Lecture 23
– Quiz next Mon. on Pentose Phosphate
Pathway
– Metabolic regulation and control of
glycolysis/gluconeogenesis
Hydrolytic reactions bypass PFK and
Hexokinase
Page 602
Instead of generating ATP by reversing the glycolytic
reactions, FBP and G6P are hydrolyzed to release Pi in an
exergonic reaction.
Page 848
Glycolysis
Glucose + 2ADP + 2Pi + 2NAD+
Gluconeogenesis
2 Pyruvate + 4ATP + 2GTP
2NADH + 4H+ + 6H2O
2 Pyruvate + 2ATP
+ 2NADH + 4H+ +
2H2O
Glucose + 4ADP
+2GDP + 6Pi + 2NAD+
Net reaction
2ATP + 2GTP + 4H2O
2ADP + 2GDP + 4Pi
Control Points in
Glycolysis
1st reaction of glycolysis (Gº’ = -4 kcal/mol)
HO
O
5
4

6
1
OH
*

2
HO
3
OH
Hexokinase (HK) I, II, II
Muscle(II), Brain (I)
Glucose
OH
Glucokinase (HK IV)
in liver
ATP Mg2+
ADP Mg2+
-2O
3P-O
6
O
5
4
OH
2
HO
3
OH

1
*

OH
Glucose-6-phosphate
(G6P)
Regulation of Hexokinase
• Glucose-6-phosphate is an allosteric inhibitor of
hexokinase.
• Levels of glucose-6-phosphate increase when
downstream steps are inhibited.
• This coordinates the regulation of hexokinase with
other regulatory enzymes in glycolysis.
• Hexokinase is not necessarily the first regulatory step
inhibited.
Types of regulation
1. Availability of substrate Glucokinase (KM 12 mM) vs.
HK (KM = 0.01 - 0.03 mM)
2. Compartmentalization -Brain vs. Liver vs. Muscle
(type I mitochondrial membrane, type II cytoplasmic)
3. Allosteric regulation - feedback inhibition by G-6-P,
overcome by Pi in type I (Brain/ mitochondrial controlled
by Pi levels)
4. Hormonal regulation. Liver has HK as fetal tissue.
Changes to glucokinase after about 2 weeks. If there is
no dietary carbohydrate, no glucokinase. Must have
both insulin and carbohydrates to induce.
2 places where there is no net reaction
PFK
1. ATP + F-6-P
2. F-1,6-P2
Net: ATP
Mg2+
F-1,6-P2 + ADP
F-phosphatase
F-6-P + Pi
Mg2+
ADP + Pi + heat
Similar reaction occurs with hexokinase and G-6-phosphatase.
Generally regulated so this does not occur (futile cycle).
May function in hibernating animals to generate heat.
Primary regulation - reciprocal with energy charge
Enzyme
+
Hexokinase
PFK
F-6phosphatas
e
Pyruvate
kinase
Pyruvate
carboxylase
G-6-P
Pi, ADP,
AMP, F-6-P,
F-2,6-P2
ATP
ATP, citrate,
NADH
K+, AMP, F2,6-P2
Acetyl-CoA
ATP, acetylCoA, cAMP
AMP, F-2,6P2
Major regulation is through energy charge
ATP
ATP
ADP
Gluconeogenesis
Glycolysis
Same reactions make AMP or ADP (primarily in lipid and
nucleotide metabolism)
Adenylate kinase
AMP + ATP
Energy charge
2 ADP
[ATP] +1/2[ADP]
[AMP] + [ADP] + [ATP]
1.0 = 100% ATP Body generally likes it close to 0.9
0.5 = 100% ADP
0 = 100% AMP
Control Points in
Glycolysis
Primary regulation - reciprocal with energy charge
Enzyme
+
Hexokinase
PFK
F-6phosphatas
e
Pyruvate
kinase
Pyruvate
carboxylase
G-6-P
Pi, ADP,
AMP, F-6-P,
F-2,6-P2
ATP
ATP, citrate,
NADH
K+, AMP, F2,6-P2
Acetyl-CoA
ATP, acetylCoA, cAMP
AMP, F-2,6P2
Regulation of PhosphoFructokinase (PFK-1)
• PKF-1 has quaternary structure
• Inhibited by ATP and Citrate
• Activated by AMP and Fructose-2,6bisphosphate
• Regulation related to energy status of cell.
