1. GLUCONEOGENESIS Student Edition 5/30/13 Version
Dr. Brad Chazotte
213 Maddox Hall
Web Site:
Original material only © B. Chazotte
Pharm. 304 Biochemistry
Fall 2014

2. Goals Learn the enzymes and step of the gluconeogenesis pathway.
Learn the similarities and difference the between gluconeogenesis and glycolysis pathways.
Understand the principles for biosynthetic (anabolic) pathways vs catabolic.
Understand how the gluconeogenesis pathway is regulated and how it is regulated vs glycolysis.
Understand the concept and benefits of metabolic burden sharing
Be aware the Cori cycle.
Understand how substrate cycles can amplify metabolic signals and/or produce heat.

3. Biosynthetic (Anabolic) Pathways
Use chemical energy (ATP, NADH, NADPH) to synthesize cellular components from simple precursor molecules.
Generally reductive rather than oxidative.
Anabolism and catabolism proceed simultaneously in a dynamic steady-state to maintain the “intricate orderliness of living cells”.
Lehninger 2000 p722

4. Organizing Principles of Biosynthesis 1
1. Molecules are synthesized and degraded by different pathways.
a. Two opposing anabolic and catabolic pathways may share many reversible reactions.
b. Each pathway has at least one unique and essentially irreversible reaction.
c. This insures that the carbon flow through these pathways is dictated by the cell’s changing requirements for energy, precursors, and macromolecules rather than simple mass action.
Lehninger 2000 p722

5. Organizing Principles of Biosynthesis 2
2. Corresponding anabolic and catabolic pathways are controlled by one or more reactions unique to each pathway.
a. Opposing pathways are regulated in a coordinated, reciprocal manner so that the stimulation of the anabolic pathway is accompanied by the inhibition of the catabolic one and vice versa.
b. Biosynthetic pathways are typically regulated at an early, exergonic step that commits intermediates to the pathway and does not waste energy by making unneeded intermediates.
Lehninger 2000 p722

6. Organizing Principles of Biosynthesis 3
3. Energy requiring biosynthetic processes are coupled to the breakdown of ATP in such a way that the overall process is essentially irreversible in vivo.
a. Thus the total amount of energy from ATP and NAD(P)H used always exceeds the minimum free energy needed to convert the precursor into the biosynthetic product.
b. Consequently the biosynthetic process is thermodynamically favorable (G < 0) even for low precursor concentrations.
Lehninger 2000 p722

7. Carbohydrate Biosyntheses from Simple Precursors
The pathway from PEP to glucose-6-P is common to the biosynthetic conversion of many different precursors in animals and plants.
Gluconeogenesis, meaning “formation of new sugar”, occurs in all animals, plants, fungi, and microorganisms. In most cases the reactions are essentially the same.
Gluconeogenesis
Lehninger 2000 Fig 20.1

8. Importance of Gluconeogenesis
The brain depends on glucose as its primary fuel using ~120g/day out of ~160g/day for the typical adult human.
Red blood cells use only glucose as a fuel.
Only 20g are present in the body fluids and ~190 g are available from glycogen storage.
Therefore, total reserves of glucose are about a single day’s supply.
For longer periods of starvation, glucose must be formed from noncarbohydrate sources.

9. Noncarbohydrate Precursors for Gluconeogenesis
Pyruvate
Any metabolite that can be converted to pyruvate or oxaloacetate can be a glucose precursor
Lactate is formed by active skeletal muscle when glycolysis > oxidative metabolism. Lactate is converted by lactate dehydrogenase to pyruvate.
Pyruvate
(Certain) Amino acids are derived from protein in the diet or from muscle protein catabolism during starvation. Carbon skeletons of most amino acids are catabolized to pyruvate or citric acid cycle intermediates
Glycerol is the result of a breakdown of triacylglycerols in fat cells. Fatty acids also result, but cannot be used by animals to make glucose. Glycerol enters glycolysis or gluconeogenesis at dihydroxyacetone phosphate

10. Glycerol’s Entry in Gluconeogenesis or Glycolysis
Berg, Tymoczko & Stryer, 2012 Chap. 16 np.481

11. The Gluconeogenesis Balanced Equation
2Pyruvate + 2NADH +4H+ + 4ATP +2GTP +6H2O
Glucose +2NAD+ + 4ADP + 2GDP + 6Pi
Gº = –16 kJ/ mol
Because of the presence of separate gluconeogenic enzymes at the three irreversible steps in the glycolytic pathway that converts glucose to pyruvate, glycolysis and gluconeogenesis both are rendered THERMODYNAMICALLY favorable.
Gluconeogenesis
Voet & Voet 1995 Chap 21 P.604

