›

Phosphofructokinase

Notes

Phosphofructokinase

Sections

PHOSPHOFRUCTOKINASE-1 (PFK-1)

  • Catalyzes rate-limiting step in glycolysis
  • Catalyzes irreversible phosphorylation of F6P to F1,6P.
  • Allosterically regulated (hormonally regulated in liver)

PFK-1 INHIBITION

  • Citrate, intermediate of citric acid cycle
  • ATP, final product of glycolysis and cellular respiration
  • H+, symptom of lactic acid buildup in exercising muscle

PFK-1 ACTIVATION

  • AMP, marker of ATP depletion.
  • Fructose-2,6-bisphosphate (special case)

PFK-2/FBP-2 (BIFUNCTIONAL ENZYME)

  • PFK-2: F6P --> F2,6P (activates PFK-1)
  • FBP-2 (fructose-2,6-bisphosphatase): F2,6P --> F6P (deactivates PFK-1)
  • PFK-2/FBP-2 regulated differently in different tissues

Skeletal muscle

  • Feed-forward activation
  • Substrate-level regulation: F6P
  • When F6P HIGH: FBP-2 inactive and PFK-2 active (activate PFK-1)
  • When F6P LOW: FBP-2 active and PFK-2 inactive (inhibits PFK-1 activity)

Liver

  • Hormonal regulation: PFK-2 has phosphorylation site (unlike muscle)
  • PFK-2 is INACTIVE when phosphorylated
  • Glucagon activates protein kinase A (PKA), which phosphorylates PFK-2
  • Insulin activates phosphoprotein phosphatase (PPP), which dephosphorylates PFK-2

FED STATE

  • HIGH blood glucose
  • INSULIN secreted --> PPP activated --> PFK-2 dephosphorylated (ACTIVE)
  • HIGH F2,6P activates PFK-1
  • Promotes glycolysis

FAST

  • LOW blood glucose
  • GLUCAGON secreted --> PKA activated --> PFK-2 phosphorylated (INACTIVE)
  • LOW F2,6P deactivates PFK-1
  • NO glycolysis

Liver responds to entire body's glucose needs

  • Site of gluconeogenesis: glucose synthesized from non-carbohydrate precursors and released into the bloodstream

Full-Length Text

  • Here, we will learn how phosphofructokinase-1, the rate-limiting enzyme in glycolysis, is regulated.
  • To begin, start a table to learn some key features of phosphofructokinase-1 (PFK-1).
  • Denote that it catalyzes the rate-limiting step in glycolysis; it is therefore the most important point of control.
    • It catalyzes the irreversible phosphorylation of fructose-6-phosphate to form fructose 1,6 bisphosphate.
    • It is allosterically regulated.
  • Specifically, denote a special case in its allosteric regulation: fructose 2,6 bisphosphate, which we will learn about in detail.

Let's start by illustrating the reaction that PFK-1 catalyzes.

  • First, show that it phosphorylates fructose-6-phosphate to form fructose-1,6-bisphosphate via an irreversible reaction (use a single-headed arrow).
  • Indicate that one molecule of ATP is consumed in this reaction.
    • PFK-1 marks the last reaction in the energy investment phase of glycolysis.

Now, let's include the molecules that allosterically regulate PFK.

  • Show that PFK-1 is inhibited by:
    • Citrate, which is an intermediate of the citric acid cycle.
    • ATP, which is a final product of glycolysis and cellular respiration.
    • H+, a symptom of lactic acid buildup, which occurs in exercising muscle.
  • This feedback inhibition keeps glycolysis in sync with the next stages of glucose metabolism, which are the citric acid cycle and oxidative phosphorylation or anaerobic lactic acid fermentation in exercising muscle.
  • Now, show that PFK-1 is activated by:
    • AMP, which is a marker of ATP depletion.
    • Fructose-2,6-bisphosphate, which is different than fructose-1,6-bisphosphate. Fructose-2,6-bisphosphate is a special case of PFK-1 regulation.

To show its mechanism, let's see how fructose 2,6 bisphosphate is made.

  • Draw an enzyme with two separate catalytic sites.
  • Label one PFK-2 phosphofructokinase-2; it is a kinase and adds a phosphate group to its substrate.
  • Label the other FBP-2 for fructose-2,6-bisphosphatase; it is a phosphatase and removes a phosphate group from its substrate.
  • Indicate that the entire enzyme, including both catalytic sites is named PFK-2/FBP-2.
  • Now, draw the substrate fructose-6-phosphate below PFK-2/FBP-2.
    • Recall, that it is also the substrate of PFK-1, thus fructose 6-phosphate can enter either reaction.
  • Next, draw fructose 2,6 bisphosphate above PFK-2/FBP-2.
  • Use an arrow to show that the kinase portion of the enzyme catalyzes its formation.
  • Show that one ATP is consumed in this reaction.
  • Use another arrow to show that FBP-2 (the phosphatase portion of the enzyme) catalyzes the reverse reaction.

Now, we've learned how PFK-2/FBP-2 works, let's learn how fructose 2,6 bisphosphate concentrations are regulated in two different organs: the skeletal muscle and the liver. Let's start with skeletal muscle, because this mechanism is less complex.

  • Draw skeletal muscle.
  • Here, indicate that PFK-2/FBP-2 is regulated by the concentration of substrate: fructose-6-phosphate.
  • Show that when fructose-6-phosphate is high: FBP-2 is inactive and PFK-2 is active.
  • Show that when fructose-6-phosphate is low: FBP-2 is active and PFK-2 is inactive.
  • Label this mechanism feed-forward activation: as fructose 6-phosphate accumulates, it drives its own metabolism.

Now, let's learn the regulatory mechanism in the liver.

