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Chapter 22
Gluconeogenesis,
Glycogen Metabolism,
and the Pentose Phosphate Pathway
Biochemistry
by
Reginald Garrett and Charles Grisham
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?
•
•
•
•
Generation (genesis) of "new (neo) glucose"
from common metabolites
Humans consume about 160 g of glucose per
day, 75% of that is in the brain
Body fluids contain only 20 g of free glucose
Glycogen stores can provide 180-200 g of
glucose
So the body must be able to make new glucose
from noncarbohydrate precursors—
gluconeogenis
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? (chap 19)
Substrates for Gluconeogenesis
1.
2.
3.
4.
Lactate
Amino acids
Glycerol
Propionyl-CoA
FAs with Odd numbers of C
Gluconeogenesis
•
•
Occurs mainly in liver (90%) and kidneys (10%)
Not the mere reversal of glycolysis for 2
reasons:
1. Energetics: must change to make gluconeogenesis
favorable (DG of glycolysis = -74 kJ/mol)
2. Reciprocal regulation: Gluconeogenesis is turned
on, and when glycolysis is turned off, and vice
versa
The new reactions provide for a spontaneous
pathway (DG negative in the direction of sugar
synthesis), and they provide new mechanisms
of regulation
Something Borrowed,
Something New
1. Seven steps of glycolysis
are retained:
Steps 2 and 4-9
2. Three steps are replaced:
Steps 1, 3, and 10 (the
regulated steps!)
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).
Hexokinase
Phosphofructokinase
1. Pyruvate Carboxylase
Pyruvate is converted to oxaloacetate
• The reaction requires ATP and bicarbonate as
substrates
• Biotin is covalently linked to a
lysine residue at the active site
and as a coenzyme
• The mechanism is typical of biotin
carbonylphosphate
pyruvate
carbanion
Figure 22.3 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.)
• The conversion is in mitochondrial matrix
• Acyl-CoA is an allosteric activator
• Regulation:
– If levels of ATP and/or acetyl-CoA are low
Pyruvate is converted to acetyl-CoA
Enters TCA cycle
– If levels of ATP and/or acetyl-CoA are high
Pyruvate is converted to oxaloacetate
Enters gluconeogenesis  glucose
• Pyruvate carboxylase is found
only in mitochondrial matrix
• Oxaloacetate cannot be
transported across the
mitochondrial membrane
Figure 22.4 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.
2. Phosphoenolpyruvate Carboxykinase
Conversion of oxaloacetate to PEP
• Lots of energy is needed to drive this reaction
• Energy is provided in 2 ways:
1. Decarboxylation is a favorable reaction and helps
drive the formation of the very high-energy enol
phosphate
2. 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
3. Fructose-1,6-bisphosphatase
Hydrolysis of F-1,6-bisP 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)
4. Glucose-6-Phosphatase
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 after uptake into the ER
Figure 22.5 Glucose-6-phosphatase is localized in the endoplasmic reticulum.
Conversion of glucose-6-phosphate to glucose occurs following transport into the ER.
• The glucose-6-phosphatase system includes the
phosphatase itself and three transport proteins,
T1, T2, and T3.
• T1 takes glucose-6-P into the ER, where it is
hydrolyzed by the phosphatase
• T2 and T3 export glucose and Pi, respectively,
to the cytosol
• Glucose is exported to the circulation by
GLUT2
Gluconeogenesis Inhibitors and Other
Diabetes Therapy Strategies
Diabetes is the inability to assimilate and metabolize
blood glucose
• Metformin improves sensitivity to insulin by
stimulating glucose uptake by glucose transporters
• Gluconeogenesis inhibitors may be the next wave of
diabetes therapy
• 3-Mercatopicolinate and hydrazine inhibit PEP
carboxykinase
• Clorogenic acid (natural product found in the skin of
peaches) inhibits transport activity by the glucose-6phosphatase system. S-3483 does the same, but
binds a thousand times more tightly to the
transporter
Lactate Recycling – Cori cycle
Lactate formed in muscles is recycled to glucose
in the liver
• 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 22.7
The Cori cycle.
22.2 – How Is Gluconeogenesis Regulated?
