1. Chapter 22 Gluconeogenesis, Glycogen Metabolism,
and the Pentose Phosphate Pathway
Biochemistry by
Reginald Garrett and Charles Grisham

2. Outline of Chapter 22
What Is Gluconeogenesis, and How Does It Operate?
How Is Gluconeogenesis Regulated?
How Are Glycogen and Starch Catabolized in Animals?
How Is Glycogen Synthesized?
How Is Glycogen Metabolism Controlled?
Can Glucose Provide Electrons for Biosynthesis?

3. 22.1 – What Is Gluconeogenesis, and How Does It Operate?
Generation (genesis) of "new (neo) glucose" from common metabolites
Humans consume about 160 g of glucose per day, 75% of that is in the brain
Body fluids contain only 20 g of free glucose
Glycogen stores can provide g of glucose
So the body must be able to make new glucose from noncarbohydrate precursors—gluconeogenis

4. Substrates for Gluconeogenesis
Pyruvate, lactate, glycerol, amino acids and all TCA intermediates can be utilized
Fatty acids cannot! Why?
Most fatty acids yield only acetyl-CoA
Except fatty acids with odd numbers of carbons
Acetyl-CoA (through TCA cycle) cannot provide for net synthesis of sugars in animal, but plants do! Why? (chap 19)

5. Substrates for Gluconeogenesis
Lactate
Amino acids
Glycerol
Propionyl-CoA
FAs with Odd numbers of C

6. Gluconeogenesis Occurs mainly in liver (90%) and kidneys (10%)
Not the mere reversal of glycolysis for 2 reasons:
Energetics: must change to make gluconeogenesis favorable (DG of glycolysis = -74 kJ/mol)
Reciprocal regulation: Gluconeogenesis is turned on, and when glycolysis is turned off, and vice versa
The new reactions provide for a spontaneous pathway (DG negative in the direction of sugar synthesis), and they provide new mechanisms of regulation

7. Something Borrowed, Something New
Seven steps of glycolysis are retained:
Steps 2 and 4-9
Three steps are replaced:
Steps 1, 3, and 10 (the regulated steps!)
Figure 22.1 The pathways of gluconeogenesis and glycolysis. Species in blue, green, and peach-colored shaded boxes indicate other entry points for gluconeogenesis (in addition to pyruvate).

8. Hexokinase
Phosphofructokinase

9. Pyruvate kinase

10. Pyruvate is converted to oxaloacetate
1. Pyruvate Carboxylase
Pyruvate is converted to oxaloacetate
The reaction requires ATP and bicarbonate as substrates
Biotin is covalently linked to a lysine residue at the active site and as a coenzyme

11. The mechanism is typical of biotin
carbonylphosphate
pyruvate carbanion
Figure A mechanism for the pyruvate carboxylase reaction. Bicarbonate must be activated for attack by the pyruvate carbanion. This activation is driven by ATP and involves formation of a carbonylphosphate intermediate—a mixed anhydride of carbonic and phosphoric acids. (Carbonylphosphate and carboxyphosphate are synonyms.)

12. The conversion is in mitochondrial matrix
Acyl-CoA is an allosteric activator
Regulation:
If levels of ATP and/or acetyl-CoA are low
Pyruvate is converted to acetyl-CoA
Enters TCA cycle
If levels of ATP and/or acetyl-CoA are high
Pyruvate is converted to oxaloacetate
Enters gluconeogenesis  glucose

13. Pyruvate carboxylase is found only in mitochondrial matrix
Oxaloacetate cannot be transported across the mitochondrial membrane
Figure 22.4 Pyruvate carboxylase is a compartmentalized reaction. Pyruvate is converted to oxaloacetate in the mitochondria. Because oxaloacetate cannot be transported across the mitochondrial membrane, it must be reduced to malate, transported to the cytosol, and then oxidized back to oxaloacetate before gluconeogenesis can continue.

