Transcript GLUCONEOGENESIS, GLYCOGEN SYNTHESIS & DEGRADATION

MIA KUSMIATI
Departemen BIOKIMIA FK UNISBA
 The
stimulation of gluconeogenesis by high
energy charge and high concentrations of
citrate and acetyl-CoA is counterintuitive.
 Gluconeogenesis is active in the fasting
state.
 the energy for gluconeogenesis is supplied
by fatty acid oxidation.
 During overnight fast: 90 % gluconeogenesis
hepar, 10% gluconeogenesis kidney
 Prolonged fasting: kidney becomes major
glucose producing organ (40% total glucose
production)
Synthesis of glucose from pyruvate utilizes many of
the same enzymes as Glycolysis.
Three Glycolysis reactions have such a large
negative DG 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
hydrolysis reactions.
 (1),
Glucokinase
 (2), phosphofructokinase;
 (3), pyruvate kinase;
 (4), pyruvate carboxylase;
 (5), phosphoenolpyruvate
 (PEP)-carboxykinase;
 (6), fructose-1,6-bisphosphatase;
 (7), glucose-6-phosphatase
STIMULATION
 INHIBITION
A, Substrate flow during fasting and in the
well-fed state, and the effects of hormones
on the amounts of glycolytic and
gluconeogenic enzymes.
Regulation of enzyme synthesis and
degradation is the most important long-term
(hours to days) control mechanism. In most
cases, the hormone acts by changing the rate
of transcription (insulin)
 B,
Short-term regulation of glycolysis and
gluconeogenesis by reversibly binding
effectors and by:
- Phosphorylation/dephosphorylation
- Allosteric and competitive effects
- phosphorylation.
Only pyruvate kinase and phosphofructo-2kinase/fructose-2,6-bisphosphatase are
regulated by cAMP-dependent
phosphorylation.
 Synthesis
and degradation of fructose-2,6bisphosphate, the most important
regulator of phosphofructokinase and
fructose-1,6-bisphosphatase.
 This regulatory metabolite is synthesized
and degraded by a bifunctional enzyme
that combines the kinase and phosphatase
activities on the same polypeptide.
 cAMP-induced
phosphorylation inhibits the
kinase activity and stimulates the
phosphatase activity of the bifunctional
enzyme.
, Phosphorylation;
, dephosphorylation;
, allosteric effect;
, stimulation;
, inhibition
Lactat
Pyruvate
Glycerol
Αlfa
keto acid (oxaloacetat, a
ketoglutarat)
7 glycolytic Rx are irreversible & are used
in the synthesis of glucose from lactat or
pyruvate:
Carboxylation of pyruvate:
A.
biotin is a coenzyme

Allosteric regualtion
B. Transport of oxaloacetate to the cytosol
C. Decaboxylation of cytosolic oxaloacetate
D. Dephosporilation of Fructose 1,6 biP  fructose
6P
E. Isomerisasi Fructose 6P Glucose 6P
F. Convert glucose 6P  free glucose

Glucose-6-phosphatase
6 CH OPO 2
2
3
5
O
H
4
OH
H
OH
3
H
H
2
CH2OH
1
OH
OH
glucose-6-phosphate
O
H
H
H2O
H
OH
H
+ Pi
H
OH
OH
H
OH
glucose
Hexokinase or Glucokinase (Glycolysis)
catalyzes:
glucose + ATP  glucose-6-phosphate + ADP
Glucose-6-Phosphatase (Gluconeogenesis)
catalyzes:
glucose-6-phosphate + H2O  glucose + Pi
Glucose-6-phosphatase
6 CH OPO 2
2
3
5
O
H
4
OH
H
OH
3
H
H
2
CH2OH
1
OH
OH
glucose-6-phosphate
O
H
H
H2O
H
OH
H
+ Pi
H
OH
OH
H
OH
glucose
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.
Phosphofructokinase 
6 CH OPO 2
2
3
O
5
H
H
4
OH
6 CH OPO 2
2
3
1CH2OH
ATP
HO
3 OH
H
fructose-6-phosphate
O
ADP
2
5
Pi
H2O
1CH2OPO32
H
H
HO
3 OH
4
OH
2
H
fructose-1,6-bisphosphate
 Fructose-1,6-biosphosphatase
Phosphofructokinase (Glycolysis) catalyzes:
fructose-6-P + ATP  fructose-1,6-bisP + ADP
Fructose-1,6-bisphosphatase (Gluconeogenesis)
catalyzes:
fructose-1,6-bisP + H2O  fructose-6-P + Pi
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.

DG for cleavage of one ~P bond of ATP is insufficient to
drive synthesis of phosphoenolpyruvate (PEP).

