Pyrophosphate-dependent Phosphofructokinase Research Articles

Purification and properties of a pyrophosphate-dependent phosphofructokinase from Toxoplasma gondii

Inorganic pyrophosphate-dependent phosphofructokinase was identified in Toxoplasma gondii and purified to near homogeneity from the crude extracts. The purified enzyme displayed one major protein band which coincided with enzyme activity on nondenaturing polyacrylamide gel electrophoresis. This phosphofructokinase had a molecular weight of 100 000 determined by gel filtration and was composed of one type of subunit with the molecular weight of 45 000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme is a homodimer. Some kinetic parameters of the purified enzyme were investigated in the forward and the reverse directions. The substrate saturation curves for fructose 6-phosphate and pyrophosphate were all hyperbolic. The apparent K m values for fructose 6-phosphate and for pyrophosphate were 2.7 × 10 −4 M and 3.3 × 10 −5 M respectively. Kinetics for Fru-1,2-P 2 and for P i in the reverse reaction were also hyperbolic. The activity of this enzyme was magnesium-dependent. Nucleoside triphosphates and polyphosphates did not serve as phosphate donor and the enzyme activity was not altered in the presence of any of these nucleotides. As in the case of pyrophosphate-dependent phosphofructokinases from other anaerobic eukaryotes the Toxoplasma enzyme was not activated by fructose 2,6-biphosphate.

Cytokinins and leaf development in sweet pepper (Capsicum annuum L.)

Stimulation of leaf expansion by an exogenous cytokinin was studied in isolated leaf discs of sweet pepper with emphasis on the assimilate utilization of the tissue. Leaf discs were floated on solutions containing sucrose and plant growth regulators. Benzyladenine (BA) promoted the area expansion rate of the leaf discs. Sucrose at 100 mM resulted in increased area expansion rate compared with 10 mM sucrose. However, the increased sucrose concentration had no influence on the effect of BA. Over a period of 24 h, treatment with BA did not result in any change of sucrose uptake nor of the partitioning of assimilated carbon in the leaf discs. Neither did BA treatment affect the activity of acid invertase (EC 3.2.1.26) or pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) in the leaf discs. We conclude that the observed promotion of leaf area expansion by exogenous BA is not mediated through the uptake of sucrose or the carbohydrate metabolism of the leaf tissue.

Expression of the Genes for the α- and β-Subunits of Pyrophosphate-Dependent Phosphofructokinase in Germinating and Developing Seeds from Ricinus communis

Various tissues from both germinating and developing castor seeds (Ricinus communis L.) have been analyzed for the level of expression of the genes for the alpha- and beta-subunits of pyrophosphate-dependent phosphofructokinase (PFP). In tissues in which PFP is expressed, there is a single mRNA species of approximately 2 kilobases for each of the subunits. In germinating endosperm, the gene for the alpha-subunit is expressed at an earlier time after imbibition than that for the beta-subunit, whereas in developing castor seed endosperm, both genes are highly and coordinately expressed. During seedling development, there is tissue-specific expression of the two genes. Tissues in which there is a high level of mRNA correspond with tissues in which both subunits of PFP can be detected. The differential expression of the two subunit genes in germinating endosperm does not result in the presence of the alpha-subunit polypeptide in the absence of the beta-subunit polypeptide. Southern analysis of castor genomic DNA indicates the presence of a single gene for both the alpha- and beta-subunits of PFP in contrast with potato, in which there are at least two genes for each subunit.

Enzymic potential for fructose 6-phosphate phosphorylation by guard cells and by palisade cells in leaves of the broad bean Vicia faba L.

Guard cells and palisade cells were dissected from freeze-dried leaflets of the broad bean, Vicia faba L. Individual cell samples (6-12 ng) were assayed for ATP-dependent and pyrophosphate-dependent phosphofructokinases. The assay indicator, NADH loss, was monitored in real time in oil droplets with a computer-driven microfluorometer. On a protein basis, both activities were 10-fold higher in guard cells than in palisade cells, indicating (i) elevated carbon metabolism in guard cells to meet demands for energy and carbon skeletons required during stomatal opening, and (ii) parallel glycolytic pathways in guard cells, one responsive to the potent regulatory metabolite fructose 2,6-bisphosphate and the other not. Future work will be devoted to clarifying the roles of the cytosolic and chloroplastic compartments in guard cells.

