Biochemistry I Fall Term, 2000

 November 20, 2000

Lecture 31: Glycogen Metabolism/Gluconeogenesis

Assigned reading in Campbell: Chapter Chapter 14.1-14.3 (& 20.4)

Key Terms:
Glycogen phosphorylase a & b
Glycogen synthase
UDP-glucose
Phosphorylation cascade
Gluconeogenesis
Fructose-2,6-bisphosphate (F2,6P)
Substrate cycling		 Hormonal control:
    Insulin, Glucagon, & Epinephrine
Adenyl cyclase & cAMP
Protein kinase A
Phosphorylase kinase
Phosphoprotein phosphatase
 

Take the Review Quiz on Lecture 31 concepts.

Regulation of anabolic (synthesis) and catabolic (degradation) pathways

  1. Synthetic and degradative pathways usually use the same enzyme for steps in which DG = 0.
  2. Synthetic and degradative pathways always utilize different enzymes for steps in which D G << 0.
  3. Regulatory steps (DG << 0) are often the same in both pathways.
  4. Consequently, regulatory enzymes are always different and they can respond to regulatory signals in different ways.
  5. Flux through pathways is often controlled by competing rates of synthesis and degradation.

Glycogen Synthesis and Degradation

Glycogen synthesis is catalyzed by glycogen synthase.
Glycogen degradation is catalyzed by glycogen phosphorylase.
Phosphorolysis is cleavage of an ester by a phosphate group (instead of water).

The major regulation of these two pathways is by phosphorylation of the enzymes involved. The phosphorylation changes the structure of the enzyme. Depending on the enzyme, either activation or inhibition can occur. Consider phosphorylation as functionally equivalent to an allosteric inhibitor/activator that has a very high affinity.

In the case of glycogen synthesis/degradation the regulatory signals are amplified by a "phosphorylation cascade" produced by the activation of additional kinases/phosphatases that act on the enzymes of glycogen synthesis/degradation.

Enzyme Active Form Inactive Form
Glycogen phosphorylase Ser/Thr-phosphate Ser/Thr (no phosphate)
Glycogen synthase Ser/Thr (no phosphate) Ser/Thr-phosphate

Thus, phosphoryation leads to glycogen degradation, and dephosphorylation leads to glycogen synthesis.

Gluconeogenesis

Gluconeogenesis, the formation of glucose from 3- and 4-carbon carboxylic acids, is essential for the maintenance of constant blood glucose levels. The liver, and to a lesser extent the kidneys, are the only organs that carry out this process. All of our other tissues and organs (especially the brain) require this newly-synthesized glucose during periods of fasting, i.e. between meals and during sleep.
All of the reversible reactions of glycolysis are used in gluconeogenesis, however, the three irreversible steps require alternative reactions. These reactions are catalyzed and regulated by the enzymes described here.

1. Pyruvate carboxylase

  • Carboxylation is coupled to ATP hydrolysis.
        Allosterically activated by acetyl coenzyme A.
        Biotin is the covalently attached cofactor.
  • Found in the mitochondria.
    (where it also functions to provide oxaloacetate for the TCA cycle.)
  • The oxaloacetate formed by pyruvate carboxylase is converted to PEP in the cytosol (by PEP carboxykinase).
        GTP is the phosphoryl donor.
        CO2 is a product.
(The steps from PEP --> F-1,6-DP are a reversal of glycolysis.)

2. Fructose-1,6-bisphosphatase

  • Allosterically activated by ATP; inhibited by AMP./
  • Inhibited by fructose-2,6-bisphosphate (F2,6P).
        F2,6P is synthesized by [PFK-2]; phosphorylation inhibits.
        F2,6P is hydrolyzed by [FBPase-2]; phosphorylation activates.
        Both of these enzyme activities reside on the same polypeptide and are sensitive to phosphorylation.
3. Glucose-6-phosphatase
Hydrolyzes G-6-P to glucose + Pi.

Overall, the "cost" of making glucose from pyruvate is two ATP equivalents. Although this corresponds to the net yield from anaerobic glycolysis, the price is cheap when ATP is plentiful.

Regulation of PFK (glycolysis) by fructose-2,6-bisphosphate (F2,6P)

  • High glucose levels: increase in glycolysis
  • Low glucose levels: decrease in glycolysis, increase in the synthesis of glucose.
  • Fructose-2,6-bisphosphate activates PFK, thus increasing glycolysis.
When blood sugar is low, glucose must be mobilized from the liver stores; therefore glycolysis must be inhibited and glucose must be produced from glycogen. Thus, protein kinases will be active.
Phosphorylation of PFK-2/FBPase in the liver: inhibits PFK-2 activity and activates FBPase activity. Thus the levels of F2,6P decrease and glycolysis is inhibited.
F2,6P also activates the synthesis of glucose from pyruvate.
When blood sugar is high, glucose can be used to generate energy and NADH for other metabolic pathways. In addition, the excess glucose is stored as glycogen. During this time protein phosphatases are active:

Dephosphorylation of PFK-2/FBPase in the liver activates PFK-2 and inhibits FBPase. Thus, the levels of F2,6P increase and glycolysis is activated.

Regulation of Biochemical Pathways

  1. Change in levels of enzymes by regulation of their synthesis/degradation (slow)
  2. Change in the activity of enzymes by covalent modification (moderately fast)
  3. Change in the activity of enzymes by feedback inhibition (fast)
  4. Product inhibition (very fast)
The following material from Campbell, Chapter 20.4, will be covered later. But since it so closely linked to the biochemistry described above, this is also an appropriate place to compare and contrast the levels of regulation involved.

Hormonal Regulation of Protein Kinases and Protein Phosphatases.
(Campbell, Chapter 20.4)

Steps in the activation of glycogen phosphorylase

  • Glucagon and/or epineprine bind to receptors on the surface of liver and muscle cells
  • The receptor-ligand (ligand=glucagon, epineprine) complex activates adenyl cyclase
  • Adenyl cyclase converts ATP to cAMP (cyclic AMP = 3',5'- cyclic AMP)

    cAMP is called a "2nd messenger."
  • cAMP activates protein kinase A
  • Protein kinase A phosphorylates:
    1. phosphorylase kinase (activating kinase activity)
    2. glycogen synthase (inactivating it).
  • Phosphorylase kinase phosphorylates glycogen phosphorylase, activating it.
  • Phosphoprotein phosphatase (which would remove phosphates) is also phosphorylated to inactivate it.

Steps in the activation of Glycogen Synthase
To reverse the above effects we need to dephosphorylate the proteins that were phosphorylated in the above reactions. This occurs by activation of phosphoprotein phosphatase. This enzyme removes phosphate groups from proteins.

During glycogen degradation, phosphoprotein phosphatase is inactive because:

  1. It is not phosphorylated on the correct location.
  2. It is complexed with glycogen phosphorylase.
Phosphoprotein phosphatase becomes activated during glycogen synthesis because:
  1. Phosphorylation by insulin-stimulated protein kinase activates it.
  2. Binding of glucose to glycogen phosphorylase releases phosphoprotein phosphatase.

Once activated Phosphoprotein phosphatase does the following:

  1. Removes the phosphate group from glycogen phosphorylase, inactivating it
  2. Removes phosphate group from phosphorylase kinase, inactivating it
  3. Removes phosphate group for glycogen synthase, activating it

cAMP levels drop since they are no longer elevated due to absence of glycogen and epinephrine.


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