Transcript CHAPTER 6

Chapter 22
Gluconeogenesis,
Glycogen Metabolism,
and the Pentose Phosphate Pathway
Biochemistry
by
Reginald Garrett and Charles Grisham
Essential Question
1. What is the nature of gluconeogenesis, the
pathway that synthesizes glucose from
noncarbohydrate precursors
2. How is glycogen synthesized from glucose
3. How are electrons from glucose used in
biosynthesis?
Outline of chapter 22
1. What Is Gluconeogenesis, and How Does It
Operate?
2. How Is Gluconeogenesis Regulated?
3. How Are Glycogen and Starch Catabolized in
Animals?
4. How Is Glycogen Synthesized?
5. How Is Glycogen Metabolism Controlled?
6. Can Glucose Provide Electrons for
Biosynthesis?
22.1 – What Is Gluconeogenesis, and
How Does It Operate?
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Synthesis of "new glucose" from common
metabolites
Humans consume 160 g of glucose per day
75% of that is in the brain
Body fluids contain only 20 g of glucose
Glycogen stores yield 180-200 g of glucose
So the body must be able to make its own
glucose
Substrates for Gluconeogenesis
Pyruvate, lactate, glycerol, amino acids and
all TCA intermediates can be utilized
• Fatty acids cannot! Why?
• Most fatty acids yield only acetyl-CoA
– Except fatty acids with odd numbers of carbons
• Acetyl-CoA (through TCA cycle) cannot
provide for net synthesis of sugars in
animal, but plants do! Why?
Substrates for Gluconeogenesis
1.
2.
3.
4.
Lactate
Amino acids
Glycerol
Propionyl-CoA
Gluconeogenesis
• Occurs mainly in liver (90%) and kidneys (10%)
• Not the mere reversal of glycolysis for 2
reasons:
– Energetics: must change to make
gluconeogenesis favorable (DG of glycolysis
= -74 kJ/mol)
– Reciprocal regulation: Gluconeogenesis
must turn one on and the other off, and vice
versa
Gluconeogenesis
Something Borrowed, Something New
• Seven steps of glycolysis are retained:
– Steps 2 and 4-9
• Three steps are replaced:
– Steps 1, 3, and 10 (the regulated steps!)
• The new reactions provide for a
spontaneous pathway (DG negative in the
direction of sugar synthesis), and they
provide new mechanisms of regulation
Figure 22.1
The pathways of gluconeogenesis
and glycolysis. Species in blue,
green, and peach-colored shaded
boxes indicate other entry points for
gluconeogenesis (in addition to
pyruvate).
Pyruvate Carboxylase
Pyruvate is converted to oxaloacetate
• The reaction requires ATP and bicarbonate as
substrates
• Biotin as a coenzyme and Acetyl-CoA is an
allosteric activator
Biotin is covalently linked to the e-amino group
of a lysine residue
Figure 22.3
Covalent linkage of biotin to an activesite lysine in pyruvate carboxylase.
• The mechanism is typical of biotin
Figure 22.4 A mechanism for the
pyruvate carboxylase reaction.
Bicarbonate must be activated for
attack by the pyruvate carbanion. This
activation is driven by ATP and involves
formation of a carbonylphosphate
intermediate—a mixed anhydride of
carbonic and phosphoric acids.
(Carbonylphosphate and
carboxyphosphate are synonyms.)
Pyruvate Carboxylase
• Acyl-CoA is an allosteric activator
• The conversion is in mitochondrial matrix
• Regulation:
– If levels of ATP and/or acetyl-CoA are low, pyruvate
is converted to acetyl-CoA and enters TCA cycle
– If levels of ATP and/or acetyl-CoA are high, pyruvate
is converted to oxaloacetate and enters
gluconeogenesis  glucose
• Pyruvate carboxylase is found
only in mitochondrial matrix
• Oxaloacetate cannot be
transported across the
mitochondrial membrane
Figure 22.5
Pyruvate carboxylase is a compartmentalized reaction.
Pyruvate is converted to oxaloacetate in the mitochondria.
Because oxaloacetate cannot be transported across the
mitochondrial membrane, it must be reduced to malate, transported
to the cytosol, and then oxidized back to oxaloacetate before
gluconeogenesis can continue.
