Phosphofructokinase type 1

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The enzyme Phosphofructokinase Type-1 uses another ATP molecule to transfer a phosphate group to Fru6P to form fructose 1, 6-bisphosphate. PFK-1 is an allosteric enzyme showing cooperative behaviour with Fru6P and hyperbolic kinetics with ATP.

Chemical equation

Fru6P + ATP \rightleftharpoons Fru1,6BP + ADP

Rate equation

The concerted transition model of Monod, Wyman and Changeux (MWC model) is used as a rate equation for this tetrameric enzyme for considering exclusive ligand binding (F6P, activators and inhibitors) together with mixed type activation, (Fru2,6BP or AMP or Pi) [1].

v = Vm \left(\frac{\frac{[ATP]}{Km_{ATP}}}{1 + \frac{[ATP]}{Km_{ATP}} }\right ) \left ( \frac{ 1 + \frac{\beta[Fru2,6BP]}{ \alpha Ka_{Fru2,6BP} } }{ 1 + \frac{[Fru2,6BP]}{ \alpha Ka_{Fru2,6BP} } } \right ) \left( \frac{\frac{[Fru6P]\left(1+\frac{[Fru2,6BP]}{[\alpha Ka_{Fru2,6BP}]}\right)}{Km_{Fru6P}\left(1 + \frac{[Fru2,6BP]}{Ka_{Fru2,6BP}}\right)} \left[1 + \frac{[Fru6P]\left(1+\frac{[Fru2,6BP]}{\alpha Ka_{Fru2,6BP}}\right)}{Km_{Fru6P}\left(1 + \frac{[Fru2,6BP]}{Ka_{Fru2,6BP}}\right)} \right]^3} { \frac{L\left( 1 + \frac{[CIT]}{Ki_{CIT}}\right)^4\left(1 + \frac{[ATP]}{Ki_{ATP}}\right)^4}{\left(1+\frac{[Fru2,6BP]}{Ka_{Fru2,6BP}}\right)^4} + \left[1 + \frac{Fru6P\left(1+\frac{Fru2,6BP}{\alpha Ka_{Fru2,6BP}}\right)}{Km_{Fru6P}\left(1 + \frac{[Fru2,6BP]}{Ka_{Fru2,6BP}}\right)} \right]^4 } - \left( \frac{\frac{[ADP][Fru1,6BP]}{K_{ADP}K_{Fru1,6BP}K_{eq}}}{\frac{[ADP]}{K_{ADP}} + \frac{[Fru1,6BP]}{K_{Fru1,6BP}} + \frac{[ADP][Fru1,6BP]}{K_{ADP}K_{Fru1,6BP}} + 1 } \right) \right)

Parameter values

Parameter Value Units Organism Remarks
Vm_{r} 0.031 [1] mM \times min^{-1} HeLa cell line Moreno-Sánchez, Marín-Hernández, Encalada & Saavedra, unpublished results
Km_{Fru6P} 1.0 [1] mM
Km_{ATP} 0.021[1] mM
Ki_{ATP} 20[1] mM
Ki_{CIT} 6.8[1] mM
Ka_{Fru2,6BP} 8.4 \times 10^{-4}[1] mM
\alpha 0.32[1] Dimensionless
\beta 0.98[1] Dimensionless
L 4.1[1] Dimensionless
K_{ADP} 5[1] mM
K_{Fru1,6BP} 5[1] mM
K_{eq} 247[1] mM Recalculated from the ΔGº´ = - 31.4 KJ mol^{-1}

Parameters with uncertainty

  • The \alpha and \beta in the rate equation represents the factors by which the ligand affinity and catalytic capacity are modified in the presence of an allosteric activatory [2]. As Fru2,6BP is the only activator in our model we considered the \alpha and \beta value of Fru2,6BP, \alpha_{Fru2,6BP} = 0.75\pm 0.4 and \beta_{Fru2,6BP} = 1.18\pm 0.17. These two values are measured in the presence of 140 \text{mM K}^{+} medium [2].
  • The Vm value is reported as 0.078 \pm 0.043 U \times (mg protein)^{-1} [2]. HeLa cells were harvested at a concentration of 65 mg protein/ml. Converting Vm to mM/min becomes 5.07 \pm 2.795 again in the presence of 140 \text{mM K}^{+}.
  • Km_{ADP} = 1.4 for Thermotoga maritima is being reported in Hansen, T., M. Musfeldt et. al. [3] and Km_{Fru1,6BP} = 16.7 for Desulfurococcus amylolyticus is reported in Hansen T, Schönheit P. et. al.[4]. The mean and std. dev. is calculated as 0.945 \pm 0.454
  • Similarly for Km_{Fru1,6BP} = 7.6 in Thermotoga maritima is being reported in Hansen, T., M. Musfeldt et. al. [3] and Km_{ADP} = 0.49 in Desulfurococcus amylolyticus is reported in Hansen T, Schönheit P. et. al.[4]. The mean and std. dev. is calculated as 12.15 \pm 4.54
  • Four Keq values have been reported in the SilicoTrypWiki (Wikipedia for insilico modelling of Trypanosome) for Phosphofructokinase: 308.4, 254, 1035, 800[5]. As the K_{eq} value does not depend on the organism, the mean and the standard deviation can be calculated from these 4 values collected for Trypanosome.[6]. The mean and std. dev. of this value is 599.35 \pm 329.38


