Evolution of allosteric citrate binding sites on 6-phosphofructo-1-kinase.

As an important part of metabolism, metabolic flux through the glycolytic pathway is tightly regulated. The most complex control is exerted on 6-phosphofructo-1-kinase (PFK1) level; this control overrules the regulatory role of other allosteric enzymes. Among other effectors, citrate has been reported to play a vital role in the suppression of this enzyme's activity. In eukaryotes, amino acid residues forming the allosteric binding site for citrate are found both on the N- and the C-terminal region of the enzyme. These site has evolved from the phosphoenolpyruvate/ADP binding site of bacterial PFK1 due to the processes of duplication and tandem fusion of prokaryotic ancestor gene followed by the divergence of the catalytic and effector binding sites. Stricter inhibition of the PFK1 enzyme was needed during the evolution of multi-cellular organisms, and the most stringent control of PFK1 by citrate occurs in vertebrates. By substituting a single amino acid (K557R or K617A) as a component of the allosteric binding site in the C-terminal region of human muscle type PFK-M with a residue found in the corresponding site of a fungal enzyme, the inhibitory effect of citrate was attenuated. Moreover, the proteins carrying these single mutations enabled growth of E. coli transformants encoding mutated human PFK-M in a glucose-containing medium that did not support the growth of E. coli transformed with native human PFK-M. Substitution of another residue at the citrate-binding site (D591V) of human PFK-M resulted in the complete loss of activity. Detailed analyses revealed that the mutated PFK-M subunits formed dimers but were unable to associate into the active tetrameric holoenzyme. These results suggest that stricter control over glycolytic flux developed in metazoans, whose somatic cells are largely characterized by slow proliferation

Posttranslational Modification of 6-Phosphofructo-1-Kinase in Aspergillus niger

Two different enzymes exhibiting 6-phosphofructo-1-kinase (PFK1) activity were isolated from the mycelium of Aspergillus niger: the native enzyme with a molecular mass of 85 kDa, which corresponded to the calculated molecular mass of the deduced amino acid sequence of the A. niger pfkA gene, and a shorter protein of approximately 49 kDa. A fragment of identical size also was obtained in vitro by the proteolytic digestion of the partially purified native PFK1 with proteinase K. When PFK1 activity was measured during the proteolytic degradation of the native protein, it was found to be lost after 1 h of incubation, but it was reestablished after induction of phosphorylation by adding the catalytic subunit of cyclic AMP-dependent protein kinase to the system. By determining kinetic parameters, different ratios of activities measured at ATP concentrations of 0.1 and 1 mM were detected with fragmented PFK1, as with the native enzyme. Fructose-2,6-biphosphate significantly increased the V(max) of the fragmented protein, while it had virtually no effect on the native protein. The native enzyme could be purified only from the early stages of growth on a minimal medium, while the 49-kDa fragment appeared later and was activated at the time of a sudden change in the growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sequential purifications of PFK1 enzymes by affinity chromatography during the early stages of the fungal development suggested spontaneous posttranslational modification of the native PFK1 in A. niger cells, while from the kinetic parameters determined for both isolated forms it could be concluded that the fragmented enzyme might be more efficient under physiological conditions

Posttranslational modification of 6-phosphofructo-1-kinase as an important feature of cancer metabolism.

BACKGROUND: Human cancers consume larger amounts of glucose compared to normal tissues with most being converted and excreted as lactate despite abundant oxygen availability (Warburg effect). The underlying higher rate of glycolysis is therefore at the root of tumor formation and growth. Normal control of glycolytic allosteric enzymes appears impaired in tumors; however, the phenomenon has not been fully resolved. METHODOLOGY/PRINCIPAL FINDINGS: In the present paper, we show evidence that the native 85-kDa 6-phosphofructo-1-kinase (PFK1), a key regulatory enzyme of glycolysis that is normally under the control of feedback inhibition, undergoes posttranslational modification. After proteolytic cleavage of the C-terminal portion of the enzyme, an active, shorter 47-kDa fragment was formed that was insensitive to citrate and ATP inhibition. In tumorigenic cell lines, only the short fragments but not the native 85-kDa PFK1 were detected by immunoblotting. Similar fragments were detected also in a tumor tissue that developed in mice after the subcutaneous infection with tumorigenic B16-F10 cells. Based on limited proteolytic digestion of the rabbit muscle PFK-M, an active citrate inhibition-resistant shorter form was obtained, indicating that a single posttranslational modification step was possible. The exact molecular masses of the active shorter PFK1 fragments were determined by inserting the truncated genes constructed from human muscle PFK1 cDNA into a pfk null E. coli strain. Two E. coli transformants encoding for the modified PFK1s of 45,551 Da and 47,835 Da grew in glucose medium. The insertion of modified truncated human pfkM genes also stimulated glucose consumption and lactate excretion in stable transfectants of non-tumorigenic human HEK cell, suggesting the important role of shorter PFK1 fragments in enhancing glycolytic flux. CONCLUSIONS/SIGNIFICANCE: Posttranslational modification of PFK1 enzyme might be the pivotal factor of deregulated glycolytic flux in tumors that in combination with altered signaling mechanisms essentially supports fast proliferation of cancer cells

