Carbohydrate Biosynthesis

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Nov.21, 2007
Carbohydrate Biosynthesis
Chapter 14 Gluconeogenesis Chapter 15 Principle
of Metabolic Regulation Glucose and Glycogen
Chapter 20 Carbohydrate Biosynthesis in Plant
Lectured by Dr. Qin Yongmei (???)
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Can you tell me
Why do we need glucose in our body ?
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What is significant role of glucose in the human
body ?
Tissues that synthesize glucose liver and kidney
Tissues that use glucose as their primary energy
source brain, muscle, erythrocytes and testes
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In which kind of situation do we need
gluconeogenesis?

• Normal physiology situation
• -Between meals and during sleep
• -Exercise/work
• After heavy exercise or work (recycling of
  lactate)
• After protein-rich diet (glucogenic amino acids)
• Starvation (glucogenic amino acids)

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Contents

• Gluconeogenesis
• 2. Biosynthesis of glycogen
• 3. Analysis of metabolic control
• 4. Photosynthetic carbohydrate
• synthesis (the Calvin Cycle)
• 5. Regulation of carbohydrate
• metabolism in plants.
• 6. Biosynthesis of starch and sucrose

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Overview of Human Metabolism
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Glycogen
Glucose-6-phosphate
Glucose
Ribose-5-phosphate
Pyruvate
irreversible
Lactate
Amino acids
Acetyl-CoA
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1. Gluconeogenesis (formation of new sugar)
Definition the formation of glucose from non-
carbohydrate precursors Species
all animals, plants, fungi and microorganisms Org
ans (in higher animals) occurring largely in
liver, a small amount in kidney Daily glucose
requirement (human being) 160g (120g for
brain), glycogen can provide 190g. In fasting
conditions gluconeogenesis accounts for up to
96 of total glucose production.
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Plant
Animal
Fatty acid oxidation in mammals provide an
important energy source (and carbon source
in plants and bacteria) for gluconeogenesis
Substrates for Gluconeogenesis Lactate Amino
acids Glycerol Propionate
Glycerol
Amino acids
Lactate
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1. Gluconeogenesis refers to the metabolic
pathway that results in net glucose
production 1) from fructose and galactose 2)
from pentose phosphates 3) from glycogen 4)
from starch 5) from alanine 2. The
gluconeogenetic pathway is active 1) in
liver cells, when on a low carbohydrate diet 2)
in muscle cells during intense exercise 3) in
heart cells during starvation 4) in adipocytes
during fasting 5) in kidney medulla when
circulating Glc is low 3. It is said that
animal cells cannot use fat for net glucose
biosynthesis because 1) No part whatsoever of
a triacylglyceride molecule can be converted into
Glc 2) No part of any fatty acid whatsoever can
be made into Glc 3) Acetyl CoA is completely
degraded into CO2 and water in the TCA cycle 4)
Any substrate that replenishes the TCA cycle is
completely degraded to CO2
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Glucose
Glycolysis and gluconeogenesis have 3 different
reactions and 7 common reactions.
hexokinase
Glucose-6-phosphate
Fructose-6-phosphate
PFK-1
Fructose-1,6-bisphosphate
Glyceraldehyde 3-phosphate
1,3-bisphosphoglycerate
3-bisphosphoglycerate
2-bisphosphoglycerate
phosphoenolpyruvate
pyruvate kinase
oxaloacetate
pyruvate
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glucose
gluconeogenesis
Oxaloacetate is the starting material for
glucogneonesis
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What is role of gluconeogenesis in plant ?
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Gluconeogenesis converts fats and proteins to
glucose in germinating seeds

• Active gluconeogenesis occurs in germinating
  seeds, providing glucose for the synthesis of
  sucrose
• Plants can convert acetyl-CoA derived from fatty
  acid oxidation into glucose via glyoxylate cycle
  (occurring in glyoxysomes). The physical
  separation of these glyoxylate cycle and
  ?-oxidation enzymes from mitochondrial citric
  acid cycle enzymes prevent the further oxidation
  of acetyl-CoA to CO2.