PFK-1 regulation by adenosine
nucleotides
• ATP is substrate and inhibitor. Binds to active site and
allosteric site on PFK. Binding of ATP to allosteric site
increase Km for ATP
• AMP and ADP are allosteric activators of PFK.
• AMP relieves inhibition by ATP.
• ADP decreases Km for ATP
• Glucagon (a pancreatic hormone) produced in
response to low blood glucose triggers cAMP
signaling pathway that ultimately results in decreased
glycolysis.
Effect of ATP on PFK-1 Activity
Effect of ADP and AMP on PFK-1 Activity
Regulation of PFK by
Fructose-2,6-bisphosphate
• Fructose-2,6-bisphosphate is an allosteric activator of
PFK in eukaryotes, but not prokaryotes
•Formed from fructose-6-phosphate by PFK-2
•Degraded to fructose-6-phosphate by fructose 2,6bisphosphatase.
•In mammals the 2 activities are on the same enzyme
•PFK-2 inhibited by Pi and stimulated by citrate
Fructose-2,6-bisphosphate can override Energy charge
Produced when [glucose] is high but need glycolysis for
anabolic role.
When glucose is needed by the brain (about 120 g/day via
diet or other tissues)
Glucose
F-6-P
3 PGA cAMP PFK-2 Citrate+
Bifunctional enzyme
F-2,6-P2
ATP PFK-1
F-2,6-Pase cAMP +
F-6-P +
NTP +
AMP +
F-1,6-Pase
F-2,6-P2 +
F-1,6-P2
AMPCitrateF-2,6-P2PEP -
Glucagon Regulation of PFK-1 in Liver
•PFK-1 normally inhibited by
ATP
•G-Protein mediated cAMP
signaling pathway
•Induces protein kinase A
that activates phosphatase
activity and inhibits kinase
activity
•Results in lower F-2,6-P
levels decrease PFK-1
activity (less glycolysis)
PFK-2
1. Serves to override ATP inhibition and promote
glycolysis once intermediates build up [citrate]
[PEP][GAP]
2. Block PFK-2 activity with high [NTP] by stimulating F2,6-Pase
This will break down F-2,6-P2 and restores energy charge
regulation.
cAMP is the hormonal control. The presence of cAMP is
indicative of low blood sugar (glucagon) stimulates F2,6-Pase to increase F-6-P formtion for
gluconeogenesis (cAMP also inhibits Pyruvate Kinase).
Regulation of Pyruvate Kinase
• Allosteric enzyme
• Activated by Fructose-1,6-bisphosphate (example of
feed-forward regulation)
• Inhibited by ATP
• When high fructose 1,6-bisphosphate present plot of
[S] vs Vo goes from sigmoidal to hyperbolic.
• Increasing ATP concentration increases Km for PEP.
• In liver, PK also regulated by glucagon. Protein
kinase A phosphorylates PK and decreases PK
acitivty.
Pyruvate Kinase Regulation
Deregulation of Glycolysis
in Cancer Cells
• Glucose uptake and glycolysis is 10X faster in solid
tumors than in non-cancerous tissues.
• Tumor cells initally lack connection to blood supply so
limited oxygen supply
• Tumor cells have fewer mitochondrial, depend more
on glycolysis for ATP
• Increase levels of glycolytic enzymes in tumors
(oncogene Ras and tumor suppressor gene p53
involved)
Glycogen biosynthesis
Most important storage form of sugar
Glycogen - highly branched (1 per 10) polymer of glucose
with (1,4) backbone and (1,6) branch points. More
branched than starch so more free ends.