12. Gluconeogenesis Pathway
Voet, Voet & Pratt 2013 Figure 16.15
Berg, Tymoczko & Stryer, 2012 Fig

13. Subcellular Location of Gluconeogenic Enzymes
Gluconeogenesis enzymes are cytosolic except:
(1) Glucose 6-phosphatase (endoplasmic reticulum)
(2) Pyruvate carboxylase (mitochondria)
(3) PEPCK (cytosol and/or mitochondria)
Mitochondrial pyruvate can:
1) be converted to citrate and used in the cytosol for the synthesis of fatty acids.
2) be changed to acetyl CoA and enter the citric acid cycle.
3) be converted by pyruvate carboxylase to oxaloacetate for gluconeogenesis.
Horton et al., 2000 Chap 13.6

14. G’s of Erythrocyte Glycolytic Rxs
Lehninger 2000 Table 20.1

15. Sequential Rx in Gluconeogenesis from Pyruvate
Lehninger 2000 Table 20.2

16. Gluconeogenesis Pathway I
Berg, Tymoczko & Stryer, 2012 Fig b

17. Conversion of Pyruvate into Phosphoenolpyruvate
Pyruvate carboxylase
Pyruvate + CO2 + ATP + H2O oxaloacetate + ADP + Pi + 2H+
The bypass of glycolysis’ pyruvate kinase reaction requires two separate reactions in gluconeogenesis.
One of the anaplerotic reactions that is used to maintain the levels of intermediates in the citric acid cycle
G’ = -2.1 kJ mol -1
Gluconeogenesis
Voet, Voet & Pratt 2013 Fig 16.16

18. Synthesis or PEP from Pyruvate I
Gluconeogenesis
Lehninger 2000 Fig 203a

19. Pyruvate Carboxylase: Rx Mechanism
Pyruvate Carboxylase catalyzes the ATP-driven synthesis of oxaloacetate from pyruvate and HCO3-. This reaction occurs in two phases.
Phase I is a three step reaction sequence. Biotin is carboxylated at its N1’ position by a bicarbonate ion.
N1’
Phase II in this phase the activated carboxyl group is transferred to pyruvate from carboxybiotin in a three step reaction sequence to form oxaloacetate.
Gluconeogenesis
Voet , Voet & Pratt 2008 Fig 16.18

20. Domain Structure of Pyruvate Carboxylase
The ATP grasp domain activates HCO3- and transfers CO2 to the biotin-binding domain. From there the CO2 is transferred to pyruvate generated in the central domain.
Gluconeogenesis
Berg, Tymoczko & Stryer, 2012 Fig

21. Pyruvate Carboxylase’s Biotin-binding Domain
Key feature: biotin is on a flexible tether, attached to the -amino group of lysine, allowing it to move between the ATP-bicarbonate site and the pyruvate site.
Gluconeogenesis
Berg, Tymoczko & Stryer, 2012 Fig

22. Carboxybiotin Gluconeogenesis Voet, Voet & Pratt 2013 Fig 16.17b
Berg, Tymoczko & Stryer, 2012 Fig
Gluconeogenesis
Voet, Voet & Pratt 2013 Fig 16.17b

23. Opposing Pathways of Gluconeogenesis and Glycolysis II
Lehninger 2000 Fig 20.2b

24. Conversion of Oxaloacetate into Phosphoenolpyruvate
phosphoenolpyruvate carboxykinase
Oxaloacetate + GTP Phosphoenolpyruvate + CO2 + GDP
G’ = 2.9 kJ mol -1
Gluconeogenesis

25. Synthesis or PEP from Pyruvate II
Oxaloacetate is converted to phosphoenolpyruvate in the cytosol by PEP carboxykinase in a reaction that requires Mg2+ and GTP as the phosphoryl donor.
Voet, Voet & Pratt 2013 Figure 16.19
Gluconeogenesis

26. PEP Carboxykinase: Rx Mechanism
A monomeric, 74 kD enzyme that catalyzes a GTP-driven decarboxylation of oxaloacetate to form PEP and GDP
The CO2 that carboxylates pyruvate to synthesize oxaloacetate is eliminated in the formation of PEP.
Gluconeogenesis
Voet & Voet Biochemistry 1995 Fig. 21.5

27. Inside the mitochondrion oxaloacetate is reduced to malate in order to be transported outside the mitochondrion
Once the malate is transported outside (via the malate - -ketoglutarate shuttle) it is re-oxidized to oxaloacetate
The Mitochondrion Supplies Malate made from Pyruvate for use in Gluconeogenesis in the Cytosol
Note: the starting molecule is pyruvate.
Gluconeogenesis
Berg, Tymoczko & Stryer, 2012 Fig