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Conversion of stored fatty acids to sucrose in
germinating seeds
Glyoxysome
The integration of reaction sequences in three
subcellular compartments is required for the
production of sucrose from stored lipids.
Mitochondrion
Cytosol
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Gluconeogenesis is not a reversal of glycolysis
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In principle

• Molecules are synthesized and degraded by
• different pathways. Even though two opposing
  
• pathways may share many reversible
  reactions,
• at least, one step is unique and
  irreversible to
• that of the opposing pathway
• Corresponding anabolic and catabolic pathways
• are controlled at one or more of the
  reactions
• unique to each pathway.
• Energy-requiring biosynthetic processes are
• coupled to energy-yielding breakdown of ATP
• in such a way that the overall process is
• essentially irreversible in vivo.

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3 Irreversible steps in glycolytic pathway
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• Conversion of pyruvate to phosphoenolpyruvate
• (two exergonic reactions)

Pyruvate ATP GTP H2O ? phosphoenolpyruvat
e ADP GDP Pi 2H
Mitochondrial enzyme
Mitochondrial enzyme/cytosolic enzyme
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What shall we learn from this reaction mechanism ?

• Free energy of cleavage of one P bond of ATP is
  conserved in the carboxylation reaction and CO2
  can be removed to power the formation of PEP in
  the decarboxylation step. .
• Decarboxylations often drive reactions otherwise
  highly endergonic This metabolic motif is used in
  the citric acid cycle, the pentose phosphate
  pathway, and fatty acid synthesis.
• Cleavage of a second P bond of GTP also
  contributes to drive synthesis of PEP.

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Carboxylation of pyruvate (anaplerotic reaction)
takes place in 3 stages
HCO3- ATP ? HOCO2PO32- ADP
Biotin-enzyme HOCO2PO32- ?
CO2-biotin-enzyme (activated
carboxyl group ) Pi CO2-biotin-enzyme
pyruvate ? biotin-enzyme oxaloacetate
Biotin is a covalently attached prosthetic group,
which serves as a carrier of activated CO2
- pyruvate carboxylase - acetyl CoA
carboxylase - propionyl CoA carboxylase
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Phase I
Phase II
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The long, flexible chain formed between biotin
and the enzyme enables this prosthetic group to
rotate from one active site of the enzyme to the
other.
Pyruvate Carboxylase
Biotin carboxyl carrier domain
carrier of activated CO2
ATP-activating domain
Avidin, a protein in egg whites tightly binds
biotin (Kd 10-15). Excess consumption of raw
eggs can cause nutritional deficiency of biotin.
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B. Cytosolic NADH is required for Gluconeogenesis
?-ketoglutarate
?-ketoglutarate
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C. Conversion of fructose 1,6-bisphosphate to
fructose 6-phosphate
?G0 -16,3 kJ/mol
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D. Conversion of glucose 6-phosphate to free
glucose
?G0 -13,8 kJ/mol
The enzyme is not present in muscle or in the
brain, and gluconeogenesis does not occur in
these tissues.
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(No Transcript)
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Coordinated regulation of gluconeogenesis and
glycolysis
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Futile cycles in carbohydrate metabolism consume
ATP
Glycolysis and gluconeogenesis pathways are both
spontaneous. If both pathways were simultaneously
active within a cell it would constitute a
futile cycle that would waste energy.
Gluconeogenesis 2Pyruvate 4ATP 2GTP 2NADH
4H2O ?glucose 4ADP 2GDP 6Pi 2NAD
2H Glycolysis Glucose 2ADP 2Pi 2NAD
? 2Pyruvate
2ATP 2NADH 2H 2H2O
A futile cycle consisting of both pathways would
waste 4 P bonds per cycle.
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• Futile cycle has regulation significance
• It is a way to turn glycolysis off and
• gluconeogenesis on when it has an adequate
• ATP supply.
• Or it is a way to turn glycolysis on and
• gluconeogenesis off when ATP is in short
• supply.

