Fructose 2,6-bisphosphate and glycolytic oscillations in skeletal muscle extracts

Oscillatory behavior of glycolysis in cell-free extracts of rat skeletal muscle involves bursts of phosphofructokinase activity due to autocatalytic activation by fructose-1,6-P2. Fructose-2,6-P2 is an even more potent activator of phosphofructokinase and is competitive with fructose-1,6-P2 in binding and kinetic studies. The possible role and effects of fructose-2,6-P2 on the oscillating system were therefore examined. When muscle extracts were provided with 1 mM ATP and 10 mM glucose, fructose-2,6-P2 slowly accumulated to 50 nM in 1 h. The nearly monotonic rise, in contrast to the 50-fold oscillations in fructose-1,6-P2, indicated no involvement of fructose-2,6-P2 in the oscillatory process. Addition of 0.5 microM fructose-2,6-P2 blocked the oscillations, and there was negligible appearance of glycolytic intermediates from fructose-1,6-P2 to phosphoenolpyruvate, although similar amounts of lactate accumulated. In the presence of 0.2 microM fructose-2,6-P2, there were small, transient accumulations of fructose-1,6-P2, suggesting aborted activations of phosphofructokinase. Oscillations were not blocked by 0.1 microM fructose-2,6-P2. The average [ATP]/[ADP] ratio in the presence of 0.2 or 0.5 microM fructose-2,6-P2 was half the value in its absence, demonstrating the advantage of the oscillatory behavior in maintaining a high energy state. In the presence of higher, near physiological levels of ATP and citrate, inhibitors which reduce the affinity of phosphofructokinase for fructose-2,6-P2, glycolytic oscillations were not blocked by 1 microM fructose-2,6-P2, its approximate concentration in vivo.     

Modulation by citrate of glycolytic oscillations in skeletal muscle extracts.

Oscillatory behavior of glycolysis in cell-free extracts of skeletal muscle involves repeated bursts of phosphofructokinase activity and associated oscillations in the [ATP]/[ADP] ratio. Addition of citrate, a potent physiological inhibitor of phosphofructokinase, decreased the frequency of the oscillations and delayed the first burst of phosphofructokinase activity in a dose-dependent manner. Citrate decreased the trigger point [ATP]/[ADP] ratio at which bursts of phosphofructokinase activity were initiated but had a much smaller effect on the average [ATP]/[ADP] ratio and did not decrease the peak values of the ratio. When oscillations were prevented by addition of fructose-2,6-P2, the decrease in the [ATP]/[ADP] ratio caused by citrate in the steady state system was similar to the decrease in the trigger point [ATP]/[ADP] ratio in the oscillatory system. The decrease in the average [ATP]/[ADP] ratio was greater in the steady state system than in the oscillating system. These results demonstrate advantages of oscillatory behavior of glycolysis in the regulation of carbohydrate utilization and the maintenance of a high [ATP]/[ADP] ratio.     

A specific enzyme for glucose 1,6-bisphosphate synthesis.

The reaction: glycerate-1,3-P2 PLUS GLUCOSE-1-P YIELDS TO GLUCOSE-1,6-P2 plus glycerate-P is catalyzed by a distinct enzyme of mouse brain. A divalent metal requirement was shown when the enzyme was treated with imidazole and EDTA. Mg2+, Mn2+, Ca2+, Zn2+, Ni2+, Co2+, and Cd2+ were quite effective cofactors. The enzyme, in better than 50 percent yield, has been purified away from 99 percent of the phosphoglucomutase, phosphoglycrate mutase, and phosphofructokinase. Acetyl-P, ATP, enolpyruvate-P, creatine-P, and fructose-1,6-P2 are not phosphoryl donors. Glucose-6-P and mannose-1-P are good alternate acceptors. Mannose-6-P, galactose-Ps, and fructose-Ps have little or no acceptor activity. Strong inhibition was found with fructose-1,6-P2, glycerate-2,3-P2, enolpyruvate-P, and acetyl CoA. From the amount of activity and the kinetic constants of the purified enzyme it seems likely that this enzyme is responsible for the glucose-1,6-P2 synthesis of brain.     