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 and other metabolites are
ustilized 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 22.8 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)
Figure 22.9
Inhibition of fructose-1,6-bisphosphatase by fructose-2,6bisphosphate 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.
(AMP)
• 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 22.10 Synthesis and
degradation of fructose-2,6bisphosphate are catalyzed by the
same bifunctional enzyme.
22.3 – How Are Glycogen and Starch
Catabolized in Animals?
A balanced diet provides carbohydrate each day,
mostly in the form of starch.
∙
If too little carbohydrate is supplied by the diet,
glycogen reserves in liver and muscle tissue can also
be mobilized
The starch and glycogen are digested by amylase
∙
∙
∙
-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
Figure 22.11
Hydrolysis of glycogen and
starch by -amylase and bamylase.
• -Amylase can cleave
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
• Debranching enzyme cleaves "limit dextrins"
• Two activities of the debranching enzyme
– Oligo(1,4→1,4)glucanotransferase
– (1→6)glucosidase
Figure 22.12
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 is
Regulated
Digestive breakdown is unregulated - nearly 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
potential glycolysis substrate
Figure 22.13
The glycogen phosphorylase reaction.
22.4 – How Is Glycogen Synthesized?
Glucose units are activated for transfer by
formation of sugar nucleotides
• What are other examples of "activated form"?
– 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
Glucose-6-P
Glucose-1-P + UTP →
UDP-glucose + pyrophosphate.
Figure 22.14
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
• The glycogen polymer is built around
aprotein Glycogenin (a protein core)
– First glucose is linked to a tyrosine -OH on
glycogenin
– Sugar units can be added by the action of
glycogen synthase
• Glycogen synthase transfers glucosyl units
from UDP-glucose to C-4 hydroxyl at a
nonreducing end of a glycogen strand. (Fig.
22.15)
Figure 22.15 The glycogen
synthase reaction.
Branching enzyme:
• 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 22.16
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
• Activation of Glycogen phosphorylase (GP)
is tightly linked to inhibition of glycogen
synthase (GS), and vice versa
• Regulation involves both allosteric control
and covalent modification
22.5 – How Is Glycogen Metabolism
Controlled?
1. Allosteric control
• Glycogen phosphorylase allosterically
activated by AMP and inhibited by ATP,
glucose-6-P and caffeine
• Glycogen synthase is stimulated by
glucose-6-P
Phosphorylation of GP and GS
2. Covalent modification
• In chapter 15 (p496) showed that protein kinase
converted phosphorylase b (-OH, inactivated) to
phosphorylase a (-OP, activated)
• Glycogen synthase also exists in two distinct
forms
– Active, dephosphorylated glycogen synthase I
– Less active, phosphorylated glycogen synthase D
(glucose-6-P dependent)
Casein kinase
Glycogen Synthase
Kinase 3 (GSK 3)
PP1: Phosphoprotein phosphatase 1
SPK: Synthase-phosphorylase kinase
(phosphorylase kinase)
• Glucagon and epinephrine activate adenylyl
cyclase (chapter 15)
– cAMP activates kinases and phosphatases that
control the phosphorylation of GP and GS
– stimulate glycogen breakdown
• Dephosphorylation of both glycogen
phosphorylase and glycogen synthase is
carried out by phosphoprotein phosphatase-1
(PP1)
– PP1 inactivates glycogen phosphorylase and
activates glycogen synthase
Hormones Regulate Glycogen Synthesis
and Degradation
Storage and utilization of tissue glycogen and
other aspects of metabolism are regulated by
hormones, including insulin, glucagon,
epinephrine, and the glucocorticoids
• Insulin is a response to increased blood glucose
• Insulin triggers glycogen synthesis when blood
glucose rises
– Between meals, blood glucose is 70-90 mg/dL
– Glucose rises to 150 mg/dL after a meal and then
returns to normal within 2-3 hours
Figure 22.17 Insulin triggers protein kinase cascades that stimulate glycogen synthesis.
(glucose uptake, GLUT4)
(F-1,6-BP & PEPCK)
(PFK & PK)
Figure 22.17 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.
