As presented in Figure 3C , the KR and KA amino acid substitutions markedly decreased the inhibitory effect of citrate. The KR mutant was shown to be more sensitive to citrate inhibition than the KA mutant; they exhibited K i values of 0.
As for the native enzyme, a decreased inhibitory role of citrate was observed at increasing substrate concentrations for both mutants Fig. All transformants were also tested for growth on glucose. It is worth noting that the parental E. Previously, no PFK-M activity in these transformants grown on complex medium was detected. Surprisingly, no growth was observed with the transformant encoding the native human PFK-M, whereas a modest growth reaching an OD value of 0.
The only transformant that showed a significant growth rate with an approximate doubling time of 3 h was the one encoding the KA mutant Fig. Since the DV mutation might prevent the consolidation of monomers into an active tetrameric structure, the quaternary structures of the enzymes were assessed by running cell free homogenate through a size-exclusion column and detecting PFK-M monomers by western blot in different eluted fractions.
For the homogenate of the native PFK-M transformant, the strongest western blot signals were recorded in fractions In contrast, the strongest western blot bands in the homogenate of the DV transformant were detected in fractions These data suggested that the substitution of the aspartic acid D residue at position with valine V enabled binding of two monomers into a dimer but prevented the formation of an active tetrameric PFK-M structure.
It should be reminded that the mutations on human PFK-M enzyme were carried out by replacing corresponding residues from the fungal A.
Since the A. Although an important feature of catabolism is the generation of chemically conserved energy in the form of ATP, numerous intermediates of primary metabolism act as precursors for the formation of cellular building blocks that enable cell growth and division. Whereas in the microbial world the overall growth rate is normally controlled by the availability of nutrients from the environment, in metazoans nutrient levels in the vascular system do not vary much.
In order to control the proliferation of somatic animal cells, more strict regulation of primary metabolism must have been developed during evolution. An important point of control over the metabolic flux of primary metabolism seems to be the enzyme PFK1. Analyses of the allosteric citrate binding sites and kinetic characteristics of eukaryotic PFK1 enzymes revealed that stronger inhibition by citrate has been selected for during the development of metazoans.
The most powerful regulatory effect of citrate as a feedback inhibitor was recorded in Vertebrata , whose PFK1 isoforms have conserved amino acid residues forming the citrate allosteric sites at both the N and C-terminal regions.
Amino acid motifs responsible for citrate binding at the C-terminus are characterized by two basic residues and one acidic residue that apparently enable strong allosteric effects of the ligand on the protein. In contrast, in fungi, where less stringent control over glycolytic flux is required, only one component of allosteric site in the C-terminal part is of this basic-ionizable type while the other two are predominantly non-ionizable some are even hydrophobic.
Similarly, in lower animals invertebrates basic residues such as lysine and arginine are found at one position, while the next two components are characterized predominantly by the presence of ionizable-acidic residues. Interestingly, a single substitution of valine V for aspartic acid D at position resulted in loss of activity. As revealed by gel filtration, monomers containing this mutation were unable to form tetrameric structures and remained dimeric.
Although amino acid residues enabling association of monomers into a dimer have been suggested [21] , further studies of the residues at position and in the surrounding area might reveal grouping of dimers into active tetrameric structures.
The mechanism of citrate interaction with individual components of citrate allosteric site on PFK1 enzymes has not been studied yet on a submolecular level. However, the importance of specific amino acid residues at allosteric citrate interaction site in mammalian PFK-M was demonstrated by replacing a basic residue at position with a hydrophobic one.
This single substitution diminished the enzyme's sensitivity to citrate. Moreover, it enabled the recombinant enzyme to participate actively in bacterial metabolism, which was reflected by the growth of transformants in a glucose-containing medium.
This might be due to the high sensitivity of mammalian PFK-M enzymes to citrate inhibition. The estimated intracellular citrate concentration during the exponential growth phase on glucose was reported to be approximately 0.
The possible role of intracellular citrate concentration on growth of transformants carrying human PFK-M enzymes was further shown by both PFK-M mutants. A correlation between PFK1 activity and cell proliferation rate has been observed also in cancer cells. By inhibiting Fructose-2,6-bisphosphate formation, a potent activator of eukaryotic PFK1 enzymes, markedly attenuated proliferation of several tumorigenic cell lines was observed [24]. Data regarding accurate cytosolic citrate concentrations in eukaryotes would be very informative.
Relative high concentrations were reported for fungal cells; in Aspergillus niger the citrate concentration was between 2 mM and 30 mM [25] and in Saccharomyces cerevisiae it was between 2.