Activation of muscle phosphofructokinase by fructose 2,6-bisphosphate and fructose 1,6-bisphosphate is differently affected by other regulatory metabolites

Fructose-2,6-P2 and fructose-1,6-P2 are strong activators of muscle phosphofructokinase. They have been shown to be competitive in binding studies, and it is generally thought that they affect the physical and catalytic properties of the enzyme in the same manner. However, there are indications in published data that the effects of the two fructose bisphosphates on phosphofructokinase are not identical. To examine this possibility, the kinetics of activation of rat skeletal muscle phosphofructokinase by the two fructose bisphosphates were compared in the presence of other regulatory metabolites. Citrate greatly increased the K0.5 of the enzyme for fructose-2,6-P2, with little effect on the maximum activation. In contrast, citrate greatly decreased the maximum activation by fructose-1,6-P2, with only a small effect on the K0.5. Changes in the concentrations of the inhibitor ATP or the activator AMP similarly altered the K0.5 for fructose-2,6-P2, but altered the maximum activation by fructose-1,6-P2. Finally, when fructose-1,6-P2 was added in the presence of a given concentration of fructose-2,6-P2, phosphofructokinase activity was decreased if the activation by fructose-2,6-P2 alone was greater than the maximum activation by fructose-1,6-P2 alone. These results are consistent with competition of the two fructose bisphosphates for the same binding site, but indicate that the conformational changes produced by their binding are different.     

Increase in glucose 1,6-bisphosphate levels, activation of phosphofructokinase and phosphoglucomutase, and inhibition of glucose 1,6-bisphosphatase in muscle induced by trifluoperazine

Injection of trifluoperazine (TFP) to rats induced a significant rise in the level of glucose 1,6-bisphosphate (Glc-1,6-P2) in muscle. This increase in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, was accompanied by a marked activation of both enzymes, when assayed in the absence of exogenous Glc-1,6-P2 under conditions in which these enzymes are sensitive to regulation by endogenous Glc-1,6-P2. Glucose-1,6-bisphosphatase (the enzyme that degrades Glc-1,6-P2) was markedly inhibited following the injection of TFP, which may account for the rise in the Glc-1,6-P2 level. Previous results from this laboratory have revealed that muscle damage or weakness is characterized by a decrease in Glc-1,6-P2 levels, leading to a marked reduction in the activities of phosphoglucomutase and phosphofructokinase (the rate-limiting enzyme in glycolysis). The present results suggest that TFP treatment may have a beneficial effect on the depressed glycolysis in muscle weakness or damage.     

The effect of natural and synthetic D-fructose 2,6-bisphosphate on the regulatory kinetic properties of liver and muscle phosphofructokinases

The effect of natural "activation factor" and synthetic fructose-2,6-P2 on the allosteric kinetic properties of liver and muscle phosphofructokinases was investigated. Both synthetic and natural fructose-2,6-P2 show identical effects on the allosteric kinetic properties of both enzymes. Fructose-2,6-P2 counteracts inhibition by ATP and citrate and decreases the Km for fructose-6-P. This fructose ester also acts synergistically with AMP in releasing ATP inhibition. The Km values of liver and muscle phosphofructokinase for fructose-2,6-P2 in the presence of 1.25 mM ATP are 12 milliunits/ml (or 24 nM) and 5 milliunits/ml (or 10 nM), respectively. At near physiological concentrations of ATP (3 mM) and fructose-6-P (0.2 mM), however, the Km values for fructose-2,6-P2 are increased to 12 microM and 0.8 microM for liver and muscle enzymes, respectively. Thus, fructose-2,6-P2 is the most potent activator of the enzyme compared to other known activators such as fructose-1,6-P2. The rates of the reaction catalyzed by the enzymes under the above conditions are nonlinear: the rates decelerate in the absence or in the presence of lower concentrations of fructose-2,6-P2, but the rates become linear in the presence of higher concentrations of fructose-2,6-P2. Fructose-2,6-P2 also protects phosphofructokinase against inactivation by heat. Fructose-2,6-P2, therefore, may be the most important allosteric effector in regulation of phosphofructokinase in liver as well as in other tissues.     

