Chapter-15 Takusagawa’s Note

©
1
Chapter-15: Glycogen Metabolism

- Glycogen is the animal storage form of branched poly(glucose).

- Processes of glycogen breakdown is:
(glucose)
n
→ glucose-1-phosphate +(glucose)
n-1


- Processes of glycogen synthesis is:
(glucose)
n-1
+UDP-glucose → (glucose)
n


- Regulation of glycogen breakdown and synthesis:
is controlled by two key enzyme (glycogen phosphorylase and glycogen synthase) activities
which are activated/inactivated by allosteric regulation and phosphorylation /
dephosphorylation.

1. Glycogen breakdown
Glycogen structure
- Glucose molecules in the main chains are connected by α(1→4) glycosidic bonds.
- The branches are attached by α(1→6) glycosidic linkages.
- A glucose unit on the non-reducing ends is cleaved or attached one by one.

O
OH
OH
CH
2
O
O
OH
OH
CH
2
OH
O
OH
OH
CH
2
OH
HO O O
CH
2
OH
OH
OH
O
O
CH
2
OH
OH
OH
OH
O
O
O
OH
HO
CH
2
OH
O
O
OH
OH
CH
2
OH
O
OH
OH
CH
2
OH
HO O
Non-reducing
ends
Reducing end
α(14) linkage
α(16) linkage
Branch
point


Different pathways of glycogen breakdown
- In muscle: Glycogen → glucose-6-phosphate (G6P) → glycolysis
- In liver: Glycogen → G6P → glucose → bloodstream → various cells → glycolysis
- Because the muscle cells mainly consume glucose molecules whereas the liver cells mainly
store the glucose molecules.


1
Chapter-15 Takusagawa’s Note
©
2
Glycogen breakdown requires three enzymes
1. Glycogen phosphorylase (simply call it phosphorylase)
(Glycogen)
n
+ P
i
↔ (glycogen)
n-1
+ G1P
(n residues) (n-1 residues)
This enzyme releases a glucose unit one by one until it reaches ~five units (limit branch)
from a branch point.
- The enzyme has a crevice where 4-5 units of a left-handed helical glycogen can fit, but it is
too narrow to fit a branch point.
O


- Enzyme-catalyzed modification/demodification process yields two forms of phosphorylase
Phosphorylase a --- E-O-PO
3
2-
(attachment at Ser-14).
Phosphorylase b --- No phosphate attachment.
- Both forms of enzyme are allosterically activated or inactivated.
- Allosteric inhibitors: ATP, G6P and glucose.
- Allosteric activator: AMP, [F2,6P].

Pyridoxal phosphate (PLP) is an essential cofactor for phosphorylase
- PLP covalently bound to phosphorylase via a Schiff base to Lys-679.
- PLP’s phosphate group probably functions as an acid-base catalyst.
PLP bound to Lys 679
via Shiff base
Pyridoxal phosphate (PLP)
Phosphorylase
N
CH
OH
CH
3
H
C O P O
O
-
O
-
H
H
N
(CH
2
)
4
+
+
N
C
O H
OH
CH
3
H
C O P O
O
-
O
-
H
H


2
Chapter-15 Takusagawa’s Note
©
3
Reaction mechanism
1. Formation of an [E-PL ·

P
i
·

glycogen] ternary complex.
2. Cleavage of the glycosidic bond by an acid catalysis (the P
i
donates H to the bridge O
because the P
i
receives H from PL), and formation of oxonium ion intermediate (half-chair
conformation).
3. Reaction of P
i
with the oxonium ion to form G1P.

Half-chair oxonium ion
O
+
OH
HO
HO
CH
2
OH
H
O
-
P
O
- O
O
PL
E
O
-
P
O
O
O
-
H
H
O
P
O
- O
O
PL
E
O
P
O
O
-
O
H
HO
CH
2
OH
HO
OH O
O
(Glu)
n-2
O
O
OH
HO
HO
CH
2
OH
H
(Glu)
n-2
O
O
OH
HO
CH
2
OH O
P
O
O
-
O
-
H
CH
2
OH
HO
HO
OH
O
O
P
O
- O
O
PL
E
H

Acid catalysis
Glycogen +P
i
+Enzyme
Ternary complex

3
Chapter-15 Takusagawa’s Note
©
4
2. Glycogen debranching enzyme
- Removes branches so that glycogen phosphorylase can complete reaction.

