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

2. Essential Question
What is the nature of gluconeogenesis, the pathway that synthesizes glucose from noncarbohydrate precursors
How is glycogen synthesized from glucose
How are electrons from glucose used in biosynthesis?

3. 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?

4. 22.1 – What Is Gluconeogenesis, and How Does It Operate?
Synthesis of "new glucose" from common metabolites
Humans consume 160 g of glucose per day
75% of that is in the brain
Body fluids contain only 20 g of glucose
Glycogen stores yield g of glucose
So the body must be able to make its own glucose

5. 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?

6. Substrates for Gluconeogenesis
Lactate
Amino acids
Glycerol
Propionyl-CoA

7. 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 must turn one on and the other off, and vice versa

8. Something Borrowed, Something New
Gluconeogenesis
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!)
The new reactions provide for a spontaneous pathway (DG negative in the direction of sugar synthesis), and they provide new mechanisms of regulation

9. Figure 22. 1 The pathways of gluconeogenesis and glycolysis
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).

10. Pyruvate is converted to oxaloacetate
Pyruvate Carboxylase
Pyruvate is converted to oxaloacetate
The reaction requires ATP and bicarbonate as substrates
Biotin as a coenzyme and Acetyl-CoA is an allosteric activator

11. Biotin is covalently linked to the e-amino group of a lysine residue
Figure 22.3 Covalent linkage of biotin to an active-site lysine in pyruvate carboxylase.

12. The mechanism is typical of biotin
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.)

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

14. Pyruvate carboxylase is found only in mitochondrial matrix
Oxaloacetate cannot be transported across the mitochondrial membrane
Figure 22.5 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.

15. Conversion of oxaloacetate to PEP
PEP Carboxykinase
Conversion of oxaloacetate to PEP
Lots of energy needed to drive this reaction
Energy is provided in 2 ways:
Decarboxylation is a favorable reaction
GTP is hydrolyzed
GTP used here is equivalent to an ATP

16. 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

17. Fructose-1,6-bisphosphatase
Hydrolysis of F-1,6-bisPase 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)

18. 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 as it passes into the ER
ER vesicles filled with glucose diffuse to the plasma membrane, fuse with it and open, releasing glucose into the bloodstream.

19. Figure Glucose-6-phosphatase is localized in the endoplasmic reticulum membrane. Conversion of glucose-6-phosphate to glucose occurs during transport into the ER.
DG = -5.1 kJ/mol
Figure The glucose-6-phosphatase reaction involves formation of a phosphohistidine intermediate.

20. Lactate Recycling – Cori cycle
How your liver helps you during exercise....
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

21. (about 700)
Figure The Cori cycle.

22. 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, etc., should be used 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

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

24. 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

25. (w/o AMP) (w/ 25mM AMP) (F-2,6-BP) (F-2,6-BP) (AMP)
Figure 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.

26. 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

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

29. 22.3 – How Are Glycogen and Starch Catabolized in Animals?
Getting glucose from storage (or diet)
-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
It is active 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

30. Figure 22.14 Hydrolysis of glycogen and starch by a-amylase and b-amylase.

31. 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.

32. Metabolism of Tissue Glycogen
Digestive breakdown is unregulated - 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 "sort-of" glycolysis substrate

33. See pages 486-491 to review glycogen phosphorylase
(Phosphorolysis)
Figure The glycogen phosphorylase reaction.
See pages to review glycogen phosphorylase

34. 22.4 – How Is Glycogen Synthesized?
Glucose units are activated for transfer by formation of sugar nucleotides
What are other examples of "activation"?
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

35. Figure 22.17 The structure of UDP-glucose, a sugar nucleotide.

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
Glycogenin (a protein core) forms the core of a glycogen particle
First glucose is linked to a tyrosine -OH on glycogenin
Glycogen synthase transfers glucosyl units from UDP-glucose to C-4 hydroxyl at a nonreducing end of a glycogen strand.
oxonium ion intermediate (Fig )

38. Figure 22. 19 The glycogen synthase reaction
Figure The glycogen synthase reaction. Cleavage of the C-O bond of UDP-glucose yields an oxonium intermediate. Attack by the hydroxyl oxygen of the terminal residue of a glycogen molecule completes the reaction.