14. 2. Phosphoenolpyruvate Carboxykinase
Conversion of oxaloacetate to PEP
Lots of energy is needed to drive this reaction
Energy is provided in 2 ways:
Decarboxylation is a favorable reaction and helps drive the formation of the very high-energy enol phosphate
GTP is hydrolyzed, GTP used here is equivalent to an ATP

15. PEP Carboxykinase
The overall DG for the pyruvate carboxylase and PEP carboxykinase reactions under physiological conditions in the liver is kJ/mol
Once PEP is formed in this way, the other reactions act to eventually form fructose-1,6-bisphosphate

16. 3. Fructose-1,6-bisphosphatase
Hydrolysis of F-1,6-bisP to F-6-P
Thermodynamically favorable - G in liver is -8.6 kJ/mol
Allosteric regulation:
citrate stimulates
fructose-2,6-bisphosphate inhibits
AMP inhibits (enhanced by F-2,6-bisP)

17. 4. Glucose-6-Phosphatase
Conversion of Glucose-6-P to Glucose
G-6-Pase is present in ER membrane of liver and kidney cells
Muscle and brain do not do gluconeogenesis
G-6-P is hydrolyzed after uptake into the ER

18. Figure Glucose-6-phosphatase is localized in the endoplasmic reticulum. Conversion of glucose-6-phosphate to glucose occurs following transport into the ER.

19. The glucose-6-phosphatase system includes the phosphatase itself and three transport proteins, T1, T2, and T3.
T1 takes glucose-6-P into the ER, where it is hydrolyzed by the phosphatase
T2 and T3 export glucose and Pi, respectively, to the cytosol
Glucose is exported to the circulation by GLUT2

20. Gluconeogenesis Inhibitors and Other Diabetes Therapy Strategies

21. Diabetes is the inability to assimilate and metabolize blood glucose
Metformin improves sensitivity to insulin by stimulating glucose uptake by glucose transporters
Gluconeogenesis inhibitors may be the next wave of diabetes therapy
3-Mercatopicolinate and hydrazine inhibit PEP carboxykinase
Clorogenic acid (natural product found in the skin of peaches) inhibits transport activity by the glucose-6-phosphatase system. S-3483 does the same, but binds a thousand times more tightly to the transporter

22. Lactate Recycling – Cori cycle
Lactate formed in muscles is recycled to glucose in the liver
Recall that vigorous exercise can lead to a buildup of pyruvate and NADH, due to oxygen shortage and the need for more glycolysis
NADH can be reoxidized during the reduction of pyruvate to lactate
Lactate is then returned to the liver, where it can be reoxidized to pyruvate by liver LDH
Liver provides glucose to muscle for exercise and then reprocesses lactate into new glucose

23. (about 700)
Figure 22.7 The Cori cycle.

24. 22.2 – How Is Gluconeogenesis Regulated?
Reciprocal control with glycolysis
When glycolysis is turned on, gluconeogenesis should be turned off, and vice versa
When energy status of cell is high, glycolysis should be off and pyruvate and other metabolites are ustilized for synthesis (and storage) of glucose
When energy status is low, glucose should be rapidly degraded to provide energy
The regulated steps of glycolysis are the very steps that are regulated in the reverse direction

25. Figure 22.8 The principal regulatory mechanisms in glycolysis and gluconeogenesis. Activators are indicated by plus signs and inhibitors by minus signs.

26. Allosteric and Substrate-Level Control
Glucose-6-phosphatase
is under substrate-level control by G-6-P, not allosteric control
Pyruvate carboxylase
Activated by acetyl-CoA
The fate of pyruvate depends on acetyl-CoA; pyruvate kinase (-), pyruvate dehydrogenase (-), and pyruvate carboxylase (+)
F-1,6-bisPase
is inhibited by AMP and Fructose-2,6-bisP
Activated by citrate - the reverse of glycolysis

27. (w/o AMP) (w/ 25mM AMP) (F-2,6-BP) (F-2,6-BP) (AMP)
Figure 22.9 Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate in the (a) absence and (b) presence of 25 mM AMP. In (a) and (b), enzyme activity is plotted against substrate (fructose-1,6-bisphosphate) concentration. Concentrations of fructose-2,6-bisphosphate (in mM) are indicated above each curve. (c) The effect of AMP (0, 10, and 25 mM) on the inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate. Activity was measured in the presence of 10 mM fructose-1,6-bisphosphate.

28. Fructose-2,6-bisP
is an allosteric inhibitor of F-1,6-bisPase
is an allosteric activator of PFK
synergistic effect with AMP
The cellular levels Fructose-2,6-bisP are controlled by phosphofructokinase-2 and fructose-2,6-bisPase which is bifunctional enzyme
F-6-P allosterically activates PFK-2 and inhibits F-2,6-BisPase
Phosphorylation by cAMP-dependent protein kinase inhibits PFK-2 and activates F-2,6-bisPase