Reaction mechanism
1. Three glucose units of the branch are cleaved and reattached to the non-reducing end of the
main chain.
2. The branch point, the glucose attached to the main chain by the α(1-6) glycosidic bond is
hydrolyzed.

G1P G
G1P G1P G1P
G1P
G1P G1P G1P G1P
G1P G1P G1P
G1P G1P G1P
Limit branch

G1P
Free glucose

4
Chapter-15 Takusagawa’s Note
©
5
3. Phosphoglucomutase
- G1P produced from the glycogen breakdown must be convert to G6P in order to enter
glycolysis or to produce glucose in liver.
- Phosphoglucomutase catalyzes the conversion of G1P to G6P.
- The Ser of the enzyme is phosphorylated.

Reaction mechanism
1. The O6 of G1P attacks the phosphoenzyme to form a dephosphoenzyme-G1,6P intermediate.
2. The Ser-OH group on the dephosphoenzyme attacks the phosphoryl group at C1 to
regenerate the phosphoenzyme, and the product G6P is released.

G6P G1,6P
G1P
O
OH
OH
OH
HO
CH
2
OPO
3
2-
Ser
H
2
C O PO
3
2-
Enzyme
O
OPO
3
2-
OH
OH
HO
CH
2
OPO
3
2-
Ser
H
2
C O H
Enzyme
Enzyme
Ser
H
2
C O PO
3
2-
O
OPO
3
2-
OH
OH
HO
CH
2
OH










2. GLYCOGEN SYNTHESIS
- Biosynthetic and degradative pathways of metabolism are almost always different.
Glucose
Glycogen
Glycogen synthesis
Glycogen breakdown







5
Chapter-15 Takusagawa’s Note
©
6
A. UDP-glucose formation by UDP-glucose pyrophosphorylase
- In the glycogen synthesis pathway, at first, the uridine diphosphate (UDP) is attached to
glucose.
- This reaction is catalyzed by UDP-glucose pyrophosphorylase.

Reaction mechanism
1. The phosphoryl oxygen of G1P attacks the α-phosphorus atom of UTP to form UDP-glucose
(ΔG°

≈ 0). Since the ΔG°

≈ 0, this reaction is not proceeded without some input energy.
2. The released PP
i
is rapidly hydrolyzed by inorganic pyrophosphatase (ΔG°

<0).
Since this hydrolysis reaction is exergonic, the first phosphorylation reaction is pulled.
ΔG°’ (kJ /mol)
G1P +UTP ↔ UDP-glucose +PP
i
~0.0
H
2
O +PP
i
→ 2P
i
-33.5
Overall G1P +UTP → UDP-glucose +2P
i
-33.5









6
Chapter-15 Takusagawa’s Note
©
7
B. Glycogen synthesis by glycogen synthase
Reaction mechanism
1. The glycosidic bond between glucose and UDP in UDP-glucose is hydrolyzed. The cleaved
glucose ion takes the oxonium ion intermediate (half-chair conformation), which is stabilized
by the enzyme.
2. The glucose unit of UDP-glucose is transferred to the C4-OH group on one of glycogen’s
non-reducing ends to form an α (1→4) glycosidic bond.



- UDP is recycled by conversion to UTP with ATP.
UDP +ATP ↔ UTP +ADP

Thermodynamics
- ΔG of glycogen breakdown by glycogen phosphorylase is <0 (ΔG ≈ -5 to -8 kJ /mol).
(glucose)
n
+P
i
⎯→ G1P +(glucose)
n-1

- ΔG of glycogen synthesis by glycogen synthase is <0 (ΔG ≈ -14 kJ /mol).
(glucose)
n-1
+UDP-glucose ⎯→ (glucose)
n

- Thus both reactions takes place spontaneous under the same physiological condition.
- However, glycogen synthesis uses one ATP hydrolysis per G1P for energy source.

