorylation of the bifunctional enzyme by cAPK disrupts these autoregulatory interactions, resulting in inhibition of the kinase and activation of the bisphosphatase. Therefore, effects of cyclic AMP-dependent phosphorylation are mediated by a combination of electrostatic and autoregulatory control mechanisms.     

Nuclear magnetic resonance studies of fructose 2,6-bisphosphate and adenosine 5'-monophosphate interaction with bovine liver fructose-1,6-biphosphatase

1H and 31P nuclear magnetic resonance was used to investigate the interaction of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) with bovine liver fructose-1,6-bisphosphatase. Mn2+ bound to fructose-1,6-bisphosphatase was used as a paramagnetic probe to map the active and AMP allosteric sites of fructose-1,6-bisphosphatase. Distances between enzyme-bound Mn2+ and the phosphorus atoms at C-6 of fructose-6-P and alpha-methyl-D-fructofuranoside 1,6-bisphosphate were identical, and the enzyme-Mn to phosphorus distance determined for the C-6 phosphorus atom of Fru-2,6-P2 was very similar to these values. Likewise, the enzyme-Mn to phosphorus distances for Pi, the C-1 phosphorus atom of alpha-methyl-D-fructofuranoside 1,6-bisphosphate, and the C-2 phosphorus atom of Fru-2,6-P2 agreed within 0.5 A. The distance between enzyme-bound Mn2+ and the phosphorus atom of AMP was significantly shorter than the distances obtained for any of the aforementioned ligands, but the presence of Fru-2,6-P2 caused the enzyme-Mn to phosphorus distance for AMP to lengthen markedly. NMR line broadening of AMP protons was studied at various temperatures. The dissociation rate constant was found to be greater than 20 s-1. It was concluded that Fru-2,6-P2 strongly affects the interaction of AMP with fructose-1,6-bisphosphatase and that the sugar most likely acts at the active site of the enzyme.