PEP Carboxykinase
Conversion of oxaloacetate to PEP
• Lots of energy needed to drive this reaction
• Energy is provided in 2 ways:
– Decarboxylation is a favorable reaction
– GTP is hydrolyzed
• GTP used here is equivalent to an ATP
PEP Carboxykinase
• The overall DG for the pyruvate carboxylase
and PEP carboxykinase reactions under
physiological conditions in the liver is -22.6
kJ/mol
• Once PEP is formed in this way, the other
reactions act to eventually form fructose-1,6bisphosphate
Fructose-1,6-bisphosphatase
Hydrolysis of F-1,6-bisPase to F-6-P
• Thermodynamically favorable - DG in liver is
-8.6 kJ/mol
• Allosteric regulation:
– citrate stimulates
– fructose-2,6-bisphosphate inhibits
– AMP inhibits (enhanced by F-2,6-bisP)
Glucose-6-Phosphatase
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Conversion of Glucose-6-P to Glucose
G-6-Pase is present in ER membrane of liver
and kidney cells
Muscle and brain do not do gluconeogenesis
G-6-P is hydrolyzed as it passes into the ER
ER vesicles filled with glucose diffuse to the
plasma membrane, fuse with it and open,
releasing glucose into the bloodstream.
Figure 22.8 Glucose-6-phosphatase is localized in the endoplasmic reticulum membrane.
Conversion of glucose-6-phosphate to glucose occurs during transport into the ER.
DG = -5.1 kJ/mol
Figure 22.9 The glucose-6-phosphatase reaction involves formation of a phosphohistidine
intermediate.
Lactate Recycling – Cori cycle
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How your liver helps you during exercise....
Recall that vigorous exercise can lead to a
buildup of pyruvate and NADH, due to oxygen
shortage and the need for more glycolysis
NADH can be reoxidized during the reduction
of pyruvate to lactate
Lactate is then returned to the liver, where it
can be reoxidized to pyruvate by liver LDH
Liver provides glucose to muscle for exercise
and then reprocesses lactate into new glucose
(about 700)
Figure 22.10
The Cori cycle.
22.2 – How Is Gluconeogenesis Regulated?
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Reciprocal control with glycolysis
When glycolysis is turned on, gluconeogenesis
should be turned off, and vice versa
When energy status of cell is high, glycolysis
should be off and pyruvate, etc., should be used
for synthesis and storage of glucose
When energy status is low, glucose should be
rapidly degraded to provide energy
The regulated steps of glycolysis are the very
steps that are regulated in the reverse direction
Figure 22.11
The principal regulatory
mechanisms in glycolysis
and gluconeogenesis.
Activators are indicated by
plus signs and inhibitors
by minus signs.
Allosteric and Substrate-Level Control
• Glucose-6-phosphatase
– is under substrate-level control by G-6-P, not
allosteric control
• Pyruvate carboxylase
– Activated by acetyl-CoA
– The fate of pyruvate depends on acetyl-CoA;
pyruvate kinase (-), pyruvate dehydrogenase (-), and
pyruvate carboxylase (+)
• F-1,6-bisPase
– is inhibited by AMP and Fructose-2,6-bisP
– Activated by citrate - the reverse of glycolysis
(w/o AMP)
(w/ 25mM AMP)
(F-2,6-BP)
(F-2,6-BP)
(AMP)
Figure 22.12
Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate in the (a) absence
and (b) presence of 25 mM AMP. In (a) and (b), enzyme activity is plotted against substrate
(fructose-1,6-bisphosphate) concentration. Concentrations of fructose-2,6-bisphosphate (in
mM) are indicated above each curve. (c) The effect of AMP (0, 10, and 25 mM) on the
inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate. Activity was
measured in the presence of 10 mM fructose-1,6-bisphosphate.
• Fructose-2,6-bisP
– is an allosteric inhibitor of F-1,6-bisPase
– is an allosteric activator of PFK
– synergistic effect with AMP
• The cellular levels Fructose-2,6-bisP are
controlled by phosphofructokinase-2 and
fructose-2,6-bisPase which is bifunctional
enzyme
– F-6-P allosterically activates PFK-2 and inhibits F2,6-BisPase
– Phosphorylation by cAMP-dependent protein kinase
inhibits PFK-2 and activates F-2,6-bisPase
Figure 22.13 Synthesis and degradation of fructose-2,6-bisphosphate are catalyzed by the same
bifunctional enzyme.