1. G1P +UTP ⎯→ UDP-glucose +P
i

2. UDP-glucose +(glucose)
n-1
⎯→ (glucose)
n
+UDP
3. UDP +ATP ⎯→ UTP +ADP
_____________________________________________________________________________________
G1P +(glucose)
n-1
+ATP ⎯→ (glucose)
n
+ADP +P
i


7
Chapter-15 Takusagawa’s Note
©
8
C. Glycogen Branching
- About 7 units of the non-reducing end of α-amylose chain are removed at α(1→4) linkage,
and reattached to the C6 of other α-amylose chain by α(1→6) linkage.
- This transfer is carried out by amylo-(1,4→1,6)-transglycosylase (branching enzyme).



Note:
- ΔG of hydrolysis of α(1→4) glycosidic bond is -15.5 kJ /mol.
- ΔG of hydrolysis of α(1→6) glycosidic bond is -7.1 kJ /mol.
- In the branching process, hydrolysis of the α(1→4) glycosidic bond (ΔG =-15.5 kJ /mol)
derives the formation of the α(1→6) glycosidic bond (ΔG =+7.1 kJ /mol).
1. α(1→4) glycosidic bond hydrolysis ΔG =-15.5 kJ /mol
2. α(1→6) glycosidic bond formation ΔG =+7.1 kJ /mol
ΔG =-8.4 kJ /mol
- Since the reverse reaction (debranching) is endergonic (ΔG >0), the debranching pathway
must be different.
- In the debranching process, the breaking and reforming the α(1→4) glycosidic bond are
energetically canceling each other, and the α(1→6) bond hydrolysis (ΔG =-7.1 kJ /mol)
drives the debranching.
1. α(1→4) glycosidic bond hydrolysis ΔG =-15.5 kJ /mol
2. α(1→4) glycosidic bond formation ΔG =+15.5 kJ /mol
3. α(1→6) glycosidic bond hydrolysis ΔG = -7.1 kJ /mol
ΔG = -7.1 kJ /mol

8
Chapter-15 Takusagawa’s Note
©
9
3. CONTROL OF GLYCOGEN METABOLISM
A. Allosteric control of glycogen phosphorylase and glycogen synthase
Glycogen phosphorylase
- has two forms (a and b), and each form is activated allosterically from T-form to R-form.
- Phosphorylase b is the non-phosphorylated enzyme and its R-form is less active, but its
response is fast since its activation signals (activators) are come from the inside of cell.
- Phosphorylase a is the Ser-14 phosphorylated enzyme and its R-form is the most active
enzyme, but its response is slow since its activation signals (effectors) are come from the
outside of cell.
- The enzyme is phosphorylated and dephosphorylated by phosphorylase kinase and
phosphoprotein phosphatase, respectively. The phosphorylation is called “covalent
modification”.
- Phosphorylase kinase and phosphoprotein phosphatase are enzyme modificator and
demodificator, respectively.
- Unmodified form (phosphorylase b) is mostly T-form, whereas modified form
(phosphorylase a) is mostly in the R-form.


Allosteric
regulators

Highly active
form

- Glycogen synthase has the same forms of phosphorylase, i.e., glycogen synthase a and
b, and their T- and R-forms.
-
The allosteric activators and inhibitors of phosphorylase and synthase are listed below.
Enzyme Activators Inhibitors
Glycogen phosphorylase AMP ATP, G6P
Glycogen synthase G6P


9
Chapter-15 Takusagawa’s Note
©
10
Let us consider how these enzymes are activated or deactivated.
- At high demand of ATP, i.e., low [ATP], low [G6P] and high [AMP]: Glycogen
phosphorylase is stimulated and glycogen synthase is inhibited, so flux through this pathway
favors the glycogen breakdown.
- At high [ATP] and [G6P]: Glycogen synthesis is favored.