29. Figure Synthesis and degradation of fructose-2,6-bisphosphate are catalyzed by the same bifunctional enzyme.

30. 22.3 – How Are Glycogen and Starch Catabolized in Animals?
A balanced diet provides carbohydrate each day, mostly in the form of starch.
If too little carbohydrate is supplied by the diet, glycogen reserves in liver and muscle tissue can also be mobilized
The starch and glycogen are digested by amylase
-Amylase is an endoglycosidase -- a(1→4) cleavage
b-Amylase is an exoglycosidase (In plants)
It cleaves dietary amylopectin or glycogen to maltose, maltotriose and other small oligosaccharides

31. -Amylase can cleave on either side of a branch point
But activity is reduced near the branch points and stops four residues from any branch point
limit dextrins
Figure Hydrolysis of glycogen and starch by a-amylase and b-amylase.

32. Debranching enzyme cleaves "limit dextrins"
Two activities of the debranching enzyme
Oligo(a1,4→a1,4)glucanotransferase
a(1→6)glucosidase
Figure The reactions of glycogen debranching enzyme. Transfer of a group of three a-(1 ® 4)-linked glucose residues from a limit branch to another branch is followed by cleavage of the a-(1 ® 6) bond of the residue that remains at the branch point.

33. Metabolism of Tissue Glycogen is Regulated
Digestive breakdown is unregulated - nearly 100%
But tissue glycogen is an important energy reservoir - its breakdown is carefully controlled
Glycogen consists of "granules" of high MW range from 6 x 106 ~ 1600 x 106
Glycogen phosphorylase cleaves glucose from the nonreducing ends of glycogen molecules
This is a phosphorolysis, not a hydrolysis
Metabolic advantage: product is a glucose-1-P; a potential glycolysis substrate

34. Figure 22.13 The glycogen phosphorylase reaction.

35. 22.4 – How Is Glycogen Synthesized?
Glucose units are activated for transfer by formation of sugar nucleotides
What are other examples of "activated form"?
acetyl-CoA : acetate
Biotin and THF : one-carbon group
ATP : phosphate
Leloir showed that glycogen synthesis depends on sugar nucleotides
UDP-glucose pyrophosphorylase catalyzes the formation of UDP-glucose
ADP-glucose is for starch synthesis in plants

36. UDP-glucose + pyrophosphate.
Glucose-1-P + UTP →
UDP-glucose + pyrophosphate.
Figure The UDP-glucose pyrophosphorylase reaction is a phosphoanhydride exchange, with a phosphoryl oxygen of glucose-1-P attacking the a-phosphorus of UTP to form UDP-glucose and pyrophosphate.

37. Forms -(1 4) glycosidic bonds in glycogen
Glycogen Synthase
Forms -(1 4) glycosidic bonds in glycogen
The glycogen polymer is built around aprotein Glycogenin (a protein core)
First glucose is linked to a tyrosine -OH on glycogenin
Sugar units can be added by the action of glycogen synthase
Glycogen synthase transfers glucosyl units from UDP-glucose to C-4 hydroxyl at a nonreducing end of a glycogen strand. (Fig )

38. Figure 22.15 The glycogen synthase reaction.

39. -(1 6) linkages, which occurs every 8-12 residues
Branching enzyme:
Glycogen branches are formed by amylo-(1,4→1,6)-transglycosylase, also called branching enzyme
-(1 6) linkages, which occurs every 8-12 residues
Transfer of 6- or 7-residue segment from the nonreducing end
Figure Formation of glycogen branches by the branching enzyme. Six- or seven-residue segments of a growing glycogen chain are transferred to the C-6 hydroxyl group of a glucose residue on the same or a nearby chain.

40. 22.5 – How Is Glycogen Metabolism Controlled?
A highly regulated process
involving reciprocal control of glycogen phosphorylase and glycogen synthase
Activation of Glycogen phosphorylase (GP) is tightly linked to inhibition of glycogen synthase (GS), and vice versa
Regulation involves both allosteric control and covalent modification

41. 22.5 – How Is Glycogen Metabolism Controlled?
1. Allosteric control
Glycogen phosphorylase allosterically activated by AMP and inhibited by ATP, glucose-6-P and caffeine
Glycogen synthase is stimulated by glucose-6-P

42. Phosphorylation of GP and GS
2. Covalent modification
In chapter 15 (p496) showed that protein kinase converted phosphorylase b (-OH, inactivated) to phosphorylase a (-OP, activated)
Glycogen synthase also exists in two distinct forms
Active, dephosphorylated glycogen synthase I
Less active, phosphorylated glycogen synthase D (glucose-6-P dependent)