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Pasteur effect LOUIS PASTEUR (1822-1895 )
Pasteur observed that decrease in the rate
of carbohydrate breakdown that occurs in yeast
when switched from anaerobic to aerotic
conditions.
The basic phenomenon is a competition between
glycolysis and oxidative phosphorylation for the
available ADP and inorganic phosphate.
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High blood glucose, fed state Liver fuel
conservation Glycogen is synthesized Glycolytic
pathway pyruvate dehydrogenase are
activated FA biosynthesis and fat storage Low
blood glucose, fast state Glycogen
breakdown Gluconeogenesis
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Example ?G in the direction of gluconeogenesis
in liver under physiological conditions
Hexokinase (32,9)/glucose 6-phosphatase
(-5,1) PFK-1 (24,5)/FBP-1 (-8,6) Puruvate
kinase (26,4)/pyruvate carboxylase-PEPCK (-22,6)
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(1). Hexokinase isozymes of muscle and liver are
affected differently by G6P
glucose in blood 4-5 mM
(in muscle, low Km)
When blood glucose rises above 5 mM,
hexokinase IV increases, but hexokinase I is
getting saturated and cannot respond to an
increase in glucose concentration.
(in liver, sigmoid curve)
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Hexokinase IV is subject to inhibition by
reversible binding of a regulatory protein
specific to liver
During a fast, glucose below 5 mM, F6P triggers
inhibition of hexokinase IV by association with
the regulator protein. In this way, liver does
not compete with other organs for the glucose.
(compete with F6p for binding to hexokinase IV)
inhibited
(allosteric effector of hexokinase IV)
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(2). PFK-1 is under complex allosteric regulation

• PFK-1 allosteric enzyme
• Binding of allosteric inhibitor
• or activator does not effect
• Vmax, but does alter Km
• Allosteric enzyme does not
• follow M-M kinetics.

Allosteric regulation of muscle PFK-1 by ATP
allosteric site
ATP inhibits PFK-1 by binding to an allosteric
site and lowering the affinity of the enzyme
for F6P.
F1,6BP
Active site
ADP
ADP
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(3). Pyruvate kinase is allosterically inhibited
by ATP
Isozymes differ in their tissue distribution and
their response to modulators.
When low glucose causes glucagon release, PKA
phosphorylated the L form (inactivation). The
mechanism prevents the liver from consuming
glucose and spares it for other organs.
M form is not affected by this phosphorylation
mechanism.
cAMP-dependent protein kinase
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(4). Gluconeogenesis is regulated by acetyl-CoA
the fate of pyruvate

• acetyl-CoA
• activates pyruvate carboxylase (gluconeogenesis),
  
• inactivates the pyruvate
• dehydrogenase complex
• (glycolysis).