22.3 – How Are Glycogen and Starch
Catabolized in Animals?

Getting glucose from storage (or diet)
-Amylase is an endoglycosidase -- (1→4)
cleavage

b-Amylase is an exoglycosidase (In plants)
• It cleaves dietary amylopectin or glycogen to
maltose, maltotriose and other small
oligosaccharides
• It is active on either side of a branch point, but
activity is reduced near the branch points and
stops four residues from any branch point
• limit dextrins
Figure 22.14
Hydrolysis of glycogen and
starch by -amylase and bamylase.
• Debranching enzyme cleaves "limit dextrins"
• Two activities of the debranching enzyme
– Oligo(1,4→1,4)glucanotransferase
– (1→6)glucosidase
Figure 22.15
The reactions of glycogen debranching
enzyme. Transfer of a group of three -(1
 4)-linked glucose residues from a limit
branch to another branch is followed by
cleavage of the -(1 6) bond of the
residue that remains at the branch point.
Metabolism of Tissue Glycogen
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Digestive breakdown is unregulated - 100%!
But tissue glycogen is an important energy
reservoir - its breakdown is carefully controlled
Glycogen consists of "granules" of high MW
range from 6 x 106 ~ 1600 x 106
Glycogen phosphorylase cleaves glucose from
the nonreducing ends of glycogen molecules
This is a phosphorolysis, not a hydrolysis
Metabolic advantage: product is a glucose-1-P; a
"sort-of" glycolysis substrate
glycogen phosphorylase
(Phosphorolysis)
Figure 22.16
The glycogen phosphorylase reaction.
• See pages 486-491 to review glycogen
phosphorylase
22.4 – How Is Glycogen Synthesized?
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Glucose units are activated for transfer by
formation of sugar nucleotides
What are other examples of "activation"?
– acetyl-CoA : acetate
– Biotin and THF : one-carbon group
– ATP : phosphate
Leloir showed that glycogen synthesis depends
on sugar nucleotides
UDP-glucose pyrophosphorylase catalyzes the
formation of UDP-glucose
ADP-glucose is for starch synthesis in plants
Figure 22.17
The structure of UDP-glucose, a sugar nucleotide.
Glucose-1-P + UTP →
UDP-glucose + pyrophosphate.
Figure 22.18
The UDP-glucose
pyrophosphorylase reaction is
a phosphoanhydride
exchange, with a phosphoryl
oxygen of glucose-1-P
attacking the -phosphorus of
UTP to form UDP-glucose and
pyrophosphate.
Glycogen Synthase
Forms -(1 4) glycosidic bonds in glycogen
• Glycogenin (a protein core) forms the core of
a glycogen particle
• First glucose is linked to a tyrosine -OH on
glycogenin
• Glycogen synthase transfers glucosyl units
from UDP-glucose to C-4 hydroxyl at a
nonreducing end of a glycogen strand.
• oxonium ion intermediate (Fig. 22.19)
Figure 22.19
The glycogen
synthase reaction.
Cleavage of the C-O
bond of UDP-glucose
yields an oxonium
intermediate. Attack
by the hydroxyl
oxygen of the
terminal residue of a
glycogen molecule
completes the
reaction.
Glycogen branching occurs by
transfer of terminal chain segments
• Glycogen branches are formed by
amylo-(1,4→1,6)-transglycosylase,
also called branching enzyme
• -(1 6) linkages, which occurs
every 8-12 residues
• Transfer of 6- or 7-residue segment
from the nonreducing end
Figure 22.20
Formation of glycogen branches by the branching enzyme. Sixor seven-residue segments of a growing glycogen chain are
transferred to the C-6 hydroxyl group of a glucose residue on the
same or a nearby chain.
22.5 – How Is Glycogen Metabolism
Controlled?