B. Covalent modification of enzymes by cyclic cascades: Effector “Signal” amplification
- As described above, two enzymes (phosphorylase kinase and phosphoprotein phosphatase)
are involved in the modification of phosphorylase.
- The phosphorylation is the most effective activation process of phosphorylase.
- Let us assume that one molecule of phosphorylase kinase can phosphorylate many
phosphorylase molecules (say 10
6
molecules). Then each phosphorylated phosphorylase
molecule catalyzes the glycogen breakdown by 10
6
times.
- Thus the one activation of the kinase molecule is translated into 10
12
glycogen breakdown
reactions, i.e., production of 10
12
glucose molecules.
- These processes are called enzyme cascade.
- There are several cycles of enzyme cascades. Monocycle enzyme cascade is shown below.

Monocycle cascade
F
+
e
1 F
e
1
Less
active
More
active
E
b
E
a
OH
P
More
active
Less
active
e
2
R
e
2 +
R
ATP
ADP
H
2
O
P
i
K
1
K
2
More
active
Less
active

- F and R are kinase and phosphatase (hydrolase), respectively.
- e
1
and e
2
are allosteric effectors of F and R, respectively, and those quantities are very little.
- Activated F·e
1
and R·e
2
can catalyze the phosphorylation and dephosphorylation reactions in
many times.
- Thus, the effector’s effects are amplified very much.
- Thus, amplification potential of a “signal” of e
1
or e
2
is enormous.

10
Chapter-15 Takusagawa’s Note
©
11
Bicyclic enzyme cascade
Activation process:
1. Effector e
1
activates F
1
by forming a F
1
·e
1
complex.
2. The activated F
1
·e
1
phosphorylates F
2b
, and converts it to an active F
2a
.
3. The activated F
2a
phosphorylates E
b
, and converts it to an active E
a
.
4. The activated E
a
catalyzes the reaction such as glycogen breakdown.

Deactivation process:
1. Effector e
2
activates R
1
by forming an R
1
·e
2
complex.
2. The activated R
1
·e
2
hydrolyzes (dephosphorylates) the active F
2a
, and converts it to an
inactive F
2b
.
3. Similarly effector e
3
activates R
2
by forming an R
2
·e
3
complex.
4. The activated R
2
·e
3
hydrolyzes (dephosphorylates) the active E
a
, and converts it an inactive E
b
.
+ e
1 e
1
F
2b
F
2a
OH
P
e
2
e
2
+
ATP
ADP
H
2
O
P
i
K
1
K
2
K
3
P
i
H
2
O
ADP
ATP
+
e
3 e
3
P
OH
E
a
E
b
R
1
F
1 F
1
R
1
P
R
2 R
2

In the glycogen metabolism (bicycle enzyme cascade), the corresponding symbols are:
P =PO
3
2-
; e
1
=cAMP; F
1
=cAMP-dependent protein kinase; F
2
=Phosphorylase kinase;
E =Glycogen phosphorylase; [R
1
·e
2
and R
2
·e
3
=Phosphoprotein phosphatase-1].

11
Chapter-15 Takusagawa’s Note
©
12
C. Glycogen phosphorylase bicyclic cascade
- Epinephrine binds its receptor on the cell surface. Its signal is transferred to a G-protein.
- The activated G-protein (G
α
) activates the adenylate cyclase (AC).
- The activated AC catalyzes the cAMP formation from ATP.
- cAMP (effector e
1
) activates cAMP-dependent protein kinase (cAPK).

- Insulin activates an insulin-stimulated protein kinase.
- The activated insulin-stimulated protein kinase activates phosphoprotein phosphatase-1 (PP-
1).
- PP-1 dephosphorylates the phosphorylated proteins to inactivate them except for glycogen synthase
which is activated by PP-1.

12

Glycogen breakdown favor pathway
Glycogen synthesis favor pathway
ATP
Other
kinases
Ca
2
+
P
P
Phosphoprotein
phosphatase
inhibitor-1 a
Phosphoprotein
phosphatase
inhibitor-1 b
ADP
ATP
H
2
O P
i
Phosphoprotein
phosphatase
inhibitor-1 a
Phosphoprotein
phosphatase-1
(inactive)
Phosphoprotein
phosphatase-1
(active)
H
2
O P
i
ADP
ATP
Glycogen-P
synthase b
Glycogen
synthase a
H
2
O P
i
ADP
ATP
Glycogen
phosphorylase b
Ser 14-CH
2
O-P
Glycogen
phosphorylase a
Ser 14-CH
2
OH
H
2
O P
i
ADP
ATP
P P
(α β γ δ)
4
Phosphorylase
kinase a
Phosphorylase
kinase b
(α β γ δ)
4
R
2
C
2
2C
+ R
2
(cAMP)
4
cAMP-dependent
protein kinase
(active)
+ 4cAMP
cAMP-dependent
protein kinase
(inactive)
Epinephrin
Adenylate cyclase
Insulin
insulin-stimulated
protein kinase
Note: Phosphorylation
activates phosphorylase
but inactivates synthase