Parameter Value Units Organism
Vm_{f} 5.07 \pm 2.795 mM \times min^{-1} HeLa Cell line
Km_{Fru6P} 1.1 \pm 0.4 mM HeLa Cell line
Km_{ATP} 0.0292 \pm 0.0015[2] mM HeLa Cell line
Ki_{ATP} 2.0 \pm 0.1[7] mM Human muscle
Ki_{CIT} 6.7 \pm 3.8 mM HeLa Cell line
Ka_{Fru2,6BP} 0.00099 \pm 0.00014 mM HeLa Cell line
\alpha 0.75\pm 0.4 Dimensionless HeLa Cell line
\beta 1.18\pm 0.17 Dimensionless HeLa Cell line
L 6.6 \pm 5 Dimensionless HeLa Cell line
K_{ADP} 0.945 \pm 0.454 mM Thermotoga maritima & Desulfurococcus amylolyticus
K_{Fru1,6BP} 12.15 \pm 4.54 mM Thermotoga maritima & Desulfurococcus amylolyticus
K_{eq} 599.35 \pm 329.38 Trypanosome

Equilibrium constant

Equilibrium constant Conditions Source
308.4 pH=7, T=25°C Lehninger, (2008)[8] p 553:

\Delta G' = -14.2\ kJ.mol^{-1}, Keq = exp(-\frac{\Delta G'}{RT}) = exp(\frac{14200}{8.31*298.15}) \approx 308.4

254 pH=7, T=25°C Lehninger, (1975)[9] p 396.
1035 pH=7, T=25°C Voet et al.[10] from Newshole et al. (1973) [11]p 97:

\Delta G' = -17.2\ kJ.mol^{-1}, Keq = exp(-\frac{\Delta G'}{RT}) = exp(\frac{17200}{8.31*298.15}) \approx 1035

800 T=298.15 K; pH=7.0; Method: calorimetry; Buffer: Tris (0.1 mol dm-3) + HCl. NIST database "Thermodynamics of Enzyme-Catalyzed Reactions" entry [75BOH/SCH_551] from Bvhme et al. (1975)[12]
  • The Average from these values are 599.35 \pm 380.33

References

  1. ↑ 1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 1.12 Marín-Hernández A, Gallardo-Pérez JC, Rodríguez-Enríquez S et al (2011). Modeling cancer glycolysis. Biochim Biophys Acta, 1807:755–767 (doi)
  2. ↑ 2.0 2.1 2.2 2.3 R. Moreno-Sánchez, A. Marín-Hernández, J.C. Gallardo-Pérez, H. Quezada, R. Encalada, S. Rodríguez-Enríquez et al. (2012), Phosphofructokinase type 1 kinetics, isoform expression, and gene polymorphisms in cancer cells, J Cell Biochem, 113, pp. 1692–1703
  3. ↑ 3.0 3.1 Hansen, T., M. Musfeldt, and P. Schonheit (2002), ATP-dependent 6-phosphofructokinase from the hyperthermophilic bacterium Thermotoga maritima: characterization of an extremely thermophilic, allosterically regulated enzyme. Arch. Microbiol. 177:401-409
  4. ↑ 4.0 4.1 Hansen T, Schönheit P. (2003),Purification and Characterization of the MQH2:NO Oxidoreductase from the Hyperthermophilic Archaeon Pyrobaculum aerophilum, J Biol Chem, 278 (38), 35861-35868
  5. ↑ [Silicotryp]
  6. ↑ F. Achcar, E.J. Kerkhoven, B.M. Bakker, M.P. Barrett, R. Breitling (2012), Dynamic modelling under uncertainty: the case of Trypanosoma brucei energy metabolism, PLoS Comput Biol, 8, p. e1002352
  7. ↑ Brueser, A.; Kirchberger, J.; Kloos, M.; Straeter, N.; Schoeneberg, T. (2012), Functional linkage of adenine nucleotide binding sites in mammalian muscle 6-phosphofructokinase, J. Biol. Chem. 287, 17546-17553 (2012)
  8. ↑ David L. Nelson, Michael M. Cox (2008), Lehninger Principles of Biochemistry (5th edn), W. H. Freeman and Company
  9. ↑ Lehninger, A.L. (1975) Biochemistry (2nd edn), Worth
  10. ↑ Voet, D., Voet., J.G. and Pratt, C. W. (1999) Fundamentals of biochemistry, Wiley
  11. ↑ Newshole, E.A. and Stuart, C. (1973) Regulation in Metabolism, Wiley
  12. ↑ Bvhme, H.-J., Schellenberger, W. and Hofmann, E. (1975) Acta Biol. Med. Germ. 34(1):15-20. (pmid: 241184)
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