A highly regulated process, involving reciprocal
control of glycogen phosphorylase and glycogen
synthase
• Glycogen phosphorylase, allosterically activated
by AMP and inhibited by ATP, glucose-6-P and
caffeine
• Glycogen synthase, is stimulated by glucose-6-P
• Both enzymes are regulated by covalent
modification - phosphorylation
Phosphorylation of GP and GS
Covalent modification
• In chapter 15 showed that protein kinase
converted phosphorylase b (-OH) to
phosphorylase a (-OP)
• Glycogen synthase also exists in two distinct
forms
– Active, dephosphorylated glycogen synthase I
– Less active, phosphorylated glycogen synthase D
(glucose-6-P dependent)
• Nine Ser residues on GS are phosphorylated
SPK: Synthasephosphorylase kinase
( phosphorylase b kinase)
PP1:
Phosphoprotein
phosphatase 1
Enzyme Cascades and GP/GS
Hormonal regulation ( insulin, Glucagon,
epinephrine, and glucocorticoids)
• Glucagon and epinephrine activate adenylyl
cyclase
• cAMP activates kinases and phosphatases that
control the phosphorylation of GP and GS
– GTP-binding proteins (G proteins) mediate the
communication between hormone receptor and
adenylyl cyclase
• Dephosphorylation is carried out by
phosphoprotein phosphatase-I (PP-I)
Hormonal Regulation
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of Glycogen Synthesis and Degradation
Insulin is secreted from the pancreas (to
liver) in response to an increase in blood
glucose
Insulin into the portal vein and traverses the
liver
Insulin stimulates glycogen synthesis and
inhibits glycogen breakdown
Other effects of insulin (Figure 22.22)
Figure 22.21
The portal vein
system carries
pancreatic secretions
such as insulin and
glucagon to the liver
and then into the rest
of the circulatory
system.
(glucose uptake)
(F-1,6-BP & PEPCK)
(PFK & PK)
Figure 22.22
The metabolic effects of insulin. As described in Chapter 32, binding of insulin to
membrane receptors stimulates the protein kinase activity of the receptor.
Subsequent phosphorylation of target proteins modulates the effects indicated.
Hormonal Regulation
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Glucagon and epinephrine
Glucagon and epinephrine stimulate glycogen
breakdown - opposite effect of insulin
Glucagon (29 AA-res) is also secreted by
pancreas and acts in liver and adipose tissue
only
Epinephrine (adrenaline) is released from
adrenal glands and acts on liver and muscles
A cascade is initiated that activates glycogen
phosphorylase and inhibits glycogen synthase
Figure 22.23 The amino acid sequence of glucagon.
Figure 22.24 Epinephrine
Hormonal Regulation
• The phosphorylase cascade amplifies the signal
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10-10 to 10-8 M epinephine
10-6 M cAMP
Protein kinase
30 molecules of phosphorylase b kinase
800 molecules of phosphorylase a
Catalyzes the formation of many molecules of
glucose-1-P
• The result of these actions is tightly coordinated
stimulation of glycogen breakdown and
inhibition of glycogen synthesis
SPK: Synthasephosphorylase kinase
( phosphorylase b kinase)
PP1:
Phosphoprotein
phosphatase 1
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The difference between Epinephrine and Glucagon
Both are glycogenolytic but for different reasons
Epinephrine is the fight or flight hormone
– Rapid breakdown of glycogen
– Inhibition of glycogen synthesis
– Stimulation of glycolysis
– Production of energy
Glucagon is for long-term maintenance of steadystate levels of glucose in the blood
– activates glycogen breakdown
– activates liver gluconeogenesis
Glucagon do not activate the phsphorylase cascade
in mucle
Cortisol and glucocorticoid
• Cortisol is a typical glucocoticoid
• In muscle (catabolic)
– promotes protein breakdown
– decrease protein synthesis
• In liver
– stimulates gluconeogenesis
– increases glycogen synthesis
– amino acid catabolism
Figure 22.25
The effects of cortisol on carbohydrate and protein metabolism in the liver.
22.6 – Can Glucose Provide Electrons
for Biosynthesis?
Pentose Phosphate Pathway
Hexose monophosphate shunt
Phosphogluconate pathway
• Provides NADPH for biosynthesis
• Produces ribose-5-P for nucleotide synthesis
• Several metabolites of the pentose
phosphate pathway can also be shuttled into
glycolysis
Pentose phosphate pathway
• Begins with glucose-6-P, a six-carbon, and
produces 3-, 4-, 5-, 6, and 7-carbon sugars,
some of which may enter the glycolytic
pathway
• Two oxidative processes followed by five nonoxidative steps
• Operates mostly in cytoplasm of liver and
adipose cells, but absent in muscle
• NADPH is used in cytosol for reductive
reaction-- fatty acid synthesis
Figure 22.26
The pentose phosphate
pathway. The numerals in the
blue circles indicate the steps
discussed in the text.