Chapter-15 Takusagawa’s Note
©
13
Adenosine-3’,5’-cyclic monophosphate (cAMP)
- The primary intracellular signal e
1
is adenosine-3’,5’-cyclic monophosphate (cAMP) in both
glycogen phosphorylase and glycogen synthase cascades.
N
N
N
N
O
OH
O
C
P
O
H
NH
2
H
H
H
O
-
O
ATP
PP
i
Adenylate
cyclase
H
2
O
phosphodiesterase
AMP

- cAMP is absolutely required for the activity of cAMP-dependent protein kinase (cAPK).
- cAPK phosphorylates specific Ser and/or Thr residues of numerous cellular proteins.
- These protein have cAPK’s consensus recognition sequence, Arg-Arg-X-Ser/Thr-Y (X:
small residue; Y: Large hydrophobic residue).
- cAPK is tetramer composed of two regulatory and two catalytic subunits, R
2
C
2
.
- cAMP binds to the regulatory subunits → dissociate active catalytic monomers (2C).
- Protein kinases play key roles in the signaling pathways by hormones, growth factors,
neurotransmitters and toxins.

Phosphorylase kinase
- is activated by Ca
2+
(as low as 10
-7
M) and by covalent modification.
- is (αβγδ)
4
.
- The γ subunit has full catalytic activity (ability to convert phosphorylase b to phosphorylase a).
- The α, β, and δ subunits are inhibitors of the catalytic reaction.
- The phosphorylation of α and β-subunits reduces the inhibitory activities of α and β-
subunits.
- The δ subunit is calmodulin (CaM), which is a ubiquitous eukaryotic Ca
2+
-binding protein.
- When Ca
2+
binds to CaM’s 4 Ca
2+
-binding sites, CaM undergoes an extensive
conformational change that activates phosphorylase kinase. Actually,
- Ca
2+
binds to the Ca
2+
-binding domain → conformational change exposes a hydrophobic
patch → CaM binds to the CaM-binding domain of phosphorylase kinase γ subunit at the
hydrophobic patch section → binding of CaM activates phosphorylase kinase (also other
protein kinases).
Active
Inactive
hydrophobic section
P
P
δ
γ
β
α
Ca
2+
ADP ATP
cAPK
P
P
Hydrophobic
patch
Inactive
α
β
γ
δ
α
β
γ
δ


13
Chapter-15 Takusagawa’s Note
©
14
X-ray and NMR studies on the structure of CaM indicate that CaM changes its structure
significantly when it binds to the specific oligopeptide of the γ-subunit of phosphorylase
kinase.




14
Chapter-15 Takusagawa’s Note
©
15
Muscle contraction processes
Initial muscle contraction by nerve impulses → release Ca
2+
from reservoir to cytosol → Ca
2+

bind to CaM → activate phosphorylase kinase → phosphorylate (activate) glycogen
phosphorylase → glycogen breakdown → ATP synthesis → ATP is used for contraction of
muscle.

Phosphoprotein phosphatase-1 (PP-1)
- catalyzes hydrolytic dephosphorylation (remove the phosphate group by hydrolysis).
- is only active when it is bound to glycogen through its glycogen-binding G-subunit.
- has two phosphorylation sites.
1. Activation site (P1) by insulin-stimulated protein kinase.
2. Deactivation site (P2) by cAPK. When the P2 site is phosphorylated by cAPK, PP-1 is
released from G-subunit. The PP-1 itself is inactive.

Insulin and epinephrine have antagonistic effects on glycogen metabolism.