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glucose 6- phosphatase
hexokinase
Fructose 1,6-bisphosphatase (FBPase-1)
phosphofructokinase (PFK-1)
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(5). F2, 6BP is a potent regulator of glycolysis
and gluconeogenesis
F2,6BP has opposite effects on the enzymatic
activities of PFK-1 and FBPase.
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Fructose-2,6-bisphosphate is synthesized and
degraded by PFK2/FBPase2
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PFK2/FBPase2 is a Bifunctional Protein
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The PFK2/FBPase2 probably arose by the fusion of
genes encoding the kinase and phosphatase
domains
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Carbohydrate metabolism as a specific example of
signal transduction
?-adrenergic receptor
cAMP
second messenger
Gluconeogenesis?
Glycolysis ?
Protein kinase A
fructose 2,6bisphosphate (stimulate PFK-1)
fructose 6-phosphate (no PFK-1 stimulation)
PFK2/FBPase2
PFK-1
PFK2/FBPase2
fructose 2,6-bisphosphate
fructose 6-phosphate
Phosphoprotein Phosphatase (activated by
xylulose 5P)
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Reciprocal regulation by fructose-2,6-bisphosphate
cAMP-dependent phosphorylation of PFK2/FBPase2
activates FBPase2 and inhibits PFK2.
Fructose-2,6-bisphosphate decreases in liver
cells in response to a glucagon-activated cAMP
signal cascade. Downstream effects
include Glycolysis slows because
fructose-2,6-bisphosphate is not available to
activate PFK-1. Gluconeogenesis increases
because of the decreased concentration of
fructose-2,6-bisphosphate, which would stimulate
the fructose-1,6-bisphosphatase.
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(6). Xylulose 5-phosphate is a key regulator of
carbohydrate and fat metabolism.
In mammalian liver X5P, involved in pentose
phosphate pathway (production of NADPH),
mediates the increase in glycolysis that follows
an ingestion of a high-carbohydrate meal.
It was also found that X5P increased the
synthesis of all the enzymes required for fatty
acid synthesis.
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PFK-1
FBPase-1
glucagon
The distinctive enzymes are regulated. Certain
effectors activate an enzyme of one pathway, but
inhibit an enzyme of the other pathway to avoid
substrate futile cycle.
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High NADH/NAD ratio
Low NADH/NAD ratio
Cori cycle the liver furnishes glucose to
contracting skeletal muscle, which derives ATP
from the glycolytic conversion of glucose into
lactate. Contracting skeletal muscle supplies
lactate to the liver, which use it to synthesize
glucose.
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Glucose-alanine cycle
Alanine brings both carbon and nitrogen from
muscle to liver.
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What is a common feature for Cori cylcle and
Glc-Ala Cycle ?
The mechanisms allow muscle cells to produce
ATP with high rates at the expense of
regenerating glucose from lactate and alanine in
the liver.
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2. Biosynthesis of glycogen
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Animals Glycogen is the storage form of
glucose. Glycogen is synthesized and stored
mainly in the liver and the muscles. Plants
Plants make starch and cellulose through the
photosynthesis processes.
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Starch vs. Glycogen Animals and
human eat plant materials and products. Digestion
is a process of hydrolysis where the starch is
broken ultimately into the various
monosaccharides. A major product is of course
glucose which can be used immediately for
metabolism to make energy. The excess glucose
is converted in the liver and muscles into
glycogen for storage. Any glucose in excess of
the needs for energy and storage as glycogen is
converted to fat.
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Glycogen is a D-glucose polymer ? (1?4)
linkage ? (1?6) linkage Branches
every 8-14 residues
Glycogen Synthesis
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Glycogen Synthesis
Glycogen synthesis is not a direct reverse of the
phosphorolysis reaction.
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Formation of a sugar nucleotide which is the
substrate for polymerization of monosaccharides
into polysaacharides
A sugar nucleotide is formed through a
condensation reaction between a NTP and a sugar
phosphate.
Hexose 1-P
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Several important properties of sugar nucleotides

• The formation of sugar nucleotide is
  metabolically
• irreversible, contributing to the
  irreversibility of
• the synthetic pathways in which they are
  intermediates.
• The nucleotide moiety has many groups that can
  undergo
• noncovalent interactions with enzymes the
  additional
• free energy of binding can contribute
  significantly
• to catalytic activity.
• The nucleotidyl group (UMP, AMP) is an excellent
  leaving
• group, facilitating nucleophilic attack by
  activating the
• sugar carbon to which it is attached.
• Cell can set sugar nucleotide aside for one
  purpose (glycogen synthesis), to distinguish from
  sugar phosphate used for glycolysis.