The synthesis of mannose 1-phosphate in brain

The interconversion of mannose-6-P and mannose-1-P in brain has been shown to be catalyzed by a distinct enzyme. The enzyme has been separated from most of the phosphoglucomutase activity of the brain. The residual phosphoglucomutase activity (less than 1%) may be associated with phosphomannomutase itself. Mannose-1,6-P2 or glucose-1,6-P2 is required for the reaction as well as a divalent cation (Mg2+ greater than Co2+ greater than Ni2+ greater than Mn2+). Glucose-1-P, glucose-6-P, and 2-deoxyglucose-6-P are also substrates or inhibitors. Other phosphorylated sugars tested, glucosamine-6-P, N-acetylglucosamine-6-P, galactose-6-P, fructose-6-P, ribose-5-P, and arabinose-5-P, do not affect the rate of the reaction when assayed in the presence of mannose-6-32P.     

Exogenous ATP antagonizes the actions of phospholipase A2, local anesthetics, Ca2+ ionophore A23187, and lithium on glucose-1,6-bisphosphate levels and the activities of phosphofructokinase and phosphoglucomutase in rat muscle

ATP, added externally to the incubation medium of rat diaphragm muscles, abolished the decrease in the levels of glucose-1,6-bisphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, induced by phospholipase A2, local anesthetics, Ca2+ ionophore A23187, or lithium. Concomitantly to the changes in Glc-1,6-P2, the potent activator of phosphofructokinase (the rate-limiting enzyme in glycolysis) and phosphoglucomutase, the activities of these enzymes were reduced by the myotoxic agents and restored by exogenous ATP, when assayed under conditions in which these enzymes are sensitive to regulation by Glc-1,6-P2. These findings suggest that ATP may have broad therapeutic action, as it may stimulate the impaired glycolysis in muscle induced by various drugs and conditions which cause muscle weakness or damage.     

Complementarity in the regulation of phosphoglucomutase, phosphofructokinase and hexokinase; the role of glucose 1,6-bisphosphate.

ATP and citrate, the well known inhibitors of phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), were found to inhibit the activities of the multiple forms of phosphoglucomutase (alpha-D-glucose 1,6-bisphosphate: alpha-D-glucose 1-phosphate phosphotransferase, EC 2.7.5.1) from rat muscle and adipose tissue. This inhibition could be reversed by an increase in the glucose 1,6-bisphosphate (Glc-1,6-P2) concentration. Other known activators (deinhibitors) of phosphofructokinase, viz. cyclic AMP, AMP, ADP or Pi, had no direct deinhibitory action on the ATP or citrate inhibited multiple phosphoglucomutases. Cyclic AMP and AMP, could however lead indirectly to deinhibition of the phosphoglucomutases, by activating phosphofructokinase which catalyzes the ATP-dependent phosphorylation of glucose 1-phosphate to form Glc-1,6-P2, the la-ter then released the multiple phosphoglucomutases from ATP or citrate inhibition. The Glc-1,6-P2 was also found to exert a selective inhibitory effect on hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) type II, the predominant form in skeletal muscle. This selective inhibition by Glc-1,6-P2 was demonstrated on the multiple hexokinases which were resolved by cellogel electrophoresis or isolated by chromatography on DEAE-cellulose. Based on the in vitro studies it is suggested that during periods of highly active epinephrine-induced glycogenolysis in muscle, the Glc-1,6-P2, produced by the cyclic AMP-stimulated reaction of phosphofructokinase with glucose 1-phosphate, will release the phosphoglucomutases from ATP or citrate inhibition, and will depress the activity of muscle type II hexokinase.     

The enzymic synthesis of ribose-1,5-bisphosphate: studies of its role in metabolism

Ribose-1,5-bisphosphate is synthesized in a reaction that uses ribose-1(or 5)-P as the phosphoryl acceptor and the acyl-P of 3-phosphoglyceryl phosphate as the donor. Glucose-1,6-bisphosphate is synthesized in a similar reaction. The relative activity with the two substrates remains unchanged over almost 300-fold purification of the enzyme, indicating that glucose-1,6-bisphosphate synthase catalyzes both reactions. The relative V/Km values for alternative phosphoryl acceptors are ribose-1-P (1); glucose-1-P (0.30); mannose-1-P and ribose-5-P (0.11); glucose-6-P (0.10); 2-deoxyglucose-6-P (0.03); and 2-deoxyribose-5-P (0.02). Fructose-1- and 6-phosphates are not substrates. The synthesis of both ribose-1,5-bisphosphate and glucose-1,6-bisphosphate is inhibited by physiologically significant levels of fructose-1,6-bisphosphate, glycerate-2,3-bisphosphate, glycerate-3-phosphate, citrate, and inorganic phosphate. Ribose-1,5-bisphosphate is a strong activator of brain phosphofructokinase.     