However, these values do not take into consideration the compartmentalization of citrate into mitochondria and cytosol. It is worth noting that citrate is formed in the mitochondrial matrix, while PFK1 is strictly cytosolic. However, a portion of the mitochondrial citrate is regularly transferred into the cytosol and used for lipid acid synthesis after being converted to acetyl-CoA by ATP-citrate lyase [29].
This effect appears to be of moderate physiological relevance, since intracellular concentrations of F6P are relatively low.
The intracellular concentration of F6P is reported to be 0. Feedback inhibition of PFK1 and concomitant regulation of metabolic flux through the glycolytic pathway obviously fails in mammalian tumor cells. Dysregulation of glycolysis, also known as the Warburg effect, is characteristic of cancer cells [31].
There have been several reports of PFK1 isoforms with atypical kinetic characteristics in tumors. A PFK1 enzyme with similar kinetic characteristics was observed in the fast growing rodent hepatoma cell line ASD; this form of PFK1 showed complete insensitivity toward its allosteric inhibitors citrate and ATP in the presence of physiological concentrations of F2,6P.
However, the nature of PFK1 isoforms exhibiting changes in enzyme kinetics has not been studied in detail. In conclusion, these results indicate that amino acid residues of allosteric binding site for citrate at the C-terminus of PFK1 enzymes determine the strength of inhibition by citrate.
By substituting a specific amino acid residue, the level of inhibition of the enzyme can be modulated. Analyses of the variations in allosteric binding sites among different eukaryotic organisms revealed that stronger inhibition of PFK1 enzymes by citrate has developed during evolution, enabling better control over glucose consumption in the slowly growing somatic cells of higher metazoans.
On the other hand, no downregulation of PFK1 isoforms by feedback inhibition has been described in cancer tissues, which are characterized by rapid cell growth and proliferation. We thank Dr. Gregory Reinhart for providing the double pfk deficient strain of E.
Conceived and designed the experiments: ML AU. Performed the experiments: AU. Analyzed the data: AU ML. Wrote the paper: ML AU.
Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract As an important part of metabolism, metabolic flux through the glycolytic pathway is tightly regulated. Expression and purification of recombinant enzymes Plasmids were initially propagated in the E. Testing transformants for growth in glucose-containing medium Transformed E. Immunoblotting Aliquots of supernatants prepared from cell-free homogenates of E. Results Citrate binding sites in various organisms Analyses of citrate binding sites on the PFK1 enzymes of various eukaryotic organisms revealed that identical amino acid residues were found at the N-terminus in all examined species Fig.
Download: PPT. Figure 1. Multiple sequence alignment of amino acid residues at the N and C-termini of PFK1 proteins that form allosteric citrate binding sites. Figure 2. Western blot analyses of E. Once PFK converts F6P to F1,6P, the reaction will not be easily reversed because of the high amount of energy that must be overcome to go backward.
This energy barrier makes sense seeing as pyruvate kinase catalyzes the final reaction 10 and hexokinase 1 is not involved in glycolysis at all when the process is begun from glycogen. While ATP binds at the active site equally well in both R and T states, it preferentially binds the allosteric site of the T state [10] This preferential binding causes a shift from equilibrium of the two states, to a greater amount of T state [11] , which decreases the affinity for F6P.
Allosteric activator ADP also binds to the allosteric site to increase the ratio of R state phosphofructokinase. As can be seen from the graph below, the plots for the activity of PFK are sigmoidal. This further demonstrates the cooperative nature of the enzyme.
The system of regulation matches well with the function of PFK. The opposite holds true as well, because high ATP concentration inhibits protein activity.
And yet, this explanation cannot completely account for the regulation of PFK, because the levels of ATP do not vary greatly enough between active and resting muscles.
Another means of allosteric regulation must be found. This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. KineMage currently not supported The first view, 1: PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pink tint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu red and Arg cyan , which are part of the F6P binding site in the T and R states, srespectively see below.
In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu and Arg in both subunits are red and cyan as before only the Ca and Cb atoms of the Arg side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed.
The T state enzyme binds the inhibitor 2-phosphoglycolate gold; "PGC" , a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate PEP.
Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three residues 2 arg and 1 Lys; not shown.
In the high-activity R state, the positively charged side chain of Arg forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P white dashed lines , an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.
F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P gray; viewed by clicking on "F6P site". Phosphofructokinase-1 PFK-1 : glycolysis, irreversible. The concentration of AMP increases when energy is low. Excess phosphate also signals low energy via increase in ATP use. Fructose 1,6-bisphosphatase : gluconeogenesis, irreversible.
The two enzymes are reciprocally regulated or ATP would be lost without energy conservation. Prevents futile cycling like using a stationary bike. Fructose-2,6-bisphosphate :.
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