Pyruvate Kinase

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PYRUVATE KINASE

• Last enzyme in glycolysis
• Irreversibly dephosphorylates phosphoenolpyruvate (PEP) to form pyruvate
• 1 ATP produced by substrate level phosphorylation
• Several isozymes: M-type (muscle) and L-type (liver)
• All isozymes allosterically regulated (L-type also hormonally regulated)

M-TYPE ISOZYMES

Allosteric regulation

Activation

• AMP: marker of ATP depletion or low energy
• Fructose 1,6-bisphosphate: product of rate-limiting reaction in glycolysis
  (feed-forward activation: stimulates downstream glycolytic enzymes)

Inhibition

• ATP: sufficient energy
• Acetyl CoA: first intermediate of citric acid cycle
• Alanine: can be produced from pyruvate; sufficient pyruvate in the cell

L-TYPE ISOZYME

• Allosteric and hormonal regulation (similar to PFK-2)

Hormonal regulation

Activation

• Insulin activates phosphatases, which remove phosphate from PK
• Makes PK susceptible to positive allosteric regulators

Inhibition

• Glucagon promotes phosphorylation of PK via cAMP-dependent pathway
• Makes PK susceptible to negative allosteric regulators

CLINICAL CORRELATION

Pyruvate kinase deficiency

• Produce hemolytic anemia (spiculated RBC's)
• RBC biconcave shape maintained by sodium-potassium pumps (require ATP)
• RBC's do not have mitochondria: rely on glycolysis for ATP

Full-Length Text

• Here we will learn about pyruvate kinase, the last regulated enzyme in glycolysis.

• To begin, start a table to learn some key features of pyruvate kinase.

• Denote that it is the last enzyme in glycolysis.
  • It catalyzes the irreversible dephosphorylation of phosphenolpyruvate (PEP) to form pyruvate.
  • It has several isozymes in the body that are encoded by different genes.
  • The M-type occurs in the muscle and brain, the L type in the liver.
  • All the isozymes are allosterically regulated.
  • The L-type is also under hormonal regulation, which we will illustrate shortly.

Now, let's draw the reaction catalyzed by pyruvate kinase.

• Draw a portion of skeletal muscle.

• Draw pyruvate kinase.

• Show that it dephosphorylates phosphoenolpyruvate to form the final product of glycolysis: pyruvate.

• Indicate that one ATP is produced via substrate level phosphorylation.
  • This is the second substrate level phosphorylation in the energy payoff phase of glycolysis.

Now, let's illustrate the regulation of pyruvate kinase.

• Indicate that M-type isozymes are controlled by allosteric regulation.

• Show that the enzyme is activated by the following:
  • AMP, which a marker of ATP depletion or low energy conditions.
  • Fructose 1,6 bisphosphate, which is the product of the rate-limiting reaction in glycolysis. Its mechanism is feed-forward activation: it stimulates downstream glycolytic enzymes to promote its own metabolism.,

• Show that the enzyme is inhibited by the following:
  • ATP, which signals that there is enough energy in the system.
  • Acetyl CoA, which is the first intermediate of the citric acid cycle.
  • Alanine, which can be produced from pyruvate and signals that there is sufficient pyruvate in the cell.
  • This is the same feedback inhibition that we have seen with both hexokinase and phosphofructokinase-1.

• Next, draw a liver.

• Indicate that the L-type isozyme experiences both allosteric and hormonal regulation, the mechanism of which is very similar to PFK-2.
  • Recall, PFK-2 catalyzes the formation of fructose 2,6 bisphosphate, an allosteric activator of PFK-1.

Let's illustrate this now.

• Draw the active form of pyruvate kinase.

• Show that it catalyzes the same reaction that we drew in the muscle.

• Next, draw an inactive form of pyruvate kinase above it with a phosphate group attached to it.
  • The M-type pyruvate kinase does not have a phosphorylation site.,

• Use an arrow to show that, much like PFK-2, the liver isozyme of pyruvate kinase can be inactivated by phosphorylation.

• Show that this process consumes ATP.

• Now, above the liver, draw a representative blood vessel.

• Show that the glucose concentration in the vessel can either be high, as in during the fed state, or low, as in fasting conditions.

• Now, illustrate that glucagon is secreted when glucose is low.
  • Pancreatic alpha cells secrete glucagon into the blood stream.
  • It binds a surface liver cell receptor and activates a cascade of reactions.

• Indicate that via a cAMP dependent pathway, glucagon promotes the phosphorylation of pyruvate kinase.

• Next, illustrate that insulin is secreted when glucose is high.
  • Pancreatic beta cells secrete insulin.

• Show that insulin activates phosphatases within the cell.

• Indicate that these phosphatases remove the phosphate from pyruvate kinase and activate the enzyme.

• Show that this reaction consumes water.

• Finally, recall that the L-type isozyme also experiences the same allosteric regulation that occurs in the muscle.

• Write that phosphorylation makes pyruvate kinase more susceptible to negative allosteric regulators and dephosphorylation does the opposite.

• As a clinical correlation, write that pyruvate kinase deficiencies produce hemolytic anemia, in which red blood cells swell and lyse.

Let's draw this mechanism as follows:

• Draw a red blood cell.

• Now, draw an amorphous red blood cell with a spiky appearance.

• Label it "spiculated."

• Use an arrow to show that pyruvate kinase deficiency leads to spiculated red blood cells.

Why?

• Draw a sodium potassium pump on the surface of the normal red blood cell.

• Indicate that these pumps maintain its biconcave shape.

• Show that they require ATP.
  • Now, write that red blood cells do not have mitochondria and rely on glycolysis for ATP.

• Without pyruvate kinase, glycolysis cannot proceed and sodium potassium pumps lack the ATP required to maintain the cell's shape.

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).