KINETIC MECHANISM OF PYROPHOSPHATE-DEPENDENT PHOSPHOFRUCTOKINASE FROM PROPIONIBACTERIUM FREUDENREICHII AT EXTREMELY HIGH CONCENTRATION (> 0.5 mM) OF PYROPHOSPHATE

ABSTRACT A new region for enzyme activity of inorganic pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii was detected at extremely high concentration (>0.5 mM) of pyrophosphate, where the maximum velocity increased about 3 times. At a relatively low concentration (<0.1 mM) of pyrophosphate, the kinetic mechanism is rapid equilibrium random sequential with E:F6P:Pi, E:MgPPi:Pi and E:FBP:MgPPI being dead-end complexes. However, the data from initial velocity and product inhibition patterns at extremely high concentration of pyrophosphate are consistent with a ping-pong mechanism with E:MgPPi:Pi as a dead-end complex, where MgPPi binds first.

Pyrophosphate-dependent Phosphofructokinase: Its Oligomeric Forms in Mature Green Tomato Fruit

Pyrophosphate-dependent phosphofructokinase (PFP: EC 2.7.1.90) from mature green tomato fruit was purified by a DEAE-Sephacel column and preparative gel electrophoresis. Tomato PFP was composed of oligomeric forms and the tetramer of 243–262 kDa molecular mass was predominant. Treatment of PFP with 20 mM pyrophosphate effectively dissociated oligomers into monomers as shown by the gel filtration patterns of a Sepharose 6B column. Kinetic analysis indicated that tomato PFP had a Km of 0.3 mM for glucose-6-phosphate and a Ki of 1.3 mM for pyrophosphate. About 11-fold activation was attained by fructose-2,6-bisphosphate, though there was no activation in the direction of the reverse, gluconeogenesis reaction. Various effectors had a role to control the PFP activity in the direction of either the forward or reverse reaction.

Effect of Anoxia on the Expression and Molecular Form of the Pyrophosphate Dependent Phosphofructokinase

Effect of Anoxia on the Expression and Molecular Form of the Pyrophosphate Dependent Phosphofructokinase

The substrate reactivity of .mu.-monothiopyrophosphate with pyrophosphate-dependent phosphofructokinase: evidence for a dissociative transition state in enzymic phosphoryl group transfer

mu-Monothiopyrophosphate (MTP), an analogue of pyrophosphate (PPi) with sulfur in place of oxygen in the bridge position, is a substrate for the enzyme pyrophosphate-dependent phosphofructokinase. At pH 9.4 and 6 degrees C, the maximal velocity for the phosphorylation of fructose 6-phosphate (F6P) by MgMTP is about 2.8% of that with MgPPi as the phosphoryl donor. The kinetic mechanism is equilibrium random with rate-limiting transformation of the substrate ternary complex to the product when either MgMTP or MgPPi is the phosphoryl donor. This is known from independent studies to be kinetic mechanism at pH 8.0 and 25 degrees C [Bertagnolli, B. L., & Cook, P. F. (1984) Biochemistry 23, 4101-4108]. The dissociation constant of MgPPi is 14 microM, that of MgMTP is 64 microM, and that of F6P from the enzyme is about 5 mM. The Km values for MgPPi and MgMTP are 14.5 and 173 microM, respectively. MgMTP competes with MgPPi for binding to the enzyme. The values of kcat are 3.4 s-1 and 140 s-1 for MgMTP and MgPPi, respectively, at pH 9.4 and 6 degrees C. The estimated rate enhancement factors are 3.6 x 10(5) and 1.4 x 10(14) for the reactions of MgMTP and MgPPi, respectively. Therefore, MgMTP is a reasonably good substrate for PPi-dependent PKF, on the basis of comparisons of kcat. However, the rate enhancement factors show that the enzyme is a poor catalyst for the reaction of MgMTP. Lesser enzymatic catalysis in the reaction of MgMTP compared with MgPPi is largely compensated for by the greater intrinsic reactivity of MgMTP. Thus, the larger substrate MgMTP is well accommodated in the active site, and the dissociative reaction of MgMTP is well accommodated in the transition state. The results are interpreted to indicate a dissociative transition state for phosphoryl group transfer by PPi-dependent PFK. A modified synthesis and purification of MTP are described, in which (trimethylsilyl)trifluoromethanesulfonate and tetra-N-butylammonium iodide are used in place of iodotrimethylsilane to dealkylate tetramethyl-MTP.