43. Casein kinase
Glycogen Synthase Kinase 3 (GSK 3)
SPK: Synthase-phosphorylase kinase
(phosphorylase kinase)
PP1: Phosphoprotein phosphatase 1

44. Glucagon and epinephrine activate adenylyl cyclase (chapter 15)
cAMP activates kinases and phosphatases that control the phosphorylation of GP and GS
stimulate glycogen breakdown
Dephosphorylation of both glycogen phosphorylase and glycogen synthase is carried out by phosphoprotein phosphatase-1 (PP1)
PP1 inactivates glycogen phosphorylase and activates glycogen synthase

45. Hormones Regulate Glycogen Synthesis and Degradation
Storage and utilization of tissue glycogen and other aspects of metabolism are regulated by hormones, including insulin, glucagon, epinephrine, and the glucocorticoids
Insulin is a response to increased blood glucose
Insulin triggers glycogen synthesis when blood glucose rises
Between meals, blood glucose is mg/dL
Glucose rises to 150 mg/dL after a meal and then returns to normal within 2-3 hours

46. Figure 22.17 Insulin triggers protein kinase cascades that stimulate glycogen synthesis.

47. (glucose uptake, GLUT4)
(F-1,6-BP & PEPCK)
(PFK & PK)
Figure The metabolic effects of insulin. As described in Chapter 32, binding of insulin to membrane receptors stimulates the protein kinase activity of the receptor. Subsequent phosphorylation of target proteins modulates the effects indicated.

48. Insulin
Insulin is secreted from the b-cells in the pancreas into the pancreatic vein, empties into the portal vein system (to liver)
Figure The portal vein system carries pancreatic secretions such as insulin and glucagon to the liver and then into the rest of the circulatory system.

49. Glucagon and epinephrine
Hormonal Regulation
Glucagon and epinephrine
Glucagon and epinephrine stimulate glycogen breakdown - opposite effect of insulin
Glucagon (29 AA-res) is also secreted from a-cells in pancreas and acts in liver and adipose tissue only
Epinephrine (adrenaline) is released from adrenal glands and acts on liver and muscles
A cascade is initiated that activates glycogen phosphorylase and inhibits glycogen synthase

50. Figure Glucagon and epinephrine activate a cascade of reactions that stimulate glycogen breakdown and inhibit glycogen synthesis in live and muscles, respectively.
PFK-2 isoforms in liver (ser32) and heart (ser466 & ser483) responds oppositely to PKA

51. Hormonal Regulation The phosphorylase cascade amplifies the signal
10-10 to M epinephine
10-6 M cAMP
Protein kinase (PK)
30 molecules of phosphorylase b kinase / PK
800 molecules of phosphorylase a
Catalyzes the formation of many molecules of glucose-1-P
The result of these actions is tightly coordinated stimulation of glycogen breakdown and inhibition of glycogen synthesis

52. The difference between Epinephrine and Glucagon
Both are glycogenolytic but for different reasons
Epinephrine is the fight or flight hormone
Rapid breakdown of glycogen
Inhibition of glycogen synthesis
Stimulation of glycolysis
Production of energy
Glucagon is for long-term maintenance of steady-state levels of glucose in the blood
activates glycogen breakdown
activates liver gluconeogenesis
Glucagon do not activate the phsphorylase cascade in muscle

53. Cortisol and glucocorticoid
Glucocorticoids are steroid hormones that exert distinct effects on liver, skeletal muscle, and adipose tissue
Cortisol is a typical glucocoticoid
In skeletal muscle (catabolic)
promotes protein breakdown
decrease protein synthesis
In liver
stimulates gluconeogenesis
increases glycogen synthesis
Activates amino acid catabolism & urea cycle

54. Figure 22.21 The effects of cortisol on carbohydrate and protein metabolism in the liver.

55. 22.6 – Can Glucose Provide Electrons for Biosynthesis?
Pentose Phosphate Pathway
Hexose monophosphate shunt
Phosphogluconate pathway
Provides NADPH for biosynthesis
Produces ribose-5-P for nucleotide synthesis
Several metabolites of the pentose phosphate pathway can also be shuttled into glycolysis
Operates mostly in cytoplasm of liver and adipose cells, but absent in muscle

56. Pentose phosphate pathway
Begins with glucose-6-P, a six-carbon, and produces 3-, 4-, 5-, 6, and 7-carbon sugars, some of which may enter the glycolytic pathway
Two oxidative processes followed by five non-oxidative steps
NADPH is used in cytosol for reductive reaction-- fatty acid synthesis