  • Draw a representative liver.
  • Write that here, the reaction is under hormonal control. We will learn why shortly.
  • Draw the enzyme and show that the PFK-2 catalytic site is active.
  • Show that the FBP-2 site is inactive.
  • Now, redraw the enzyme.
  • This time indicate that FBP-2 is active.
  • Draw a phosphate group on PFK-2.
    • The skeletal muscle isozyme of PFK-2 does not have a phosphorylation site.
  • Now, indicate that the PFK-2 catalytic site is inactive.
    • This is an important point: PFK-2 is INACTIVE when it is phosphorylated.

Now, let's add the enzymes that phosphorylate and dephosphorylate PFK-2.

  • Show that protein kinase A (PKA) phosphorylates PFK-2.
  • Indicate that phosphoprotein phosphatase (PPP) removes a phosphate from PFK-2.

Now, lets add the hormones: insulin and glucagon.

  • Show that insulin activates PPP, and thus ACTIVATES PFK-2.
  • Show that glucagon activates protein kinase A and thus INACTIVATES PFK-2.
    • Thus, by extension, fructose 2,6 bisphosphate concentration is under hormonal control in the liver.

Why is this?

  • The liver is also the site of gluconeogenesis, in which glucose is synthesized from non-carbohydrate precursors and released into the blood stream.
    • Thus, the liver must respond to the entire body's glucose needs, while skeletal muscle functions independently.

Let's illustrate this now.

  • First, write that fructose 2,6 bisphosphate promotes glycolysis but inhibits gluconeogenesis.
  • Now, draw two columns next to the liver and label them feed and fast.
  • Indicate that in the fed state; blood glucose is high, and the insulin:glucagon ratio is also high.
    • Step 1: Insulin binds a receptor on the liver cell.
    • Step 2: Via a cascade of intracellular events, insulin activates PPP.
    • Step 3: PPP removes the phosphate group from the PFK-2 catalytic site. PFK-2 is now active and FBP-2 is inactive.
    • Step 4: PFK-2 catalyzes the addition of a phosphate group to fructose-6-phosphate, and fructose 2,6 bisphosphate levels increase. This promotes glycolysis and inhibits gluconeogenesis during the fed state.

The exact opposite series of events occurs during fast.

  • Indicate that during the fasting state, blood glucose is low and the insulin to glucagon ratio is low (glucagon is high).
    • Step 1: Glucagon is released in response to low blood glucose.
    • Step 2: It activates PKA
    • Step 3: PKA adds a phosphate group to PFK-2 and inactivates it. FBP-2 is now active.
    • Step 4: FBP-2 removes a phosphate from fructose 2,6 bisphosphate, and fructose 2,6 bisphosphate levels decrease.
  • This inhibits glycolysis and promotes gluconeogenesis.
    • Gluconeogenesis supplies the body with glucose, so that tissues such as skeletal muscle can continue to produce ATP.
  • Thus, the rate-limiting step in glycolysis is controlled differently in different tissues to meet all of the body's metabolic needs

UNIT CITATIONS

  1. Nelson, D. L., Lehninger A.L. & Cox, M. M. Lehninger Principles of Biochemistry, 5th ed. (Macmillan, 2008).
  2. McKee T. & McKee, J. Biochemistry: The Molecular Basis of Life, 6th ed. (Oxford University Press, 2015).
  3. Champe, P.C., Harvey R.A. & Ferrier, D.R. Biochemistry. (Lippincott Williams & Wilkins, 2005).
  4. Berg, J.M., Tymoczko, J.L. & Stryer, L. Biochemistry, 7th ed. (W.H. Freeman and Company, 2010).
  5. Campbell, N. A. & Reece, J.B. Biology, 7th ed. (Benjamin Cummings, 2004).
  6. Watson, C. Human Physiology. (Jones and Bartlett Learning, 2014).
  7. Salway, J.G. Metabolism at a Glance, 49-52 (John Wiley & Sons, 2013).
  8. Lodish, H., Berk, A., Kaiser, C. A., Krieger, M., Scott, M. P., Bretscher, A., Ploegh, H. & Matsudaira, P. Molecular Cell Biology, 6th ed., 483-484 (W. H. Freeman and Company, 2008).
  9. Stipanuk M.H., Caudill, M. A. Biochemical, Physiological, and Molecular Aspects of Human Nutrition, 226-233 (Elsevier Health Sciences, 2013).
  10. LeRoith, D., Taylor, S. I. & Olefsky, J. M. Diabetes Mellitus: A Fundamental and Clinical Text, 6th ed., 1033 (Lippincott Williams & Wilkins, 2004).
  11. Puri, D. Textbook of Medical Biochemistry, 166 (Elsevier Health Sciences, 2014).
  12. Naik, P. Biochemistry, 164-166 (JP Medical Ltd, 2015).
  13. Snape, A., Papachristodoulou, D., Elliott, W. H. & Elliott, D. C. Biochemistry and Molecular Biology, 5th ed., 319 (OUP Oxford, 2014).
  14. Rider MH, Bertrand L, Vertommen D, Michels PA, Rousseau GG, Hue L. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis. Biochemical Journal. 2004;381(Pt 3):561-579. doi:10.1042/BJ20040752.
  15. Pretlow, T. G. & Pretlow, T. P. Biochemical and Molecular Aspects of Selected Cancers, 330 (Academic Press, 2014).
  16. Lammert, E. & Zeeb, M., Metabolism of Human Diseases: Organ Physiology and Pathophysiology, 284(Springer Science & Business Media, 2014).
    Ferrier, D. Lippincott's Illustrated Reviews: Biochemistry, 6th ed. (Lippincott Williams & Wilkins, 2014).