Chapter 16

Glycolysis and
Gluconeogenesis

© 2023 W. H. Freeman and Company

 CHAPTER 16
Glycolysis and Gluconeogenesis
 Ch.16 Learning Goals
By the end of this chapter, you should be able to:
1. Describe how ATP is generated in glycolysis.
2. Explain why the regeneration of NAD+ is crucial to
fermentations.
3. Describe how gluconeogenesis is powered in the
cell.
4. Describe the coordinated regulation of glycolysis
and gluconeogenesis.
 Ch.16 Outline

• 16.1 Glycolysis Is an Energy-Conversion Pathway in

Most Organisms
• 16.2 Glycolysis Can Be Divided into Two Parts
• 16.3 The Glycolytic Pathway Is Tightly Controlled
• 16.4 Glucose Can Be Synthesized from
Noncarbohydrate Precursors
• 16.5 Gluconeogenesis and Glycolysis Are
Reciprocally Regulated
 Section 16.1 Glycolysis Is an
Energy-Conversion Pathway in Most
Organisms
• glycolysis = the sequence of reactions that
metabolizes one molecule of glucose to two molecules
of pyruvate with the concomitant net production of two
molecules of ATP
– anaerobic process (does not require O2)
– common to both prokaryotic and eukaryotic cells

• gluconeogenesis = the process by which metabolic

products are salvaged to synthesize glucose
 Glucose Can Be Metabolized to
Different Products Depending on the
Presence or Absence of Oxygen
 Glucose Is Generated from Dietary
Carbohydrates
• Glucose is typically consumed as starch and glycogen
and must be broken down for absorption and transport.
• α-amylase = pancreatic enzyme that digests starch and
glycogen
– cleaves α-1,4 bonds, but not α-1,6 bonds
– produces maltose and maltotriose

• α-glucosidase (maltase) = intestinal surface enzyme

cleaves maltose, maltotriose, and other α-1,4-linked
oligosaccharides into glucose molecules
• sucrase = intestinal surface enzyme that degrades
ingested sucrose to fructose and glucose
 Transport of Monosaccharides

• Monosaccharides are transported into endothelial cells

lining the intestine by active transporters.
• Glucose moves passively down its concentration
gradient into the bloodstream and passively into cells
that will utilize it.
 A Family of Transporters Enables
Glucose to Enter and Leave Animal
Cells
• Glucose transporters (GLUT1 to GLUT5) mediate the
thermodynamically downhill movement of glucose across
animal plasma membranes.
– have a 12-transmembrane-helix structure
– each member of the family has a distinctive role
 Family of Glucose Transporters

TABLE 16.1 Family of glucose transporters

Name Tissue location KM Comments
GLUT1 All mammalian tissues 1 mM Basal glucose uptake
GLUT2 Liver and pancreatic β 15–20 mM In the pancreas, plays a role in the
cells regulation of insulin
In the liver, removes excess glucose from
the blood
GLUT3 All mammalian tissues 1 mM Basal glucose uptake
GLUT4 Muscle and fat cells 5 mM Amount in muscle plasma membrane
increases with endurance training
GLUT5 Small intestine — Primarily a fructose transporter
 Section 16.2 Glycolysis Can Be
Divided into Two Parts
• 10 enzyme-catalyzed reactions make up glycolysis.
• Stage 1: traps glucose in the cell and modifies it so that it
can be cleaved into phosphorylated three-carbon units
– does not generate ATP

• Stage 2: oxidizes three-carbon unit to pyruvate

– generates 2 molecules of ATP
The Glycolytic Pathway—Stage 1
The Glycolytic Pathway—Stage 2
The First Stage of Glycolysis Uses the
Energy from Two Molecules of ATP
 Stage 1 Begins: Hexokinase Traps
Glucose in the Cell and Begins
Glycolysis
• kinases = enzymes that catalyze the transfer of a
phosphoryl group from ATP to an acceptor, or to ADP
from a phosphoryl donor
• Hexokinase uses ATP to phosphorylate glucose to
glucose 6-phosphate (G-6P).
– requires Mg2+ or Mn2+ as a cofactor
– traps glucose in the cell because G-6P is negatively
charged and is not a substrate for glucose transporters
– facilitates the metabolism of glucose into three-carbon
molecules with high phosphoryl-transfer potential
The Hexokinase Reaction
 Induced Fit of Hexokinase

