BIOCHEMISTRY

1. GLUCONEOGENESIS
V.S.RAVIKIRAN, MSc.

2. V.S.RAVIKIRAN, MSc.,
Department of Biochemistry,
ASRAM Medical college,
Eluru-534005.AP, India.
vsravikiran2013@gmail.com

3. GLUCONEOGENESIS
The de novo synthesis of glucose and its role in
preventing hypoglycemia

4. Gluconeogenesis occurs mainly in liver.
Gluconeogenesis occurs to a more limited extent in
kidney & small intestine under some conditions.
Synthesis of glucose from pyruvate utilizes many of
the same enzymes as Glycolysis.
Three Glycolysis reactions have such a large
negative ∆G that they are essentially irreversible.
 Hexokinase (or Glucokinase)
 Phosphofructokinase
 Pyruvate Kinase.
These steps must be bypassed in
Gluconeogenesis.
Two of the bypass reactions involve simple

5. Gluconeogenesis: Overview

6. General Features
 Tissues:
liver (80%)
kidneys (20%)
 Subcellular location of
enzymes
pyruvate carboxylase:
mitochondrial
glucose-6-phosphatase:
ER
all other enzymes
cytoplasmic

7. Pathway of Gluconeogenesis
The distinctive reactions and
enzymes of this pathway are shown
in red.
The other reactions are common to
glycolysis. The enzymes for
gluconeogenesis are located in the
cytosol, except for pyruvate
carboxylase (in the mitochondria)
and glucose 6-phosphatase
(membrane bound in the
endoplasmic reticulum).
The entry points for lactate,
glycerol, and amino acids are
shown.

8. Compartmental Cooperation
Oxaloacetate utilized in the cytosol
for gluconeogenesis is formed in
the mitochondrial matrix by
carboxylation of pyruvate.
Oxaloacetate leaves the
mitochondrion by a specific
transport system (not shown) in the
form of malate, which is reoxidized
to oxaloacetate in the cytosol.

9. Generation of Glucose from Glucose 6-Phosphate
Several endoplasmic reticulum (ER) proteins play a role in the generation of
glucose from glucose 6-phosphate. T1 transports glucose 6-phosphate into
the lumen of the ER, whereas T2 and T3 transport Pi and glucose,
respectively, back into the cytosol. Glucose 6-phosphatase is stabilized by a
Ca2+-binding protein (SP).

10. Glyceraldehyde-3-phosphate
Dehydrogenase
Phosphoglycerate Kinase
Enolase
PEP Carboxykinase
glyceraldehyde-3-phosphate
NAD+
+ Pi
NADH + H+
1,3-bisphosphoglycerate
ADP
ATP
3-phosphoglycerate
Phosphoglycerate Mutase
2-phosphoglycerate
H2O
phosphoenolpyruvate
CO2 + GDP
GTP
oxaloacetate
Pi + ADP
HCO3
−
+ ATP
pyruvate
Pyruvate Carboxylase
Gluconeogenesis
Summary of
Gluconeogenesis
Pathway:
Gluconeogenesis
enzyme names in
red.
Glycolysis enzyme
names in blue.

11. Glucose-6-phosphatase
Fructose-1,6-bisphosphatase
glucose Gluconeogenesis
Pi
H2O
glucose-6-phosphate
Phosphoglucose Isomerase
fructose-6-phosphate
Pi
H2O
fructose-1,6-bisphosphate
Aldolase
glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate
Triosephosphate
Isomerase
(continued)

12. Malate Shuttle
 OAA produced in
mitochondria
 mitochondrial membrane
impermeable to OAA
 malate transporter in mito.
Membrane
 malate dehydrogenase in
both mito and cyto
 NADH produced in cyto
also used in
gluconeogenesis.