57. Figure 22. 22 The pentose phosphate pathway
Figure The pentose phosphate pathway. The numerals in the blue circles indicate the steps discussed in the text.
Transketolase: 6 & 8
Transaldolase: 7

58. Begins with Two Oxidative Steps
Glucose-6-P Dehydrogenase
Begins with the oxidation of glucose-6-P
The products are a cyclic ester (the lactone of phosphogluconic acid) and NADPH
Irreversible 1st step and highly regulated
Inhibited by NADPH and acyl-CoA
Gluconolactonase
Gluconolactone hydrolyzed →6-phospho-D-gluconate
Uncatalyzed reaction happens too
Gluconolactonase accelerates this reaction

59. Figure The glucose-6-phosphate dehydrogenase reaction is the committed step in the pentose phosphate pathway.
Figure The gluconolactonase reaction.

60. 6-Phosphogluconate Dehydrogenase
An oxidative decarboxylation of 6-phosphogluconate
Yields ribulose-5-P and NADPH
Releases CO2
Figure The 6-phosphogluconate dehydrogenase reaction.

61. The Nonoxidative Steps
Five steps, only 4 types of reaction...
Phosphopentose isomerase
converts ketose to aldose
Phosphopentose epimerase
epimerizes at C-3
Transketolase (TPP-dependent)
transfer of two-carbon units
Transaldolase (Schiff base mechanism)
transfers a three-carbon unit

62. Glucose-6-P + 2 NADP+ + H2O → ribose-5-P + 2 NADPH + 2 H+ + CO2
Phosphopentose isomerase
converts ketose to aldose
Ribose-5-P is utilized in the biosynthesis of coenzymes, nucleotides, and nucleic acids
Glucose-6-P NADP+ + H2O → ribose-5-P NADPH H+ + CO2
Figure The phosphopentose isomerase reaction involves an enediol intermediate.

63. Phosphopentose epimerase An inversion at C-3
Figure The phosphopentose epimerase reaction interconverts ribulose-5-P and xylulose-5-phosphate. The mechanism involves an enediol intermediate and occurs with inversion at C-3.

64. Transketolase (TPP-dependent) transfer of two-carbon units
The donor molecule is a ketose and the recipient is an aldose
Figure The transketolase reaction of step 6 in the pentose phosphate pathway.

65. Figure 22.29 The transketolase reaction of step 8 in the pentose phosphate pathway.

66. Figure The mechanism of the TPP-dependent transketolase reaction. Ironically, the group transferred in the transketolase reaction might best be described as an aldol, whereas the transferred group in the transaldolase reaction is actually a ketol. Despite the irony, these names persist for historical reasons.

67. Transaldolase (Schiff base, imine) transfers a three-carbon unit
Yields erythrose-4-P & Fructose-6-P
Figure The transaldolase reaction.

68. Figure The transaldolase mechanism involves attack on the substrate by an active-site lysine. Departure of erythrose-4-P leaves the reactive enamine, which attacks the aldehyde carbon of glyceraldehyde-3-P. Schiff base hydrolysis yields the second product, fructose-6-P.

69. Variations on the Pentose Phosphate Pathway
1) Both ribose-5-P and NADPH are needed
2) More ribose-5-P than NADPH is needed
3) More NADPH than ribose-5-P is needed
4) NADPH and ATP are needed, but ribose-5-P is not
Figure When biosynthetic demands dictate, the first four reactions of the pentose phosphate pathway predominate and the principal products are ribose-5-P and NADPH.

70. 2) More Ribose-5-P than NADPH is needed by the cell
2) More Ribose-5-P than NADPH is needed by the cell. Synthesis of ribose-5-P can be accomplished without making NADPH, by bypassing the oxidative reactions of the pentose phosphate pathway

71. 3) More NADPH than ribose-5-P is needed by the cell
3) More NADPH than ribose-5-P is needed by the cell. This can be accomplished if ribose-5-P produced in the pentose phosphate pathway is recycled to produce glycolytic intermediates

72. 4) Both NADPH and ATP are needed by the cell, but ribose-5-P is not
4) Both NADPH and ATP are needed by the cell, but ribose-5-P is not. This can be done by recycling ribose-5-P, as in case 3 above, if fructose-6-P and glyceraldehyde-3-P made in this way proceed through glycolysis to produce ATP and pyruvate, and pyruvate continues through the TCA cycle to make more ATP

73. Figure 21.25 The Calvin-Benson Cycle of reactions