Biochemical and transcript level differences between the three human phosphofructokinases show optimisation of each isoform for specific metabolic niches

6-Phosphofructokinase-1-kinase (PFK) tetramers catalyse the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate (F16BP). Vertebrates have three PFK isoforms (PFK-M, PFK-L, and PFK-P). This study is the first to compare the kinetics, structures, and transcript levels of recombinant human PFK isoforms. Under the conditions tested PFK-M has the highest affinities for F6P and ATP (K0.5ATP 152 µM; K0.5F6P 147 µM), PFK-P the lowest affinities (K0.5ATP 276 µM; K0.5F6P 1333 µM), and PFK-L demonstrates a mixed picture of high ATP affinity and low F6P affinity (K0.5ATP 160 µM; K0.5F6P 1360 µM). PFK-M is more resistant to ATP inhibition compared with PFK-L and PFK-P (respectively, 23%, 31%, 50% decreases in specificity constants). GTP is an alternate phospho donor. Interface 2, which regulates the inactive dimer to active tetramer equilibrium, differs between isoforms, resulting in varying tetrameric stability. Under the conditions tested PFK-M is less sensitive to fructose 2,6-bisphosphate (F26BP) allosteric modulation than PFK-L or PFK-P (allosteric constants [K0.5ATP+F26BP/K0.5ATP] 1.10, 0.92, 0.54, respectively). Structural analysis of two allosteric sites reveals one may be specialised for AMP/ADP and the other for smaller/flexible regulators (citrate or phosphoenolpyruvate). Correlations between PFK-L and PFK-P transcript levels indicate that simultaneous expression may expand metabolic capacity for F16BP production whilst preserving regulatory capabilities. Analysis of cancer samples reveals intriguing parallels between PFK-P and PKM2 (pyruvate kinase M2), and simultaneous increases in PFK-P and PFKFB3 (responsible for F26BP production) transcript levels, suggesting prioritisation of metabolic flexibility in cancers. Our results describe the kinetic and transcript level differences between the three PFK isoforms, explaining how each isoform may be optimised for distinct roles.


SUPPLEMENTARY TABLE 1: Summary of natural and artificial modulators of PFK activity in the published literature
PFK is allosterically regulated by many compounds ("+" indicates activator; "-" indicates inhibitor), with selected references.

Effector
Effect IC50 or AC50 Reference

SUPPLEMENTARY TABLE 2: Comparison of physiological and tested effector concentrations
The concentrations selected was based on two factors: the normal physiological concentrations of the effector, and the chance of seeing an effect on activity at a given concentration. There were often discrepancies between these two criteria; a compromise was made in these cases, with preference given to concentrations which were likely to demonstrate effects over and above the signal-to-noise threshold for this assay. For some effectors there is significant uncertainty -and sometimes even controversy -about physiological concentrations e.g. citrate.

SUPPLEMENTARY TABLE 3: Comparison of Interface 1 (D-C) amino acid sequence between PFK-M, PFK-L and PFK-P
Interface 1 (D-C interface) amino acid sequence comparison between PFK-M, PFK-L, and PFK-P, incorporating all residues contributing more than 4 Å 2 buried surface area (BSA) to the interface based on the X-ray structure of PFK-P (PDB code 4xz2). Column 1 has the name of the residue in the X-ray structure. Yellow highlights non-conserved amino acids across the three isoforms. Blue indicates interface residues that also interact with FBP. HB indicates the residue forms a hydrogen bond across the interface.
PFK-M PFK-L PFK-P BSA (Å 2 ) (D-C)  G  G  G  13  D:THR 91  T  T  T  21 HB  D:ILE 92  V  I  I  20  D:GLY 94  G  G  G  15  D:SER 95  S  S  S  31 HB  D:ALA 96  A  A  A  6  D:VAL 197  V  I  V  21  D:ALA 200  A  A  A  55 HB  D:ILE 201  I  I  I  26  D:THR 203  T  T  T  42 HB  D:THR 204  T  T  T  The C-D interface comprises 75 residues from chain D and 71 from chain C. The BSA D = 2164 Å 2 and for chain C = 2196 Å 2 with 40 h-bonds including 9 salt bridge interactions. The A-B interface comprises 73 residues from chain B and 67 from chain A. The BSA B = 2113 Å 2 and for chain A = 2158 Å 2 with 34 h-bonds including 7 salt bridge interactions. There are 66 residues that contribute to this interface with a BSA of at least 4 Å 2 : PFK-P differs from PFK-M by 6/66 residues; PFK-P differs from PFK-L by 7/66 residues; PFK-L differs from PFK-M by 8/66 residues. Between P and M most of the changes (I90V, V91I, D448E) are conservative. A677S, S571A, I476K are the only three difference between isoforms P and M that are not conservative.

SUPPLEMENTARY TABLE 4: Interface 2 (D-A and B-C) amino acid sequence comparison
Interface 2 amino acid sequence comparison. The output from PISA (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html) using the coordinates from PFK-P (PDB code 4xz2) was used to determine BSA values for each of the 19 residues associated with the D-A and B-C interfaces. All residues contributing a BSA value greater than 0.5 Å 2 were included in the analysis. Yellow highlighting indicates non-conserved amino acid. HB indicates the residues forms a hydrogen bond across the interface.

SUPPLEMENTARY TABLE 6: Comparison of F26BP binding sites between PFK-M, PFK-L, and PFK-P.
Output from PISA (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html) using the coordinates from PFK-P (PDB code 4xz2) was used to determine Buried Surface Area (BSA) values for each of the residue close to F26BP (results are very similar for all chains in the tetramer and only one set is shown). All residues contributing a BSA value greater than 0.5 Å 2 were included in the analysis. 5/19 residues are involved in both binding F26BP and Interface 1 (coloured blue).
No residues that bind F26BP are involved in Interface 2. B indicates Hydrogen Bond.

SUPPLEMENTARY TABLE 7
Supplementary Table 4 shows the residues identified within 4Å from ADP in the X-ray structure of EcPFK (PDB code 1PFK). Structural and sequence comparisons were used to identify the corresponding residues in the two pockets (labelled Site A and Site B in Figure 1C) in PFK using the numbering in PFK-P (PDB 4xz2). Shaded boxes indicate which residues at the ADP binding site in EcPFK are type-conserved among the isoforms. Residues shown in red are amino acids identified by PISA (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html) to be involved in binding phosphate. T628  T  T  T  T629  T  D  T  I630  V  I  I  H215 L266