- PP-1 is also inhibited by phosphoprotein phosphatase inhibitor 1 (protein).
- Phosphoprotein phosphatase inhibitor 1 is an active inhibitor protein when it is
phosphorylated by cAPK.
- Thus, cAMP concentration controls not only the phosphorylation activity (stimulation) but
also dephosphorylation activity (suppression).

15
Chapter-15 Takusagawa’s Note
©
16
D. Glycogen synthase
- is inactivated by the bicycle enzyme cascade.
1. cAMP activates cAMP-dependent protein kinase.
2. cAMP-dependent protein kinase phosphorylates phosphorylase kinase a.
3. Phosphorylase kinase a phosphorylates glycogen synthase a. The phosphorylation on
glycogen synthase inactivates it, glycogen synthase b is inactive phosphorylated form.
- is activated by phosphoprotein phosphatase-1 which dephosphorylates the glycogen synthase
(b → a).

E. Integration of glycogen metabolism control mechanisms
- The rates of glycogen breakdown and synthesis largely depend on the rate of the
phosphorylation and dephosphorylation reactions of the two bicyclic cascades.
- Phosphorylation and dephosphorylation are controlled by hormones.

Hormones trigger glycogen metabolism through the intermediacy of second messengers.
In liver: Hormone glucagon controls glycogen metabolism (stimulate glycogen breakdown).
- Glucagon is a 29 amino acid polypeptide.

In muscle and various tissues: Insulin and epinephrine (adrenaline) and norepinephrine
(noradrenaline) control glycogen metabolism.

- Insulin is a polypeptide hormone. Inactive
form, proinsulin is a single peptide of 84
amino acid residues, while the active mature
hormone is two chains (30 and 21 amino
acid residues) connected by two S-S bonds.



X = CH
3
Epinephrin
X = H Norepinephrin
HO
HO
C
H
C
H
OH
H
H N
H
X



16
Chapter-15 Takusagawa’s Note
©
17
- Hormonal stimulation at their plasma membranes occurs through the mediation of
transmembrane protein, receptor (review Chapter 21b).
Cytosol
Receptor
Second messenger
Hormone
Cell membrane

- When a hormone binds at the receptor, the second messenger is released into cytosol.
- cAMP, Ca
2+
, inositol-1,4,5-triphosphate (IP
3
), diacylglycerol (DG), and NO are second
messengers.
- Hormonal stimulation by glucagon or epinephrine increases the intracellular [cAMP] →
increase cAPK activity → increases the rate of phosphorylation of many proteins and
decreases the rate of dephosphorylation.
- Cyclic cascades amplify the small change of [cAMP] to a large change in the fraction of
enzymes in their phosphorylated forms.
- Hormonal stimulation by insulin increases the phosphoprotein phosphatase-1 (PP-1) activity.
- PP-1 dephosphorylates various proteins.
- Thus epinephrine and insulin are antagonistic relation.

F. Maintenance of blood glucose levels
- Need to maintain glucose concentration at ~5 mM in blood.
When the [glucose] in blood is low:
1. Glucagon is secreted from pancreas into bloodstream.
2. Glucagon binds at glucagon receptor on the liver cells and activates adenylate cyclase,
thereby increasing intracellular [cAMP].
3. The [cAMP] increase triggers an increase in the rate of glycogen breakdown, leading to
increase intracellular [G6P].
4. Since G6P cannot pass through the cell membrane and is not major energy source of liver,
G6P is hydrolyzed to glucose by glucose-6-phosphatase. The resulting glucose enters the
blood stream.

When blood glucose level is high (immediate after meal)
1. Glucagon level decrease.
2. Insulin is secreted from pancreatic β cells.
- In muscle,
- Insulin stimulates the glucose transporter on the muscle cells. Thus, glucose in bloodstream
enters into the cells.
- Insulin stimulates insulin-stimulated protein kinase which phosphorylates G-subunit of
phosphoprotein phosphatase-1 (PP-1).
- The phosphorylated PP-1 dephosphorylates glycogen synthase b to increase glycogen
synthesis, and also dephosphorylates glycogen phosphorylase a to decrease glycogen
breakdown.