15. Energetics of Gluconeogenesis
Pyruvate
Carboxylase
 2 ATPs
PEP Carboxykinase
 2 GTPs
3-P-glycerate kinase
 2 ATPs
Glyceraldehyde-3-P
dehydrogenase

16. Glycolysis & Gluconeogenesis are both spontaneous.
If both pathways were simultaneously active in a cell, it would
constitute a "futile cycle" that would waste energy.
Glycolysis:
glucose + 2 NAD+
+ 2 ADP + 2 Pi

2 pyruvate + 2 NADH + 2
ATP
Gluconeogenesis:
2 pyruvate + 2 NADH + 4 ATP + 2 GTP 
glucose + 2 NAD+
+ 4 ADP + 2 GDP + 6 Pi
Questions:
1. Glycolysis yields how many ~P ?
2. Gluconeogenesis expends how many ~P ?
3. A futile cycle of both pathways would waste how many
~P per cycle ?
2
6
4

17. Precursors for gluconeogenesis
Glycerol
derived from adipocyte lipolysis
hepatic glycerol kinase

18. Precursers for gluconeogenesis
Lactate
RBC
muscle
the Cori Cycle

19. The lactic acid (Cori) and glucose-alanine cycles.

20. Cori Cycle
Liver Blood Muscle
Glucose Glucose
2 NAD+
2 NAD+
2 NADH 2 NADH
6 ~P 2 ~P
2 Pyruvate 2 Pyruvate
2 NADH 2 NADH
2 NAD+
2 NAD+
2 Lactate 2 Lactate

21. Precursers for gluconeogenesis
Alanine and other amino acids
 transamination of pyruvate
 pyruvate derived from glycolysis or from amino acid degradation
 alanine cycle

22. Hexokinase or Glucokinase (Glycolysis) catalyzes:
glucose + ATP  glucose-6-phosphate +
ADP
Glucose-6-Phosphatase (Gluconeogenesis)
catalyzes:
glucose-6-phosphate + H2
O  glucose + Pi
H O
OH
H
OHH
OH
CH2OH
H
OH
HH O
OH
H
OHH
OH
CH2OPO3
2−
H
OH
H
H2O
1
6
5
4
3 2
+ Pi
glucose-6-phosphate glucose
Glucose-6-phosphatase

23. H O
OH
H
OHH
OH
CH2OH
H
OH
HH O
OH
H
OHH
OH
CH2OPO3
2−
H
OH
H
H2O
1
6
5
4
3 2
+ Pi
glucose-6-phosphate glucose
Glucose-6-phosphatase
Glucose-6-phosphatase enzyme is embedded in the
endoplasmic reticulum (ER) membrane in liver cells.
The catalytic site is found to be exposed to the ER lumen.
Another subunit may function as a translocase, providing
access of substrate to the active site.

24. Phosphofructokinase (Glycolysis) catalyzes:
fructose-6-P + ATP  fructose-1,6-bisP + ADP
Fructose-1,6-bisphosphatase (Gluconeogenesis)
catalyzes:
fructose-1,6-bisP + H2
O  fructose-6-P + Pi
fructose-6-phosphate fructose-1,6-bisphosphate
Phosphofructokinase →
CH2OPO3
2−
OH
CH2OH
H
OH H
H HO
O
6
5
4 3
2
1 CH2OPO3
2−
OH
CH2OPO3
2−
H
OH H
H HO
O
6
5
4 3
2
1
ATP ADP
Pi H2O
←Fructose-1,6-biosphosphatase

25. Bypass of Pyruvate Kinase:
Pyruvate Kinase (last step of Glycolysis)
catalyzes:
phosphoenolpyruvate + ADP  pyruvate
+ ATP
For bypass of the Pyruvate Kinase reaction, cleavage of 2
~P bonds is required.
 ∆G for cleavage of one ~P bond of ATP is insufficient to
drive synthesis of phosphoenolpyruvate (PEP).
 PEP has a higher negative ∆G of phosphate hydrolysis
than ATP.