Average molecular weight -several million in liver, muscle.
1/3 in liver (more concentrated but less overall mass (5-8%)),
2/3 in muscle (1%).
Not found in brain - brain requires free glucose (120 g/ day)
supplied in diet or from breakdown of glycogen in the liver.
Glucose levels regulated by several key hormones - insulin,
glucagon.
Page 627
Figure 18-1a Structure of glycogen. (a)
Molecular formula.
Page 627
Figure 18-1bStructure of glycogen.
(b) Schematic diagram illustrating its
branched structure.
Glycogen is an efficient storage form
UDP-glucose
G-6-P
G-1-P + UTP
UDP + ATP
Glycogen + UDP + Pi
UTP + ADP
Net: 1 ATP required
90% 1,4 residues Glycogen + Pi
10% 1,6 residues
Glycogen
G-1-P
G-6-P
glucose
1.1 ATP/38 ATP so, about a 3% loss, therefore it is about 97%
efficient for storage of glucose
Glycogen biosynthesis
3 enzymes catalyze the steps involved in glycogen
synthesis:
UDP-glucose pyrophosphorylase
Glycogen synthase
Glycogen branching enzyme
Glycogen biosynthesis
MgATP
Glucose
HK
MgADP
[G-1,6-P2]
G-6-P
F-6-P
G-1-P
phosphoglucomutase
PGI
The hydrolysis of
pyrophosphate to
inorganic phosphate
is highly exergonic
and is catalyzed by
inorganic
pyrophosphatase
PPase
UTP
G-1-P
PPi
UDP-Glucose Pyrophosphorylase
2Pi
Page 633
Figure 18-6 Reaction catalyzed by UDP–glucose
pyrophosphorylase.
UDP-Glucose pyrophosphorylase
Coupling the highly exergonic cleavage of a nucleoside
triphosphate to form PPi is a common biosynthetic strategy.
The free energy of the hydrolysis of PPi with the NTP
hydrolysis drives the reaction forward.
Glycogen synthase
In this step, the glucosyl unit of UDP-glucose (UDPG) is
transferred to the C4-OH group of one of glycogen’s
nonreducing ends to form an (1,4) glycosidic bond.
Involves an oxonium ion intermediate (half-chair
intermediate)
Each molecule of G1P added to glycogen regenerated
needs one molecule of UTP hydrolyzed to UDP and Pi.
UTP is replenished by nucleoside diphosphate kinase
UDP + ATP
UTP + ADP
Figure 18-7 Reaction catalyzed by glycogen synthase.
Page 633
O
Glycogen synthase
All carbohydrate biosynthesis occurs via UDP-sugars
Can only extend an already (1,4) linked glucan change.
First step is mediated by glycogenin, where glucose is
attached to Tyr 194OH group.
The protein dissociates after glycogen reaches a minimum
size.
Glycogen branching
Catalyzed by amylo (1,41,6)-transglycosylase (branching
enzyme)
Branches are created by the terminal chain segments
consisting of 7 glycosyl residues to the C6-OH groups of
glucose residues on another chain.
Each transferred segment must be at least 11 residues.
Each new branch point at least 4 residues away from other
branch points.
Page 634
Figure 18-8
The branching of glycogen.
Glycogen Breakdown
Requires 3 enzymes:
1. Glycogen phosphorylase (phosphorylase) catalyzes
glycogen phosphorylysis (bond cleavage by the
substitution of a phosphate group) and yields glucose-1phosphate (G1P)
2. Glycogen debranching enzyme removes glycogen’s
branches, allowing glycogen phosphorylase to complete
it’s reactions. It also hydrolyzes a(16)-linked glucosyl units
to yield glucose. 92% of glycogen’s glucse residues are
converted to G1P and 8% to glucose.