17
Chapter-15 Takusagawa’s Note
©
18
- In liver,
- Glucose in blood enters freely into the liver cells when the [glucose] is high in bloodstream.
- Glucose itself inhibits glycogen phosphorylase a (R state) and shifts equilibrium to glycogen
phosphorylase b (T state). [Phosphorylase A → Phosphorylase B]
- Phosphoprotein phosphatase inhibitor-1 is dephosphorylated and is released from PP-1.
- PP-1 activates glycogen synthase and deactivates glycogen phosphorylase a. Therefore, the
liver can store the excess of glucose as glycogen.
Phosphorylase a
Glycogen synthase a
Enzyme activity in mouse liver
Time after glucose infusion (min)
0
2 4 6 8


How does liver work as a buffer of blood [glucose]?
- Liver does not have hexokinase, but glucokinase.
- Hexokinase (that is in tissues other than in liver) obeys Michaelis-Menten kinetics, and is
inhibited by product G6P.
- Glucokinase (that is in liver) displays sigmoidal kinetics, and is not inhibited by G6P at
physiological concentration.
- The K
M
of hexokinase is much smaller than K
0.5
of glucokinase, indicating that hexokinase
has stronger glucose affinity than glucokinase, i.e., hexokinase is good for glycolysis.
K
M
(Hexokinase) <<K
0.5
(Glucokinase), i.e., 0.2 mM <<5 mM.



[Glucose] in blood <5 mM: Liver releases glucose (G6P→ Glucose)
[Glucose] in blood >5 mM: Liver stores glucose (Glucose → G6P)


18
Chapter-15 Takusagawa’s Note
©
19
Liver cells are freely permeable to glucose, but other cells are not
- Thus, at low [glucose] in bloodstream, liver releases glucose whereas at high [glucose] in
bloodstream, liver cells receive glucose and convert it to glycogen since glucose freely
permeable to liver.

Other [glucose] regulator, β-D-fructose-2,6-bisphosphate (F2,6P)
- F2,6P is an extremely potent allosteric activator of PFK and inhibitor of FBPase.
- Thus, the [glucose] is regulated by the [F2,6P].
β-D-Fructose-2,6-bisphosphate
(F2,6P)
O
O
CH
2
OH
HO
OH
CH
2
O
-2
O
3
P
PO
3
2-


- F2,6P is formed from fructose-6-phosphate (F6P) catalyzed by phosphofructokinase-2 (PFK-2)
and is hydrolyzed to F6P by fructose bisphosphatase-2 (FBPase-2).

Phosphofructokinase-2 (PFK-2) and Fructose bisphosphatase-2 (FBPase-2)
- The 100kD homodimeric protein carries both PFK-2 and FBPase-2 activities which are
located on different domains.
PFK-2
FBPase-2
Phosphorylation sites

- Isozymes in liver and heart muscle have the phosphorylation site, whereas the isozyme in
skeletal muscle does not have the phosphorylation site.


19
Chapter-15 Takusagawa’s Note
©
20
Liver isozyme
- Phosphorylation by cAPK effects:
- Inhibition of PFK-2 activity. ⎬ F2,6P is hydrolyzed → [F2,6P]↓
- Activation of FBPase-2 activity. ⎭
- Thus, increase FBPase activity and decrease PFK since the allosteric activator F2,6P is
not available to activate PFK and to inhibit FBPase.
- Therefore, the glycolysis is inversed, and G6P is produced. Then G6P hydrolysis
produces free glucose that is secreted into bloodstream.

FBPase-2
FBPase
PFK-2
PFK
[F1,6P]↓ [F6P]↑ [F2,6P]↓
G6P
G1P
Glycogen synthesis pathway
Glycogen Glucose Bloodstream


Heart muscle isozyme
- Phosphorylation by cAPK effects:
- Activation of PFK-2 activity ⎬ F2,6P is produced → [F2,6P]↑
- Inhibition of FBPase-2 activity ⎭
- Thus, increase PFK activity and decrease FBPase since F2,6P activates PFK and inhibits
FBPase.
- Therefore, the glycolysis pathway is activated.