Influence of bradykinin on glucose 1,6-bisphosphate and cyclic GMP levels and on the activities of glucose 1,6-bisphosphatase, phosphofructokinase and phosphoglucomutase in muscle

The intracellular concentration of glucose-1,6-bisphosphate (Glc-1,6-P2) in rat tibialis anterior muscle was markedly decreased following the injection of bradykinin. Injection of bradykinin also induced a significant increase in the level of cyclic GMP in muscle. The activity of glucose-1,6-bisphosphatase, the enzyme that degrades Glc-1,6-P2, was markedly enhanced by bradykinin, which may account for the decrease in the level of Glc-1,6-P2. The decrease in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, was accompanied by a concomitant reduction in these enzymes' activities. The bradykinin-induced decrease in Glc-1,6-P2 and in the activity of phosphofructokinase, the rate-limiting enzyme in glycolysis, may be involved in the pathogenic influences of this hormone in various clinical conditions.     

A binding study of the interaction of beta-D-fructose 2,6-bisphosphate with phosphofructokinase and fructose-1,6-bisphosphatase

The binding of beta-D-fructose 2,6-bisphosphate to rabbit muscle phosphofructokinase and rabbit liver fructose-1,6-bisphosphatase was studied using the column centrifugation procedure (Penefsky, H. S., (1977) J. Biol. Chem. 252, 2891-2899). Phosphofructokinase binds 1 mol of fructose 2,6-bisphosphate/mol of protomer (Mr = 80,000). The Scatchard plots of the binding of fructose 2,6-bisphosphate to phosphofructokinase are nonlinear in the presence of three different buffer systems and appear to exhibit negative cooperativity. Fructose 1,6-bisphosphate and glucose 1,6-bisphosphate inhibit the binding of fructose-2,6-P2 with Ki values of 15 and 280 microM, respectively. Sedoheptulose 1,7-bisphosphate, ATP, and high concentrations of phosphate also inhibit the binding. Other metabolites including fructose-6-P, AMP, and citrate show little effect. Fructose-1,6-bisphosphatase binds 1 mol of fructose 2,6-bisphosphate/mol of subunit (Mr = 35,000) with an affinity constant of 1.5 X 10(6) M-1. Fructose 1,6-bisphosphate, fructose-6-P, and phosphate are competitive inhibitors with Ki values of 4, 2.7, and 230 microM, respectively. Sedoheptulose 1,7-bisphosphate (1 mM) inhibits approximately 50% of the binding of fructose 1,6-bisphosphate to fructose bisphosphatase, but AMP has no effect. Mn2+, Co2+, and a high concentration of Mg2+ inhibit the binding. Thus, we may conclude that fructose 2,6-bisphosphate binds to phosphofructokinase at the same allosteric site for fructose 1,6-bisphosphate while it binds to the catalytic site of fructose-1,6-bisphosphatase.     

Oscillations of the glycolytic pathway and the purine nucleotide cycle.

In a treatment modeled after the oscillatory behavior of the glycolytic pathway and the purine nucleotide cycle observed in skeletal muscle extracts, it is shown that the basis of the oscillations is the AMP-dependent activation of phosphofructokinase by fructose diphosphate. Control of phosphofructokinase by the adenine nucleotides alone leads to the establishment of a steady state. Whether steady state or oscillatory behavior occurs depends in part on the activity of glyceraldehyde-3-phosphate dehydrogenase, which controls the rate of removal of fructose diphosphate. Under appropriate conditions oscillatory behavior can maintain a higher [ATP]/[ADP] ratio than steady state behavior. Viewed in the context of conditions that may be encountered in skeletal muscle in vivo, oscillatory behavior of glycolysis is shown to have additional advantages for maintaining a high [ATP]/[ADP] ratio.     