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UDP-glucose pyrophosphorylase
UDP-glucose is formed from Glc-1-P and UTP
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(substrate)
glycogen synthase
Glycogen synthesis
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Initiation of a glycogen particle by glycogenin
catalyzing its own glycosylation
protein-tyrosine- Glucosyltransferase (glycogenin)
glycogenin
complex of glycogenin and glycogen synthase
glycogenin
glycogen synthase
glycogen synthase, glycogen-branching enzymes
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Glycogenin has glucosyltransferase activity which
catalyzes the assembly of the first 8 residues
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Structure of Glycogen Particle
about 55,000 glucose residues in a molecule of
about 21 nm diameter and Mr 107
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Coordinated regulation of glycogen synthesis and
breakdown
Glycogen synthase and glycogen phosphorylase are
reciprocally regulated allosterically and
hormonally. when one process is stimulated and
the other is inhibited, if not, resulting in a
futile cycle.
Glycogen phosphorylase enzyme used in
glycogenolysis (glycogen break-down)-stimulated
by low boold glucose Glycogen synthase enzyme
used in glycogenesis (glycogen synthesis)-
stimulated by high blood glucose
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Allosteric regulation of muscle glycogen
phosphorylase
The glycogen phosphorylases of liver and muscle
are isozymes, encoded by different genes and
differing in their regulation properties.
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Ca 2 binds to and activates phosphorylase b
kinase through ? subunit that is calmodulin.
AMP binds and activates phosphorylase, speeding
the release of G1P from glycogen.
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Glycogen phosphorylase of liver as a glucose
sensor, regulated hormonally and allosterically.
activate
indirectly
glucose as an allosteric inhibitor
glycogen breakdown decrease
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Carl F. Cori (1896-1984) Gerty T. Cori
(1896-1957)
Polysaccharide phosphorylase
Nobel Prize in Medicine and Physiology in 1947
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Carl F. Cori (1896-1984) Gerty T. Cori
(1896-1957)
Polysaccharide phosphorylase
Nobel Prize in Medicine or Physiology in 1947
Original publications
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"for his discoveries concerning the mechanisms of
the action of hormones"
The Nobel Prize in Physiology and Medicine 1992
Nobel Prize in Medicine or Physiology in 1971
After reading a book about Louis Pasteur in high
school he decided to go into medical research.
He isolated a previously unknown compound, called
cyclic adenine monophosphate (cAMP) and proved
that it had an intermediary role in many hormonal
functions.
Earl W. Sutherland
USA
1915-1974 (trained in Coris lab)
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Leloir discovered in 1949 that one sugar was
transformed to another sugar via sugar
nucleotide, later, he found that glycogen was
synthesized from UDP-glucose in 1959.
(trained in Coris lab)
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They showed that epinephrine and cAMP stimulate
glycogen breakdown by activating glycogen
phosphorylase via a protein kinase
The Nobel Prize in Physiology and Medicine 1992
Nobel Prize in Medicine or Physiology 1992
Edmond H. Fischer
Eldwin G. Krebs
USA
USA
1920- (trained in Coris lab)
1918-
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Glycogen synthase is also regulated by
phosphorylation and dephosphorylation
GSK3 action requires prior phosphorylation (primi
ng) by casein kinase (CKII).
GSK3 (glycogen synthase kinase) adds phosphoryl
group to three Ser residues, strongly inactivatin
g it.
PP1 (in liver) phosphoprotein phosphatase
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Priming of GSK3 phosphorylation of glycogen
synthase
C-terminus
N-terminus
phosphorylated by PKA or PKB
Activation of GSK3 requires removal of the
priming phosphoryl group by PP1.
(inactivate form)
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GSK3 mediates the action of insulin
The inactivation of GSK3 allows PP1 to
dephosphorylate glycogen synthase, converting it
to its active form.
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PP1 is central to glycogen metabolism
A single enzyme can remove phosphoryl groups from
all three enzymes phosphorylated in response to
glucagon (liver) and epinephrine (liver
muscle) phosphorylase kinase, glycogen
phosphorylase and glycogen synthase (all form a
big complex).
activate
activate
GM-P
Insulin
PP1
GM glycogen-targeting protein
Epinephrine
PKA
GM-2P
Inhibitor-P
Dissociation of PP1 from the complex
Inhibitor-P binds and inactivates PP1
PP1 does not exist free in the cytosol.
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The balance between glycogen synthesis and
breakdown in liver is controlled by the hormones
glucagon,epinephrine and insulin.
Enzyme Stimulated by Inhibited
by Glycogen phosphorylase
glucagon, epinephrine, Insulin,
ATP cAMP, Ca2,
AMP glucose
phosphorylation Glycogen synthase
Insulin,
glucagon,
glucose-6-phosphate epinephrine