PEP has a higher negative DG of phosphate hydrolysis than
ATP.
Pyruvate Carboxylase
PEP Carboxykinase
O
O
O
O
C
ATP ADP + Pi
C
C
C
O
CH3
C
O
pyruvate
O
GTP GDP
CH2
HCO3
O
O
C
CO2
O
oxaloacetate
C
OPO 32
CH2
PEP
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
Pyruvate Carboxylase
PEP Carboxykinase
O
O
O
O
C
ATP ADP + Pi
C
C
C
O
CH3
GTP GDP
O
C
C
CO2
C
O
pyruvate
O
CH2
HCO3
O
O

oxaloacetate
OPO 32
CH2
PEP
Contributing to spontaneity of the 2-step process:
Free energy of one ~P bond of ATP is conserved in
the carboxylation reaction.
Spontaneous decarboxylation contributes to
spontaneity of the 2nd reaction.
Cleavage of a second ~P bond of GTP also
contributes to driving synthesis of PEP.
Pyruvate
Carboxylase
uses biotin
as prosthetic
group.
N subject to
carboxylation
O
C
HN
NH
CH CH
H2C
CH
S
lysine
biotin
H
H3N+
C
CH2
CH2
CH2
CH2

NH3
COO
lysine
residue
(CH2)4
O
O
C
NH
C
(CH2)4 CH
NH
Biotin has a 5-C side chain whose terminal
carboxyl is in amide linkage to the e-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.
O
O

O
P
-O
O
O
C
O
OH
carboxyphosphate
C
C
O
N
NH
lysine
residue
CH CH
H2C
CH
S
(CH2)4
O
O
C
NH
C
(CH2)4 CH
carboxybiotin
NH
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
of a ureido group of the biotin ring. Overall:
biotin + ATP + HCO3  carboxybiotin + ADP + Pi
N
At the other
active site of
Pyruvate
Carboxylase the
activated CO2 is
transferred from
biotin to
pyruvate:
carboxybiotin
+ pyruvate

biotin +
oxaloacetate
O
O
O
-O
C
C
C
C
O
CH3
pyruvate
O
N
H2C
O
CH
S
(CH2)4
C
NH
R
O
C
C
C
O
CH2
HN
NH
biotin
CH CH
H2C
C
O
carboxybiotin
CH CH
O
O
NH
O
oxaloacetate
CH
S
(CH2)4
O
C
NH R
Pyruvate
Carboxylase
(pyruvate 
oxaloactate)
is allosterically
activated by
acetyl CoA.
[Oxaloacetate]
tends to be
limiting for Krebs
cycle.
Glucose-6-phosphatase
glucose-6-P
glucose
Gluconeogenesis
Glycolysis
pyruvate
fatty acids
acetyl CoA
oxaloacetate
ketone bodies
citrate
Krebs Cycle
When gluconeogenesis is active in liver, oxaloacetate is
diverted to form glucose. Oxaloacetate depletion hinders
acetyl CoA entry into Krebs Cycle. The increase in [acetyl
CoA] activates Pyruvate Carboxylase to make oxaloacetate.
 The
main stores of glycogen in the body:
1. Liver to mantain the blood glucose
level
2. Skeletal muscleto serve as a fuel
reserve for synthesis of ATP during
muscle contraction
 Glycogen
is a branched polymer of between
10,000 and 40,000 glucose residues held
together by α-1,4 glycosidic bonds
 Glucose-6-phosphate
is isomerized to
glucose-1-phosphate by
phosphoglucomutase.
 Glucose-1-phosphate then reacts with uridine
triphosphate (UTP) to form UDP-glucose.
 UDP is attached to C-1 of glucose, and it is
therefore this carbon that forms the
glycosidic bond. The bond between glucose
and UDP is energy rich

Metabolic fates of glycogen in the liver (A) and in muscle
(B). Note that the liver possesses glucose-6-phosphatase,
which forms free glucose both in gluconeogenesis and from
glycogen. This enzyme is not present in muscle tissue.
 Glycogen
breakdown serves different
purposes in liver and muscle.
 The liver synthesizes glycogen after a
carbohydrate meal and degrades it to free
glucose during fasting.
 The glucose-6-phosphate from glycogen
breakdown is cleaved to free glucose by
glucose-6-phosphatase.
 The liver releases this glucose into the blood
for use by needy tissues, including brain and
blood cells
 Skeletal
muscle synthesizes glycogen at rest
and degrades it during exercise.
 Muscles cannot produce free glucose because
they have no glucose-6-phosphatase.
 Because glycogen degradation produces
glucose-6-phosphate without consuming any
ATP, anaerobic glycolysis from glycogen
produces three rather than two molecules of
ATP for each glucose residue.
The phosphorylation state of the enzymes is
regulated by hormones and their second
messengers.
1. Insulin stimulates glycogen synthesis both in
the liver and in skeletal muscle. It ensures that
excess carbohydrate is stored away as glycogen
after a meal.
2. Glucagon stimulates glycogen degradation in
liver but not muscle during fasting when the
blood glucose level is low.
3. Norepinephrine and epinephrine are powerful
activators of glycogen breakdown both in
muscle and liver. They mobilize glycogen when
glucose is needed to fuel muscle contraction.

 A,
Hormonal effects on the phosphorylation
of the glycogen-metabolizing enzymes by
protein kinases in the liver. ER, endoplasmic
reticulum; GSK3, glycogen synthase kinase-3;
 B, Hormonal effects on the
dephosphorylation of the glycogenmetabolizing enzymes by protein
phosphatase-1, and the effects of allosteric
effectors.
 Note
that the hormones affect glycogen
synthase and glycogen phosphorylase through
the protein kinases and the protein
phosphatase (phosphatase-1) that regulate
their phosphorylation state. , Allosteric
effects; , phosphorylation; ,
dephosphorylation; , activation; , inhibition.