28. Alternate Paths from Pyruvate to PEP
This oxaloacetate is converted directly to PEP by a mitochondrial isozyme of PEP carboxykinase
Pathway predominant when the starting molecule is pyruvate
When lactate is the precursor this pyruvate to PEP bypass is predominant
Gluconeogenesis
Lehninger 2000 Fig 20.4

29. PEP & Oxaloacetate Transport Cytosol  Mitochondria
PEP has a direct transporter, whereas oxaloacetate does not and must be converted for transmembrane passage.
Route 2 involves the conversion of oxaloacetate to malate AND involves NADH.
Route 1 uses aspartate amino transferase. In this case oxaloacetate is converted to aspartate for transport.
PEP is transported between the mitochondrion and the cell cytosol by a specific membrane transporter so it can move between the two compartments depending on the equilibrium.
Voet, Voet & Pratt 2013 Figure 16.20

30. Sequential Reactions in Gluconeogenesis (Reprise)
Lehninger 2000 Table 20.2

31. Gluconeogenesis Pathway II
Berg, Tymoczko & Stryer, 2012 Fig a

32. Conversion of Fructose 1,6-bisphosphate into Fructose -6P and PPi: An Irreversible Step
Fructose 1,6-biphosphatase
Fructose 1,6-bisphosphate + H2O fructose 6-phosphate + Pi
G’ = kJ mol -1
Gluconeogenesis

33. Conversion of Glucose-6-phosphate into Glucose: An Irreversible Step
Glucose-6-phosphatase
Glucose-6-phosphate + H2O Glucose + Pi
G’ = kJ mol -1
Gluconeogenesis

34. Glucose-6-Phosphatase
This enzyme is found primarily in the endoplasmic reticulum of liver cells. – Why?

35. Metabolic Control of Glycolysis and Gluconeogenesis
In cells gluconeogenesis and glycolysis are coordinated such that one is mainly inactive while the other in highly active. (If both were highly active at the same time the result would be a FUTILE CYCLE consuming two ATP and two GTP per reaction cycle).
The rate of glycolysis is typically controlled by the glucose concentration.
The rate of gluconeogenesis is typically controlled by the concentrations of lactate and other glucose precursors.

36. Reciprocal Regulation of Gluconeogenesis and Glycolysis in Liver
IMPORTANT!!
Berg, Tymoczko & Stryer, 2012 Fig
Gluconeogenesis
Voet, Voet & Pratt Fig ; 16.23

37. “Subtrate Cycle” can amplify metabolic signals and produce heat.
Gluconeogenesis
Berg, Tymoczko & Stryer, 2012 Fig

38. Metabolic Burden Sharing
Different organs/tissues in the body can be metabolically linked.
Lactate produced by active skeletal muscle and erythrocytes is an energy source for other organs.
Skeletal muscle during vigorous exercise produces pyruvate at a rate faster than oxidative metabolism via the citric acid cycle can use it.
Also NADH production is more rapid than its conversion to NAD+ in aerobic metabolism.
(Remember that glycolysis needs NAD+ for glyceraldehyde 3-P oxidation to proceed.)
The cell can oxidize NADH to NAD+ in a reaction that converts pyruvate to lactate via lactate dehydrogenase. The problem is that lactate can not be further metabolized, only converted back to pyruvate.
Important: Muscle’s ability to reduce pyruvate to lactate is a way shift the metabolic burden under high stress to other organs, e.g. liver, heart. (Reaction also regenerates NAD+ in muscle)

39. Cori Cycle (an interorgan metabolic “pathway”)
Lactate & pyruvate diffuse out of active muscle into blood.
Excess lactate in the blood enters the liver where it is converted to pyruvate and then to glucose via gluconeogenesis.
Gluconeogenesis: Cori Cycle
Berg, Tymoczko & Stryer, 2012 Fig
Horton et al., Fig

40. Coordination of Glycolysis and Gluconeogenesis
In liver and kidney glucose is produced by gluconeogenesis and can go out into the blood to be used by other tissues such as muscle.
In muscle alanine is formed by a transamination reaction from pyruvate. Whereas the reverse process occurs in the liver. This cycling helps to maintain the nitrogen balance
(Remember: alanine is also a major glucose precursor)
Muscles and red blood cells can produce lactate which, as we have seen in the Cori cycle can travel to the e.g. liver where it is metabolized to pyruvate, etc
Gluconeogenesis
Berg, Tymoczko & Stryer, 2002 Fig

41. Opposing Pathways of Gluconeogenesis and Glycolysis I
Lehninger 2000 Fig 20.2a

42. End of Lectures