Cloning, sequencing, and expression of pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii.

Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii is a non-allosteric enzyme with properties dissimilar to those of other described phosphofructokinases. The enzyme was cloned into pBluescript, sequenced, and expressed in Escherichia coli at levels 15 times higher than those observed in Propionibacterium. The gene consists of 1215 bases which code for a protein of 404 amino acids and a mass of 43,243 daltons. High G + C in the codon usage (66%) of the gene is consistent with the classification of Propionibacterium in the High-G + C subdivision of the Gram-positive bacteria. While showing no sequence identity to the non-allosteric ATP-dependent phosphofructokinase of E. coli, alignments of the amino acid sequence with other PFKs reveal degrees of identities among the amino halves of the proteins, from 26% between the Propionibacterium and potato PPi-PFKs, and 29% between Propionibacterium and E. coli ATP-PFKs. These levels of identities indicate that the amino halves of these proteins are homologous. Identities between the carboxyl half of Propionibacterium PFK and carboxyl halves of other sequences are below 20%, suggesting that the carboxyl half is not homologous. Despite the poor conservation, most of the residues that take part in the binding of fructose-6-P or Mg-PPi could be readily identified by analogy to the structure of the E. coli PFK. Both the fructose-6-P and ATP-binding sites are conserved, indicating that PPi binds to the homologous site of the E. coli ATP-binding site.

Pyrophosphate Dependent Phosphofructokinase of Citrullus lanatus: Molecular Forms and Expression of Subunits

During germination and seedling establishment, the total pyrophosphate-dependent phosphofructokinase (PFP) activity in the cotyledons increases. Two types of subunits with molecular weights of 68 (alpha-subunit) and 65 (beta-subunit) kilodaltons are present. The increase in activity coincides with an approximately 10-fold increase in beta-subunit and twofold increase in alpha-subunit content. Different isoforms of PFP are present at all stages of incubation, but the ratio between the isoforms significantly changes. A linear relationship exists between the ratio of the two PFP subunits and the ratio of the two isoforms of the enzyme. The more anionic (peak 2) isoform of the enzyme apparently is favored by a high ratio of total beta-subunit to alpha-subunit content. The beta- to alpha-subunit ratio of the peak 2 isoform is also approximately fivefold higher than that of the peak 1 (less anionic) isoform. It is evident that the two subunits are not coordinately expressed and the level of expression of each subunit appears to be the primary factor determining the molecular form in which the enzyme is present. In some tissues, only the 65 kilodalton polypeptide is expressed in large amounts. The peak 1 isoform has a higher affinity for pyrophosphate than the peak 2 isoform, while the affinity for fructose-6-phosphate is similar. Both molecular forms are activated by fructose-2,6-bisphosphate.

Pyrophosphate-dependent phosphofructokinase, an anaerobic glycolytic enzyme?

Recent evidence indicates that in as diverse organisms as unicellular eukaryotes, higher plants and prokaryotes, anaerobic glycolysis relies on a pyrophosphate-dependent phosphofructokinase instead of the classical ATP-dependent enzyme. This difference in phosphoryl donor specificity does not necessarily reflect a primitive metabolism, as thought earlier, but could rather be the result of convergent evolution, fostered by the energetic advantage conferred to the cell when glycolysis is the sole source of ATP.

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.

Pyrophosphate-dependent phosphofructokinase. Conservation of protein sequence between the alpha- and beta-subunits and with the ATP-dependent phosphofructokinase.

Full-length cDNA clones for the alpha- and beta-subunits of pyrophosphate-fructose 6-phosphate 1-phosphotransferase have been isolated from a cDNA expression library derived from potato tuber poly(A)+ RNA. The nucleotide sequences indicate that the alpha- and beta-subunits are related with about 40% of amino acid residues being identical. A comparison of the deduced amino acid sequences of both subunits of this enzyme with that of the major ATP-dependent fructose 6-phosphate 1-phosphotransferase from Escherichia coli (Shirakihara, Y., and Evans, P. R. (1988) J. Mol. Biol. 204, 973-994) showed little homology between the proteins except for regions involved in the binding of fructose 6-phosphate/fructose, 1,6-bisphosphate and possibly between regions binding pyrophosphate and the beta- and gamma-phosphates of ADP/ATP. A comparison of the derived secondary structures of the two subunits of the PPi-dependent enzyme with the known secondary structure of the E. coli ATP-dependent enzyme indicated that the overall structure of these enzymes is similar. These data suggest that catalytic activity resides on the beta-subunit of the pyrophosphate-dependent enzyme.