Modulation of muscle phosphofructokinase at physiological concentration of enzyme

Two approaches have been used to study the allosteric modulation of phosphofructokinase at physiological concentration of enzyme; a "slow motion" approach based on the use of a very low Mg2+/ATP ratio to conveniently lower Vmax, and the addition of polyethylene glycol as a "crowding" agent to favor aggregation of diluted enzyme. At 0.6 mg/ml muscle phosphofructokinase exhibited a drastic decrease in the ATP inhibition and the concomitant increase in the apparent affinity for fructose-6-P, as compared to a 100-fold diluted enzyme. Similar results were obtained with diluted enzyme in the presence of 10% polyethylene glycol (Mr = 6000). Results with these two approaches in vitro were essentially similar to those previously observed in situ (Aragón, J. J., Felíu, F. E., Frenkel, R., and Sols, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6324-6328), indicating that the enzyme is strongly dependent on homologous interactions at physiological concentrations. With polyethylene glycol it was observed that within the physiological range of concentration of substrates and the other positive effectors, fructose-2,6-P2 still activates the liver phosphofructokinase although it no longer significantly affects the muscle isozyme. In the presence of polyethylene glycol, muscle phosphofructokinase can approach its maximal rate even in the presence of physiologically high concentrations of ATP. Three minor activities of muscle phosphofructokinase have been studied at high enzyme concentration: the hydrolysis of MgATP (ATPase) and fructose-1,6-P2 (FBPase), produced in the absence of the other substrate, and the reverse reaction from MgADP and fructose-1,6-P2. The kinetic study of these activities has allowed a new insight into the mechanisms involved in the modulation of phosphofructokinase activity. The binding of (Mg)ATP at its regulatory site reduces the ability of the enzyme to cleave the bond of the terminal phosphate of MgATP at the substrate site. The positive effectors (Pi, cAMP, NH+4, fructose-1,6-P2, and fructose-2,6-P2) decrease the inhibitory effect of MgATP. Citrate and fructose-2,6-P2 both act as mechanistically "secondary" effectors in the sense that citrate does not inhibit and fructose-2,6-P2 does not activate the FBPase activity, requiring both the presence of ATP to affect the enzyme activity. In conclusion it appears that the regulatory behavior of mammalian phosphofructokinases is utterly dependent on the fact of their high concentrations in vivo.     

The purine nucleotide cycle. Control of phosphofructokinase and glycolytic oscillations in muscle extracts.

Linked oscillations of the glycolytic pathway and the purine nucleotide cycle were studied in particle-free extracts of rat skeletal muscle. Under the conditions used, an accumulation of about 1 muM fructose diphosphate can trigger a sudden increase in phosphofructokinase activity. The activation by fructose diphosphate depends on the presence of AMP. When the AMP concentration drops, phosphofructokinase becomes inhibited, even though the fructose disphosphate concentration remains high. It is concluded that the oscillatory behavior can be of advantage for maintaining a high average [ATP]/[ADP] ratio.     

Methylglyoxal is an intermediate in the biosynthesis of 6-deoxy-5-ketofructose-1-phosphate: a precursor for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii

A biosynthetic pathway is proposed for creating 6-deoxy-5-ketofructose-1-phosphate (DKFP), a precursor sugar for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii. First, two possible routes were investigated to determine if a modified, established biosynthetic pathway could be responsible for generating 6-deoxyhexoses in M. jannaschii. Both the nucleoside diphosphate mannose pathway and a pathway involving nucleoside diphosphate derivatives of fructose-1-P, fructose-2-P, or fructose-1,6-bisP were tested and eliminated. The established pathways did not produce the expected intermediates nor did the anticipated enzymes have the predicted enzymatic activities. Because neither anticipated pathway could produce DKFP, M. jannaschii glucose-6-P metabolism was studied in detail to establish exactly how glucose-6-P is converted into DKFP. This detailed analysis showed that methylglyoxal and a fructose-1-P- or fructose-1,6-bisP-derived dihydroxyacetone-P fragment are key intermediates in DKFP production. Glucose-6-P readily converts to fructose-6-P, which in turn converts to fructose-1,6-bisP. Fructose-6-P and fructose-1,6-bisP convert into glyceraldehyde-3-P (Ga-P-3), which converts into methylglyoxal by a 2,3-elimination of phosphate. The MJ1585-derived enzyme catalyzes the condensation of methylglyoxal with a dihydroxyacetone-P fragment, which is derived from fructose-1-P and/or fructose-1,6-bisP, generating DKFP. The elimination of phosphate from Ga-P-3 proceeds by both enzymatic and chemical routes in cell extracts, producing sufficient concentrations of methylglyoxal to support the reaction. This work is the first report of methylglyoxal functioning in central metabolism.     