Glycogen
G1P G6P
[F2,6P]↑
[F6P]↓ [F1,6P]↑
PFK
PFK-2
FBPase
FBPase-2
Glycolysis pathway
Glycolysis


- The phosphorylation of PFK-2/FBPase-2 in heart muscle activates PFK-2 rather than inhibits
PFK-2 as seen in liver.



Increase glycogen breakdown effects:
- In liver: increase glucose secretion into bloodstream, and decrease glycolysis.
- In muscle: increase glycolysis.

In skeletal muscle isozyme, there is no phosphorylation site. Thus, there is no subject to cAMP-
dependent phosphorylation control.

20
Chapter-15 Takusagawa’s Note
©
21
G. Response to stress
- Epinephrine and norepinephrine are released into the bloodstream by adrenal glands in
response to stress.
- also stimulate the pancreatic α cells to secrete glucagon.
- These hormones bind to:
- β-adrenergic receptors, stimulates adenylate cyclase, and produces cAMP.
- α-adrenergic receptors, stimulates phospholipase C, and produces inositol-1,4,5-triphosphate
(IP
3
), diacylglycerol (DG), and Ca
2+
. These second messengers act as reinforce the cells’
response to cAMP.


Muscle cells do not have
glucagons receptors.
← Liver Cell




21
Chapter-15 Takusagawa’s Note
©
22
4. GLYCOGEN STORAGE DISEASE

Enzyme deficiency Reaction Symptoms
Glucose-6-phosphatase G6P → glucose [G6P]↑ in liver, [glucose]↓ in blood
α-1,4-Glucosidase maltose → 2 glucose Large accumulation of glycogen
Amylo-1,6-glucosidase branch glycogen → glucose Accumulation of abnormal short
chain glycogen
Amylo-(1,4→1,6)-
transglycosylase
glucose → branch glycogen Very long unbranched glycogen
Glycogen phosphorylase
(muscle):
glycogen → G6P Inability of glycogen breakdown
Glycogen phosphorylase
(liver):
glycogen → G6P

Inability of glycogen breakdown
Phosphofructokinase F6P → FBP (F1,6P)

Accumulation of G6P and F6P
Phosphorylase kinase glycogen breakdown Inability to convert phosphorylase b
to a
Glycogen synthase UDP-glucose → glycogen Inability to synthesize glycogen in
liver






22
Chapter-15 Takusagawa’s Note
©
23
D. Mechanism of passive-mediated glucose transport
- Erythrocyte glucose transporter is a 55 KD protein, which is composed of 12 transmembrane helices.
- The helices form a bundle whose interior is hydrophilic and exterior is hydrophobic.
- Glucose is transport through the channel.



23
Chapter-15 Takusagawa’s Note
©
24
Glucose transport occurs via a gated pore mechanism (Erythrocytes, Liver cells)
- When glucose transports across the erythrocyte membrane, no other molecule or ion are accompanied.
- Simply glucose moves from high concentration side to low concentration side.


Insulin-sensitive exocytosis/endocytosis controls the glucose transport (Fat and muscle cells)
- Cellular glucose uptake is regulated through the insulin-
sensitive exocytosis/endocytosis of the vesicles
containing the glucose transports.
- When the [glucose] in blood is high, insulin is secreted
from pancreas.
- Insulin stimulates the exocytosis activity of the vesicle
that contains the glucose transporters.
- The glucose transporters are moved on the membrane.
- Glucose in blood is taken up and stored in cells.

- When the [glucose] in blood is low, the glucose
transporters are moved into the cell by endocytosis so that
glucose in cells cannot flow out.

- Thus glucose uptake is regulated by population of glucose
transporters.










24
Chapter-15 Takusagawa’s Note
©


25
25
A. Na
+
-Glucose symport (Small intestine)
- Glucose is absorbed from lumen of small intestine into brush border cells by Na
+
concentration gradient.
- Actually, the Na
+
is pumped out by (Na
+
-K
+
)-ATPase from the brush border cell to create the Na
+
gradient.



- Na
+
-glucose symport system is a random Bi Bi kinetic mechanism.
Note: most antiport systems are ordered sequential kinetic mechanism.