Insulin
• Insulin is secreted from
the b-cells in the
pancreas into the
pancreatic vein,
empties into the portal
vein system (to liver)
Figure 22.18
The portal vein system carries pancreatic secretions
such as insulin and glucagon to the liver and then
into the rest of the circulatory system.
Hormonal Regulation
Glucagon and epinephrine
• Glucagon and epinephrine stimulate glycogen
breakdown - opposite effect of insulin
– Glucagon (29 AA-res) is also secreted from -cells
in 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 22.20 Glucagon and
epinephrine activate a cascade
of reactions that stimulate
glycogen breakdown and inhibit
glycogen synthesis in live and
muscles, respectively.
PFK-2 isoforms in liver (ser32) and
heart (ser466 & ser483) responds
oppositely to PKA
Hormonal Regulation
• The phosphorylase cascade amplifies the signal
– 10-10 to 10-8 M epinephine
→10-6 M cAMP
→Protein kinase (PK)
→30 molecules of phosphorylase b kinase / PK
→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
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 muscle
Cortisol and glucocorticoid
• Glucocorticoids are steroid hormones that
exert distinct effects on liver, skeletal muscle,
and adipose tissue
• Cortisol is a typical glucocoticoid
• In skeletal muscle (catabolic)
– promotes protein breakdown
– decrease protein synthesis
• In liver
– stimulates gluconeogenesis
– increases glycogen synthesis
– Activates amino acid catabolism & urea cycle
Figure 22.21 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
1. Provides NADPH for biosynthesis
2. Produces ribose-5-P for nucleotide synthesis
• Several metabolites of the pentose phosphate
pathway can also be shuttled into glycolysis
• Operates mostly in cytoplasm of liver and
adipose cells, but absent in muscle
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
non-oxidative steps
• NADPH is used in cytosol for reductive
reaction-- fatty acid synthesis
Figure 22.22 The pentose
phosphate pathway. The
numerals in the blue circles
indicate the steps discussed in
the text.
Transketolase: 6 & 8
Transaldolase: 7
Begins with Two Oxidative Steps
1. 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
2. Gluconolactonase
–
–
–
Gluconolactone hydrolyzed →6-phospho-Dgluconate
Uncatalyzed reaction happens too
Gluconolactonase accelerates this reaction
Figure 22.23 The glucose-6phosphate dehydrogenase
reaction is the committed step
in the pentose phosphate
pathway.
Figure 22.24 The
gluconolactonase reaction.
3. 6-Phosphogluconate Dehydrogenase
– An oxidative decarboxylation of 6phosphogluconate
– Yields ribulose-5-P and NADPH
– Releases CO2
Figure 22.25 The 6-phosphogluconate dehydrogenase reaction.
The Nonoxidative Steps
•
•
•
•
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
Glucose-6-P + 2 NADP+ + H2O → ribose-5-P + 2 NADPH + 2 H+ + CO2
Figure 22.26 The phosphopentose isomerase reaction involves an enediol intermediate.
• Phosphopentose epimerase
– An inversion at C-3
Figure 22.27 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 22.28 The transketolase reaction of step 6 in the pentose phosphate pathway.
Figure 22.29 The transketolase reaction of step 8 in the pentose phosphate pathway.
Figure 22.30
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 22.31 The transaldolase reaction.
Figure 22.32
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
•
•
•
•
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 22.33 When biosynthetic demands dictate, the first four reactions of the pentose
phosphate pathway predominate and the principal products are ribose-5-P and NADPH.
2) More Ribose-5-P than NADPH is needed by the cell.
Synthesis of ribose-5-P can be accomplished without
making NADPH, by bypassing the oxidative reactions of
the pentose phosphate pathway
3) More NADPH than ribose-5-P is needed by the cell.
This can be accomplished if ribose-5-P produced in
the pentose phosphate pathway is recycled to
produce glycolytic intermediates
4) Both NADPH and ATP are needed by the cell, but ribose-5-P is not.
This can be done by recycling ribose-5-P, as in case 3 above, if
fructose-6-P and glyceraldehyde-3-P made in this way proceed
through glycolysis to produce ATP and pyruvate, and pyruvate
continues through the TCA cycle to make more ATP
Figure 21.25 The Calvin-Benson
Cycle of reactions