26. Bypass of Pyruvate Kinase (2 enzymes):
Pyruvate Carboxylase (Gluconeogenesis) catalyzes:
pyruvate + HCO3
−
+ ATP  oxaloacetate + ADP +
Pi
PEP Carboxykinase (Gluconeogenesis) catalyzes:
oxaloacetate + GTP  PEP + GDP + CO2
C
C
CH2
O O−
OPO3
2−
C
C
CH3
O O−
O
ATP ADP + Pi C
CH2
C
C
O
O O−
O−
O
HCO3
−
GTP GDP
CO2
pyruvate oxaloacetate PEP
Pyruvate Carboxylase PEP Carboxykinase

27. Biotin has a 5-C side chain whose terminal
carboxyl is in amide linkage to the ε-amino
group of an enzyme lysine.
The biotin & lysine side chains form a long
swinging arm that allows the biotin ring to
swing back & forth between 2 active sites.
Pyruvate
Carboxylase
uses biotin
as prosthetic
group.
CHCH
H2C
S
CH
NH
C
HN
O
(CH2)4 C NH (CH2)4 CH
CO
NH
O
biotin
N subject to
carboxylation
lysine
residue
lysine

28. Biotin carboxylation is catalyzed at one active site of
Pyruvate Carboxylase.
ATP reacts with HCO3
−
to yield carboxyphosphate.
The carboxyl is transferred from this ~P intermediate to N of
a ureido group of the biotin ring. Overall:
biotin + ATP + HCO3
−
 carboxybiotin + ADP + Pi
carboxyphosphate
CHCH
H2C
S
CH
NH
C
N
O
(CH2)4 C NH (CH2)4 CH
CO
NH
O
C
O
-O
carboxybiotin
lysine
residue

29. At the other
active site of
Pyruvate
Carboxylase the
activated CO2 is
transferred from
biotin to
pyruvate:
carboxybiotin
+ pyruvate

biotin +
oxaloacetate
CHCH
H2C
S
CH
NH
C
N
O
(CH2)4 C NH R
O
C
O
-OC
C
CH3
O O−
O
C
CH2
C
C
O
O O−
O−
O
CHCH
H2C
S
CH
NH
C
HN
O
(CH2)4 C NH R
O
carboxybiotin
pyruvate
oxaloacetate
biotin

30. PEP Carboxykinase catalyzes GTP-dependent
oxaloacetate → PEP. It is thought to proceed in 2 steps:
 Oxaloacetate is first decarboxylated to yield a pyruvate
enolate anion intermediate.
 Phosphate transfer from GTP then yields
phosphoenolpyruvate (PEP).
C
C
CH2
O O−
OPO3
2−
C
CH2
C
C
O
O O−
O−
O
CO2
C
C
CH2
O O−
O−
GTP GDP
oxaloacetate PEP
PEP Carboxykinase Reaction

31. Coordinated Regulation of
Gluconeogenesis and Glycolysis
Gluconeogenesis and
Glycolysis are regulated by
similar effector molecues but in
the opposite direction
avoid futile cycles
PK vs PC & PEPCK
PFK-1 vs FDP’tase
GK vs G6P’tase

32. Coordinated Regulation of
Gluconeogenesis and Glycolysis
Regulation of enzyme
quantity
 Fasting: glucagon, cortisol
 induces gluconeogenic
enzymes
 represses glycolytic enzymes
 liver making glucose
 Feeding: insulin
 induces glycolytic enzymes
 represses gluconeogenic
enzymes
 liver using glucose

34. Coordinated Regulation of
Gluconeogenesis and Glycolysis
 Short-term Hormonal Effects
 Glucagon, Insulin
 cAMP & F2,6P2
 PFK-2 & FBPase-2
 A Bifunctional enzyme
 cAMP
 Inactivates PFK-2
 Activates FBPase-2
 Decreases F2,6P2
 Reduces activation of PFK-1
 Reduces inhibition of FBPase-1
 Low blood sugar results in
 Hi gluconeogenesis
 Lo glycolysis

35. Coordinated Regulation of
Gluconeogenesis and Glycolysis
Allosteric Effects
 Pyruvate kinase vs Pyruvate carboxylase
PK - Inhibited by ATP and alanine
PC - Activated by acetyl CoA
Fasting results in gluconeogenesis
 PFK-1 vs FBPase-1
FBPase-1 inhibited by AMP & F2,6P2
PFK-1 activated by AMP and & F2,6P2
Feeding results in glycolysis

36. Reciprocal Regulation of Gluconeogenesis and Glycolysis in the Liver
The level of fructose
2,6-bisphosphate is
high in the fed state
and low in starvation.
Another important
control is the inhibition
of pyruvate kinase
by phosphorylation
during starvation.

37. Pathway of Gluconeogenesis
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