Enzyme Sets of Glycolysis, Gluconeogenesis, and Oxidative Pentose Phosphate Pathway Are Not Complete in Nongreen Highly Purified Amyloplasts of Sycamore (Acer pseudoplatanus L.) Cell Suspension Cultures

Differential centrifugation and Percoll-gradient centrifugation of protoplast lysates of suspension-cultured cells of sycamore (Acer pseudoplatanus L.) yielded pure amyloplasts. Contamination of the final amyloplast preparation by foreign compartments was assessed by measuring marker enzyme activities. The activity of alkaline pyrophosphatase was taken as a 100% plastid marker; relative to this marker, mitochondria (cytochrome c oxidase) averaged 0.34%, microbodies (catalase) 0.61%, and cytosol (alcohol dehydrogenase) 0.09%. Enzymatic activities of the glycolytic, gluconeogenic, pentose phosphate and the starch degradation pathways were found to be present in these amyloplast extracts in appreciable amounts. But the pyrophosphate-dependent phosphofructokinase and phosphoglyceromutase were judged to be essentially absent from amyloplasts because the activities of these enzymes were not enriched above the level of contaminating enzymatic activities in the amyloplast fractions. Additionally, the in vitro activities of starch phosphorylase, ATP dependent phosphofructokinase, NAD dependent glyceraldehyde-3 phosphate dehydrogenase, and glucose-6 phosphate dehydrogenase did not seem to support carbon fluxes from starch to triose phosphates as calculated from the rate of starch disappearance during carbon starvation of the cells. These results provide additional, indirect evidence for the recently emerged view that, in addition to the well known phosphate-triosephosphate translocator, another hexose phosphate and possibly also an ATP/ADP translocating system play major roles in nongreen plastids.

Molecular, Kinetic, and Immunological Properties of the 6-Phosphofructokinase from the Green Alga Selenastrum minutum

The ATP:d-fructose-6-phosphate 1-phosphotransferase (PFK) from Selenastrum minutum was purified to homogeneity. The purified plastid enzyme had a specific activity of 180 micromoles per milligram of protein per minute. It is a homomer with a subunit molecular weight of 70,000. The smallest enzymatically active form of the protein is a homotetramer of 280,000 daltons. The enzyme can, however, aggregate into different active forms, the largest of which has a molecular weight of more than 6 x 10(6). The pH optimum, regardless of aggregation state, is 7.25. The enzyme exhibits sigmoidal kinetics with respect to fructose-6-phosphate and hyperbolic kinetics with respect to ATP. Phosphate changes the sigmoidal fructose-6-phosphate saturation kinetics to hyperbolic. Phosphoenolpyruvate, 3-phosphoglycerate, 2-oxoglutarate, malate, citrate and ATP all inhibit the enzyme. The ratios of phosphoenolpyruvate and/or 3-PGA to phosphate are probably the most important factors regulating PFK activity in vivo. The enzyme cross-reacts with several antisera against both cytosolic and plastidic PFKs as well as against native potato pyrophosphate dependent phosphofructokinase suggesting that the algal PFK represents an evolutionarily primitive form.

Regulation of Carbon Partitioning to Respiration during Dark Ammonium Assimilation by the Green Alga Selenastrum minutum

The assimilation of NH(4) (+) causes a rapid increase in respiration to provided carbon skeletons for amino acid synthesis. In this study we propose a model for the regulation of carbon partitioning from starch to respiration and N assimilation in the green alga Selenastrum minutum. We provide evidence for both a cytosolic and plastidic fructose-1,6-bisphosphatase. The cytosolic form is inhibited by AMP and fructose-1,6-bisphosphate and the plastidic form is inhibited by phosphate. There is only one ATP dependent phosphofructokinase which, based on immunological cross reactivity, has been identified as being localized in the plastid. It is inhibited by phosphoenolpyruvate and activated by phosphate. No pyrophosphate dependent phosphofructokinase was found. The initiation of dark ammonium assimilation resulted in a transient increase in ADP which releases pyruvate kinase from adenylate control. This activation of pyruvate kinase causes a rapid 80% drop in phosphoenolpyruvate and a 2.7-fold increase in pyruvate. The pyruvate kinase mediated decrease in phosphoenolpyruvate correlates with the activation of the ATP dependent phosphofructokinase increasing carbon flow through the upper half of glycolysis. This increased the concentration of triosephosphate and provided substrate for pyruvate kinase. It is suggested that this increase in triosephosphate coupled with the glutamine synthetase mediated decline in glutamate, serves to maintain pyruvate kinase activation once ADP levels recover. The initiation of NH(4) (+) assimilation causes a transient 60% increase in fructose-2,6-bisphosphate. Given the sensitivity of the cytosolic fructose-1,6-bisphosphatase to this regulator, its increase would serve to inhibit cytosolic gluconeogenesis and direct the triosephosphate exported from the plastid down glycolysis to amino acid biosynthesis.