cAMP, Ca2, AMP

phosphorylation
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Overall shifts in carbohydrate metabolism that
occur in the well-fed state in hepatocyte
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Overall shifts in carbohydrate metabolism that
occur during fasting in hepatocyte
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Difference in the regulation of carbohydrate
metabolism in liver and muscle
Glycogenolysis glycogen ? glucose
6-phosphate Glycogenesis glucose ? glycogen
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The physiology and carbohydrate metabolism of
skeletal muscle differs from that of liver in
three ways
(1). Muscle uses its stored glycogen only for its
own needs. (2). Glycolysis occurs when muscle
goes from rest to vigorous contraction.
- Pyruvate kinase (M) is not
phosphorylated by PKA, so glycolysis is
not turn off when cAMP is high. (3). Muscle
lacks gluconeogenesis pathway. - myocytes
lack receptors for glucagon
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3. Analysis of metabolic control
For every complex problem there is a simple
solution. And it is always wrong.
- H.L. Mencken, A
Mencken Chrestomathy, 1949
Is single rate-determining step hypothesis
right ?
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(1). The control coefficient (C) quantifies the
effect of a change in enzyme activity on
metabolite flux through a pathway
C expresses the relative contribution of each
enzyme to setting the rate at which metabolites
flow through the pathway.
Range of C 0-1.0
C - not a constant - a function of the
whole system of enzymes. - depends on
the concentrations of substrates and
effectors
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(2). The contribution of each enzyme to flux
through a pathway is experimentally
measurable
Dependence of glycolytic flux in a rat liver
homogenate on added enzymes.
(C flux control coefficient)
C0.79
Both hexokinase IV and PFK-1 increase the rate
of glycolysis, and hexokinase contributes more
than PFK-1 does.
C0.21
C 0
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(3). The elasticity coefficient (?) is related to
an enzymes responsiveness to changes in
metabolite or regulator concentrations.

• - quantitatively the
• responsiveness of
• a single enzyme to
• changes in the
• concentration
• of a metabolite or
• regulator
• - a function of enzymes
• intrinsic kinetic
• properties

Hyperbolic curve
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(4). The response coefficient (R) expresses the
effect of an outside controller on flux
through a pathway
The experiment measures the flux through
the pathway at various parameter P (for
example, insulin changes) to obtain R
R C ?
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(5). Metabolic control analysis has been applied
to carbohydrate metabolism, with
surprising results
regulation of insulin in muscle

• increases glucose
• transport via GLU4
• (control)
• induce the synthesis
• of hexokinase
• (control)
• activate glycogen
• synthase
• (regulation, not
• control !)

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Summary on glucose synthesis and polymerization

• Gluconeogenesis, the synthesis of glucose from
  3-carbon compounds (mainly pyruvate) is highly
  conserved in all organisms.
• Gluconeogenesis shares most of the reactions
  occurring in glycolysis, but bypassing the three
  irreversible reactions (using different enzymes).
• Gluconeogenesis consumes more energy than
  glycolysis releases.
• Most of the amino acids, but not fatty acids can
  be used for net production of glucose in
  vertebrates.
• The gluconeogenesis and glycolysis are
  reciprocally regulated by molecules like acetyl
  CoA, AMP, fructose 1,6BP.

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• To limit futile cycling, the two pathways are
  under reciprocal allosteric control, mainly
  achieved by opposite effects of F2,6BP on PFK-1
  and FBPase-1.
• Glucagon or epinephrine decreases F2,6BP.
• The hormones do this by raising cAMP and
  bringing about phosphorylation of the
  bifunctional enzyme, PFK-2/FBPase-2.
  Phosphorylation inactivates PFK-2 and activates
  FBPase-2, leading to breakdown of F2,6BP.
• Insulin increases F2,6BP by activating a
  phosphoprotein phosphatase that dephosphorylates
  (activates) PFK-2.

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• Insulin stimulates glycogen synthesis in muscle
  and liver.
• In liver, glucagon stimulates glycogen breakdown
  and gluconeogenesis while blocking glycolysis,
  thereby sparing glucose for export to the brain
  and other tissues.
• In muscle, epinephrine stimulates glycogen
  breakdown and glycolysis, providing ATP to
  support contraction.
• Sugar nucleotides are used for biosynthesis
• Metabolic control analysis predicts that flux
  toward a desired product is most effectively
  increased by raising the concentration of all
  enzymes in the pathway.