Oxidative Steps
• Glucose-6-P Dehydrogenase
– Begins with the oxidation of glucose-6-P
– The products are a cyclic ester (the lactone of
phosphogluconic acid) and NADPH
– Irreversible 1st step and highly regulated
– Inhibited by NADPH and acyl-CoA
• Gluconolactonase
– Gluconolactone hydrolyzed →6-phospho-D-gluconate
– Uncatalyzed reaction happens too
– Gluconolactonase accelerates this reaction
Figure 22.27 The glucose-6-phosphate dehydrogenase reaction is the committed
step in the pentose phosphate pathway.
Figure 22.28
The gluconolactonase reaction.
Oxidative Steps
• 6-Phosphogluconate Dehydrogenase
– An oxidative decarboxylation of 6phosphogluconate
– Yields ribulose-5-P and NADPH
– Releases CO2
Figure 22.29 The 6-phosphogluconate dehydrogenase reaction.
The Nonoxidative Steps
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Five steps, only 4 types of reaction...
Phosphopentose isomerase
– converts ketose to aldose
Phosphopentose epimerase
– epimerizes at C-3
Transketolase (TPP-dependent)
– transfer of two-carbon units
Transaldolase (Schiff base mechanism)
– transfers a three-carbon unit
• Phosphopentose isomerase
– converts ketose to aldose
– Ribose-5-P is utilized in the biosynthesis of
coenzymes, nucleotides, and nucleic acids
Figure 22.30
The phosphopentose isomerase reaction involves an enediol intermediate.
• Phosphopentose epimerase
– An inversion at C-3
Figure 22.31
The phosphopentose epimerase reaction interconverts ribulose-5-P and xylulose-5-phosphate.
The mechanism involves an enediol intermediate and occurs with inversion at C-3.
• Transketolase (TPP-dependent)
– transfer of two-carbon units
– The donor molecule is a ketose and the
recipient is an aldose
Figure 22.32 The transketolase reaction of step 6 in the pentose phosphate pathway.
Figure 22.33
The transketolase reaction of step 8 in the pentose phosphate pathway.
Figure 22.34
The mechanism of the TPPdependent transketolase
reaction. Ironically, the group
transferred in the transketolase
reaction might best be
described as an aldol, whereas
the transferred group in the
transaldolase reaction is
actually a ketol. Despite the
irony, these names persist for
historical reasons.
• Transaldolase (Schiff base, imine)
– transfers a three-carbon unit
– Yields erythrose-4-P & Fructose-6-P
Figure 22.35 The transaldolase reaction.
Figure 22.36
The transaldolase
mechanism involves
attack on the substrate
by an active-site lysine.
Departure of erythrose4-P leaves the reactive
enamine, which attacks
the aldehyde carbon of
glyceraldehyde-3-P.
Schiff base hydrolysis
yields the second
product, fructose-6-P.
Variations on the Pentose Phosphate
Pathway
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1)
2)
3)
4)
Both ribose-5-P and NADPH are needed
More ribose-5-P than NADPH is needed
More NADPH than ribose-5-P is needed
NADPH and ATP are needed, but
ribose-5-P is not
Figure 22.37 When biosynthetic demands dictate, the first four reactions of the pentose phosphate
pathway predominate and the principal products are ribose-5-P and NADPH.
Figure 22.38
The oxidative steps of
the pentose phosphate
pathway can be
bypassed if the primary
need is for ribose-5-P.
Figure 22.39
Large amounts of NADPH can
be produced by the pentose
phosphate pathway without
significant net production of
ribose-5-P. In this version of the
pathway, ribose-5-P is recycled
to produce glycolytic
intermediates.
Figure 22.40
Both ATP and NADPH (as
well as NADH) can be
produced by this version of
the pentose phosphate and
glycolytic pathways.
(a) Contributions of the various energy sources to muscle activity during mild exercise. (b)
Consumption of glycogen stores in fast-twitch muscles during light, moderate, and heavy
exercise. (c) Rate of glycogen replenishment following exhaustive exercise. (a and c
adapted from Rhodes and Pflanzer, 1992. Human Physiology. Philadelphia: Saunders College
Publishing; b adapted from Horton and Terjung, 1988. Exercise, Nutrition and Energy Metabolism. New
York : Macmillan.)