Changes in the levels of glucose 1,6-diphosphate and cyclic GMP, and in the activities of phosphofructokinase and phosphoglucomutase induced by serotonin in muscle

Injection of serotonin (5-hydroxytryptamine) induced a marked decrease in the level of glucose 1,6-diphosphate (Glc-1,6-P2) in the rat tibialis anterior muscle. Concomitant to the decrease in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, the activities of both these enzymes were markedly reduced by serotonin. The level of Glc-1,6-P2 and the activities of phosphofructokinase and phosphoglucomutase increased with age in the tibialis anterior muscle and the effect of serotonin was more pronounced in the older animals. Serotonin also induced a significant increase in the level of cyclic GMP in muscle. The serotonin-induced changes in the normal muscle mimic the changes in carbohydrate metabolism we found previously in muscular dystrophy.     

Phosphofructokinases from Escherichia coli. Purification and characterization of the nonallosteric isozyme.

The main phosphofructokinase of Escherichia coli (PFK I) is an extensively studied allosteric enzyme specified by the pfkA gene. A nonallosteric phosphofructokinase was reported (Fraenkel, D.G., Kotlarz, D., and Bluc, H. (1973) J. Biol. Chem. 248, 4865-4866) in strains carrying the pfkB1 mutation, a suppressor of pfkA mutants, and very low levels of this enzyme have also been detected in strains not carrying the suppressor (i.e. pfkB+). The nonallosteric protein has now been prepared pure from three strains, one carrying pfkB1 and pfkA+, one carrying pfkB1 and completely deleted for pfkA, and one carrying pfkB+ and also deleted for pfkA. It is apparently the same enzyme (PFK II) in all three strains, which shows that pfkB1 is a mutation affecting the amount of a normally minor isozyme. PFK II is a tetramer of slightly larger subunit molecular weight than PFK I (36,000 and 34,000, respectively). No immunological cross-reactivity was detected between PFK II and PFK I. Unlike PFK I, PFK II does not show cooperative interactions with fructose-6-P, inhibition by P-enolpyruvate, or activation by ADP. Also unlike PFK I, PFK II is somewhat sensitive to inhibition by fructose-1,6-P2 and can use tagatose-6-P as substrate. Both enzymes can perform the reverse reaction, fructose-6-P + ATP from fructose-1,6-P2 + ADP in vitro, but not in vivo. The normal function of PFK II is not known.     

Glucose-induced accumulation of glucose-1,6-bisphosphate in pancreatic islets : its possible role in the regulation of glycolysis

A rise in the extracellular concentration of glucose from zero to 5.6 and 16.7 mM caused a graded increase in the glucose-1,6-bisphosphate content of rat pancreatic islets. Glucose-1,6-bisphosphate activated phosphofructokinase in islet homogenates, when the reaction velocity was measured at low concentrations of fructose-6-phosphate. It is postulated that glucose-1, 6-bisphosphate participates, together with fructose-2,6-bisphosphate, in the regulation of glycolysis in intact islet cells.     

Catalytic properties of phosphoglucomutase from pea chloroplasts.

Electrophoretically homogeneous phosphoglucomutase (PGM) with specific activity of 3.6 units/mg protein was isolated from pea (Pisum sativum L.) chloroplasts. The molecular mass of this PGM determined by gel-filtration is 125 +/- 4 kD. According to SDS-PAGE, the molecular mass of subunits is 65 +/- 3 kD. The Km for glucose-1-phosphate is 18.0 +/- 0.5 microM, and for glucose-1, 6-diphosphate it is 33 +/- 0.7 microM. At glucose-1-phosphate and glucose-1,6-diphosphate concentrations above 0.5 and 0.2 mM, respectively, substrate inhibition is observed. The enzyme has optimum activity at pH 7.9 and 35 degrees C. Mg2+ activates the PGM. Mn2+ activates the enzyme at concentrations below 0.2 mM, while higher concentrations have an inhibitory effect. The activity of the PGM is affected by 6-phosphogluconate, fructose-6-phosphate, NAD+, ATP, ADP, citrate, and isocitrate.