39. Glycogen branching occurs by transfer of terminal chain segments
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
Glycogen phosphorylase, allosterically activated by AMP and inhibited by ATP, glucose-6-P and caffeine
Glycogen synthase, is stimulated by glucose-6-P
Both enzymes are regulated by covalent modification - phosphorylation

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

42. SPK: Synthase-phosphorylase kinase
( phosphorylase b kinase)
PP1: Phosphoprotein phosphatase 1

43. Enzyme Cascades and GP/GS
Hormonal regulation ( insulin, Glucagon, epinephrine, and glucocorticoids)
Glucagon and epinephrine activate adenylyl cyclase
cAMP activates kinases and phosphatases that control the phosphorylation of GP and GS
GTP-binding proteins (G proteins) mediate the communication between hormone receptor and adenylyl cyclase
Dephosphorylation is carried out by phosphoprotein phosphatase-I (PP-I)

44. of Glycogen Synthesis and Degradation
Hormonal Regulation
of Glycogen Synthesis and Degradation
Insulin is secreted from the pancreas (to liver) in response to an increase in blood glucose
Insulin into the portal vein and traverses the liver
Insulin stimulates glycogen synthesis and inhibits glycogen breakdown
Other effects of insulin (Figure 22.22)

45. 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.

46. (glucose uptake)
(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.

47. 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 by 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

48. Figure 22.23 The amino acid sequence of glucagon.
Figure Epinephrine

49. Hormonal Regulation The phosphorylase cascade amplifies the signal
10-10 to M epinephine
10-6 M cAMP
Protein kinase
30 molecules of phosphorylase b kinase
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

50. SPK: Synthase-phosphorylase kinase
( phosphorylase b kinase)
PP1: Phosphoprotein phosphatase 1

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 mucle

53. Cortisol and glucocorticoid
Cortisol is a typical glucocoticoid
In muscle (catabolic)
promotes protein breakdown
decrease protein synthesis
In liver
stimulates gluconeogenesis
increases glycogen synthesis
amino acid catabolism

54. Figure 22.25 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

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
Operates mostly in cytoplasm of liver and adipose cells, but absent in muscle
NADPH is used in cytosol for reductive reaction-- fatty acid synthesis

57. Figure 22. 26 The pentose phosphate pathway
Figure The pentose phosphate pathway. The numerals in the blue circles indicate the steps discussed in the text.

58. Oxidative Steps Glucose-6-P Dehydrogenase Gluconolactonase
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.

60. Figure 22.28 The gluconolactonase reaction.

61. Oxidative Steps 6-Phosphogluconate Dehydrogenase
An oxidative decarboxylation of 6-phosphogluconate
Yields ribulose-5-P and NADPH
Releases CO2

62. Figure 22.29 The 6-phosphogluconate dehydrogenase reaction.

63. 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

64. Phosphopentose isomerase converts ketose to aldose
Ribose-5-P is utilized in the biosynthesis of coenzymes, nucleotides, and nucleic acids
Figure The phosphopentose isomerase reaction involves an enediol intermediate.

65. 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.

66. 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.

67. Figure 22.33 The transketolase reaction of step 8 in the pentose phosphate pathway.

68. 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.

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

70. 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.

71. 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.

72. Figure The oxidative steps of the pentose phosphate pathway can be bypassed if the primary need is for ribose-5-P.

73. Figure Large amounts of NADPH can be produced by the pentose phosphate pathway without significant net production of ribose-5-P. In this version of the pathway, ribose-5-P is recycled to produce glycolytic intermediates.

74. Figure Both ATP and NADPH (as well as NADH) can be produced by this version of the pentose phosphate and glycolytic pathways.

75. (a) Contributions of the various energy sources to muscle activity during mild exercise. (b) Consumption of glycogen stores in fast-twitch muscles during light, moderate, and heavy exercise. (c) Rate of glycogen replenishment following exhaustive exercise. (a and c adapted from Rhodes and Pflanzer, Human Physiology. Philadelphia: Saunders College Publishing; b adapted from Horton and Terjung, Exercise, Nutrition and Energy Metabolism. New York : Macmillan.)