PH Dependence of the kinetic parameters for the pyrophosphate-dependent phosphofructokinase reaction supports a proton-shuttle mechanism

The pH dependence of kinetic parameters for the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii suggests that the enzyme catalyzes its reaction via general acid-base catalysis with the use of a proton shuttle. The base is required unprotonated in both reaction directions. In the direction of fructose 6-phosphate phosphorylation the base accepts a proton from the hydroxyl at C-1 of F6P and then donates it to protonate the leaving phosphate. Whether this occurs in one or two steps cannot be deduced from the present data. The maximum velocity of the reaction is pH independent in both reaction directions while V/K profiles exhibit pKs for binding groups (including enzyme and reactant functional groups) as well as pKs for enzyme catalytic groups. These data suggest that reactants bind only when correctly protonated and only to the correctly protonated form of the enzyme. Specifically, the requirement for two enzyme epsilon-amino groups in the protonated form for reactant binding was detected as was the requirement for the ionized phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, MgPPi and HPO4(2-). The protonation state of enzyme and reactant binding groups is in agreement with data obtained previously [Cho, Y.-K., & Cook, P. F. (1988) J. Biol. Chem. 263, 5135].

Sucrose metabolic pathways in sweetgum and pecan seedlings.

Sucrose metabolism and glycolysis were studied in one- to two-year-old seedlings of sweetgum (Liquidambar styraciflua L.) and pecan (Curya illinoinensis (Wangenh.) C. Koch). The sucrose synthase pathway was identified as the dominant sucrose metabolic activity in sucrose sink tissues such as terminal buds and the root cambial zone. The sucrose synthase pathway was completely dependent on uridine diphosphate and pyrophosphate and it was activated by fructose 2,6-bisphosphate. Both acid and neutral invertases were less active than sucrose synthase in sucrose sink tissues. According to the magnitude of seasonal changes in activity, sucrose synthase, the pyrophosphate-dependent phosphofructokinase, and fructokinase were identified as adaptive enzymes, whereas neutral invertase, uridine diphosphate-glucopyrophosphorylase, phosphoglucomutase, and the nonspecific, nucleotide triphosphate-dependent phosphofructokinase were identified as maintenance enzymes. The periodically high activities of pyrophosphate-dependent phosphofructokinase indicate that pyrophosphate can serve as an energy source in trees. The observations support the hypothesis that sucrose glycolysis and gluconeogenesis in plants proceed by a network of alternative enzymes and substrates.

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.

Inactivation of pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii by pyridoxal 5'-phosphate. Determination of the pH dependence of enzyme-reactant dissociation constants from protection against inactivation.

The pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii is rapidly inactivated by low concentrations of pyridoxal 5'-phosphate (PLP). The inactivation is first order with respect to PLP and the rate increases linearly with PLP concentrations suggesting that over the concentration range used no significant E-PLP complex accumulates during inactivation. The rate of inactivation decreases at high and low pH and this is discussed in terms of the mechanism of Schiff base formation. The presence of any reactants decreases the rate of inactivation to 0 at infinite concentration. This protection against inactivation has been used to obtain the pH dependence of the dissociation constants of all enzyme-reactant binary complexes. Reduction of the PLP-inactivated enzyme with NaB[3H]4 indicates that about 7 lysines are modified in free enzyme and fructose 6-phosphate protects 2 of these from modification. The pH dependence of the enzyme-reactant dissociation constants suggests that the phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, inorganic phosphate, and Mg-pyrophosphate must be completely ionized and that lysines are present in the vicinity of the 1- and 6-phosphates of the sugar phosphate and bisphosphate probably directly coordinated to these phosphates.