Growth of <i>E. coli</i> RL 257 transformants encoding human native and mutant PFK-M forms.

<p>Growth was recorded in a minimal medium with glucose as the sole carbon source at 30°C. Data are presented as means ± standard deviation.</p

Kinetic measurements of recombinant human PFK-M and mutant forms of PFK-M.

<p><b>A. Fructose-6-phosphate (F6P) saturation curves for the native human and mutant forms of PFK-M.</b> Measurements were carried out at pH 7.8 in a buffer containing 5 mM Mg²⁺ and 0.5 mM ATP. Activities are expressed as a ratio of enzyme activity (v) at a specific substrate concentration to the activity detected at saturating F6P concentration (V_max). Data are presented as means ± standard deviation. <b>B. Citrate inhibition of the native human PFK-M measured at different fructose-6-phosphate (F6P) concentrations.</b> The assay was performed at pH 7.8 in a buffer containing 5 mM Mg²⁺ and 0.5 mM ATP. Data are presented as means ± standard deviation. <b>C. Citrate inhibition of the native and mutant forms of human PFK-M.</b> All measurements were conducted at 0.4 mM F6P. The assay was carried out at pH 7.8 in the presence of 5 mM Mg²⁺ and 0.5 mM ATP. Activities are expressed as a ratio of activity detected in the presence of citrate to activity measured without citrate in the system. Data are presented as means ± standard deviation. <b>D. IC₅₀ values for citrate inhibition of the native and mutant forms of human PFK-M measured at increasing concentrations of F6P.</b> The assay was carried out at pH 7.8 in the presence of 5 mM Mg²⁺ and 0.5 mM ATP. Data were obtained by determining the citrate concentration that caused inhibition of the wild type and mutated forms of PFK-M by 50%. Mean values of at least three independent measurements are reported.</p

Western blot analyses of <i>E. coli</i> transformants.

<p><b>A</b>. Western blots of inactive mutant forms of human PFK-M synthesized in <i>E. coli</i> transformants grown in LB medium. <b>B</b>. Western blots of specific fractions collected after gel filtration of homogenate prepared from <i>E. coli</i> transformants encoding wild type human PFK-M (above) and its inactive mutant D591V (below). Molecular weights of the proteins in individual fractions as determined using the calibration curve are shown in the bottom line.</p

Additional file 6: Table S1. of Effect of the cancer specific shorter form of human 6-phosphofructo-1-kinase on the metabolism of the yeast Saccharomyces cerevisiae

Pyridine nucleotide levels detected in the wild type, nPFKM and sfPFKM strain. The strains were pre-grown on glycerol/ethanol medium until the value of 1(OD600) was reached. As a control pfk null HD114-8D strain with empty plasmid has been taken. Pyridine nucleotide levels were measured 3 h after the transfer of the cells to 1% maltose or maltose with 10 mM ethanol medium. In a table NADH, NAD+, NADPH and NADP+ levels from three independent measurements are shown. Data are presented as means ± standard deviation. (DOCX 39 kb

Changes in primary metabolism leading to citric acid overflow in aspergillus niger

For citric acid-accumulating Aspergillus niger cells, the enhancement of anaplerotic reactions replenishing tricarboxylic acid cycle intermediates predisposes the cells to form the product. However, there is no increased citrate level in germinating spores and a complex sequence of developmental events is needed to change the metabolism in a way that leads to an increased level of tricarboxylic acid cycle intermediates in mycelia. A review of physiological events that cause such intracellular conditions, with the special emphasis on the discussion of hexose transport into the cells and regulation of primary metabolism, predominantly of glycolytic flux during the process, is presented