Figure 1.
Multiple sequence alignment of amino acid residues at the N and C-termini of PFK1 proteins that form allosteric citrate binding sites.
A. Amino acid residues (grey background) of citrate binding sites in the N-terminal region of PFK1 isoforms from the following species: ECO24, Escherichia coli; ASPNG Aspergillus niger; CAEEL Caenorhabditis elegans; DROME, Drosophila melanogaster; HUMAN, Homo sapiens. B. Amino acid residues (grey background) of citrate binding sites in the C-terminal region of PFK1 isoforms from the following fungi: ASPNG, Aspergillus niger; ASPFU, Aspergillus flavus; Yeast, Saccharomyces cerevisiae; PICPA, Pichia pastoris; SCHPO, Schizosaccharomyces pombe. C. Amino acid residues (grey background) of citrate binding sites in the C-terminal region of PFK1 isoforms from the following invertebrates: SCHMA, Schistosoma mansoni; HAECO, Haemonchus contortus; CAEEL, Caenorhabditis elegans; DROME, Drosophila melanogaster. D. Amino acid residues (grey background) of citrate binding sites in the C-terminal region of PFK1 isoforms from the following species: XENLA, Xenopus laevis; CHICK, Gallus gallus; PIG Sus scrofa; CANFA, Canis familiaris; HUMAN, (Homo sapiens). The components of allosteric citrate site were originally identified in the mouse PFK-C enzyme (Accession number Q9WUA3) [6], which has 69,58% of identical; 13,18% strongly similar and 6,46% weakly similar residues to the human PFK-M (Accession number P08237); however, there is a minor shift in numbering of amino acid residues between the enzymes. The mouse PFK-C enzyme has an extension of 8 amino acid residues at the N-terminal end of the enzyme and an insertion at position 349. Therefore, the corresponding ligand binding sites in the N-terminal part of human PFK-M differ by 8 amino acid residues and in the C-terminal region by 9 residues with respect to the mouse PFK-C. The numbering system for amino acids used in the entire paper, therefore reflects the positions on the human PFK-M. The alignments were generated using CLUSTAL W [34].
Figure 2.
Western blot analyses of E. coli transformants.
A. Western blots of inactive mutant forms of human PFK-M synthesized in E. coli transformants grown in LB medium. B. Western blots of specific fractions collected after gel filtration of homogenate prepared from E. coli 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.
Figure 3.
Kinetic measurements of recombinant human PFK-M and mutant forms of PFK-M.
A. Fructose-6-phosphate (F6P) saturation curves for the native human and mutant forms of PFK-M. 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 (Vmax). Data are presented as means ± standard deviation. B. Citrate inhibition of the native human PFK-M measured at different fructose-6-phosphate (F6P) concentrations. 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. C. Citrate inhibition of the native and mutant forms of human PFK-M. 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. D. IC50 values for citrate inhibition of the native and mutant forms of human PFK-M measured at increasing concentrations of F6P. 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.
Figure 4.
Growth of E. coli RL 257 transformants encoding human native and mutant PFK-M forms.
Growth was recorded in a minimal medium with glucose as the sole carbon source at 30°C. Data are presented as means ± standard deviation.