3. Phosphoglucomutase converts G1P to G6P-can either
go through glycolysis (muscle cells) or converted to
glucose (liver).
Glycogen Phosphorylase
A dimer - 2 identical 842 residue subunits.
Catalyzes the controlling step of glycogen breakdown.
Regulated by allosteric interactions and covalent modification.
Two forms of phosphorylase made by regulation
Phosphorylase a- has a phosphoryl group on Ser14 in each
subunit.
Phosphorylase b-lacks the phosphoryl groups.
Inhibitors: ATP, G6P, glucose
Activator: AMP
Glycogen forms a left-handed helix with 6.5 glucose residues
per turn.
Structure can accommodate 4-5 sugar residues only.
Pyridoxal phosphate is an essential cofactor for
phosphorylase.
Converts glucosyl units of glycogen to G1P
Page 628
Figure 18-2a X-Ray structure of rabbit muscle glycogen
phosphorylase. (a) Ribbon diagram of a phosphorylase
b subunit.
Page 630
Phosphoglucomutase
Converts G1P to G6P.
Reaction is similar to that of phosphoglycerate mutase
Difference between phosphoglycerate mutase and
phosphoglucomutase is the amino acid residue to which
the phosphoryl group is attached.
Serine in phosphoglucomutase as opposed to His imidazole
N in phosphoglycerate mutase.
G1,6P occasionally dissociates from the enzyme, so catalytic
amounts are necessary for activity. This is supplied by the
enzyme phosphoglucokinase.
Page 631
Figure 18-4 The mechanism of action of
phosphoglucomutase.
Glycogen debranching enzyme
(14) transglycosylase (glycosyl transferase) transfers a
(14) linked trisaccharide unit from a limit branch to a
nonreducing end of another branch.
Forms a new (14) linkage with three more units available
for phosphorylase.
The (16) bond linking the remaining linkage is hydrolyzed
by the same enzyme to yield glucose.
2 active sites on the same enzyme.
Page 631
Figure 18-5 Reactions catalyzed by debranching
enzyme.
Regulation of glycogen synthesis
Both synthase & phosphorylase exist in two forms.
Phosphorylated at Ser residues by synthase kinase and
phosphorylase kinase
Synthase a
Normal form
“active”
Pi
OH
OH
phosphoprotein
phosphatase
ATP
Synthase kinase
ADP
Synthase b
Requires G6P for activation
“inactive”
OP
OP
Regulation of glycogen synthesis
AMP+, ATP-, G6PPhosphorylase b
Normal form
“inactive”
Pi
phosphoprotein
phosphatase
OH
OH
ATP
phosphorylase
kinase
Ca2+
ADP
Phosphorylase a
OP
Independent of energy status
OP
active
High [ATP] (related to high G6P) inhibits phosphorylase
and stimulates glycogen synthase.
Regulation of glycogen synthesis
Process is also under hormonal control
Adrenaline (epinephrine) can regulate glycogen
synthesis/breakdown by stimulating adenylate cyclase
ATP
1. External stimulus
Adrenaline
Adenylate
cAMP
cyclase
2. R2C2
cAMP dependent
protein kinase
“inactive”
[C]2 + [R-AMP]2
“active”
ATP
ADP
Glycogen synthase b (inactive)
3a. Glycogen synthase a
[C]2
(active)
ATP
3b. Inactive
phosphorylase kinase
Phosphorylase b
(inactive)
cAMP
+ PPi
ADP
Active phosphorylase kinase
[C]2
ATP
ADP
Phosphorylase a
(active)
Consider the whole system
Resting muscle
Glycolytic pathway
pyruvate
O2
respiration
ATP
Inactive phosphorylase b, active synthase a
Muscle lacks G6 Pase, Liver PFK inhibited by ATP unless F2,6P2 present
Upon stress
Epinephrine
cAMP
Phosphorylse b
Synthase/phosphorylase kinase
Phosphorylse a
Page 863
Figure 23-25 The pentose phosphate pathway.