• Binding of glucose
causes the two lobes
of hexokinase to move
toward each other.
– makes the
environment around
glucose more
nonpolar favoring the
reaction
– excludes water from
the active site
 Fructose 6-Phosphate Is Generated
from Glucose 6-Phosphate
• Phosphoglucose isomerase catalyzes the
isomerization of G-6P to the fructose 6-phosphate
(F-6P).
– catalyzes a conversion of an aldose into a ketose
– take several steps because the enzyme must open
the G-6P ring, catalyze the isomerization, and form
the F-6P ring
– prevents the reformation of glucose 6-phosphate
– ensures that two interconvertible 3-carbon fragments
results from Stage 1
The Phosphoglucose Isomerase
Reaction
 Fructose 1,6-Bisphosphate Is
Generated from Fructose 6-Phosphate
• Phosphofructokinase (PFK) uses ATP to phosphorylate
F-6P to fructose 1,6-bisphosphate (F-l,6-BP).
– PFK is an allosteric enzyme that sets the pace of
glycolysis.
 The Six-Carbon Sugar Is Cleaved
into Two Three-Carbon Fragments
• Aldose cleaves F-l,6-BP into glyceraldehyde 3-phosphate
(GAP) and dihydroxyacetone phosphate (DHAP).
– this reaction completes Stage 1 of glycolysis
– readily reversible
 Dihydroxyacetone Phosphate Is
Isomerized to Glyceraldehyde 3-
Phosphate
• Only GAP can be processed to pyruvate to yield ATP.
• Triose phosphate isomerase (TPI) catalyzes the
isomerization of DHAP to GAP.
– readily reversible, but proceeds to GAP because
subsequent glycolysis reactions remove GAP
 Triose Phosphate Isomerase Is an
Example of a Common "Barrel" Motif

• The active site is

buried in the center.
 Mechanism: Triose Phosphate
Isomerase Salvages a Three-Carbon
Fragment
• TPI catalyzes the transfer of a hydrogen atom from
carbon 1 to carbon 2.
– This is an intramolecular oxidation–reduction.
– Isomerization proceeds through an enediol intermediate.
The Catalytic Mechanism of Triose
Phosphate Isomerase
 The Roles of Glu 165 and His 95

• Glu 165 plays the role of a general acid–base catalyst.

• His 95 assists catalysis by donating a proton to stabilize
the negative charge that develops on the C-2 carbonyl
group.
Features of Triose Phosphate Isomerase
• TPI accelerates isomerization by a factor of 1010
compared with a simple base catalyst.
– The kcat /KM ratio for the isomerization of GAP is close to
the diffusion-controlled limit.
– TPI is a kinetically perfect enzyme.

• TPI suppresses an undesired side reaction.

The first stage of glycolysis: (1 of 2)

a. produces two molecules of ATP.

b. ends with the production of two molecules of
dihydroxyacetone phosphate.
c. involves two phosphorylation reactions and an
isomerization.
d. traps fructose in the cell.
e. forms a compound that can be readily cleaved into
phosphorylated two-carbon units.

© Macmillan Learning, 2023

The first stage of glycolysis: (2 of 2)

a. produces two molecules of ATP.

b. ends with the production of two molecules of
dihydroxyacetone phosphate.
*c. involves two phosphorylation reactions and an
isomerization.
d. traps fructose in the cell.
e. forms a compound that can be readily cleaved into
phosphorylated two-carbon units.

© Macmillan Learning, 2023

The Second Stage of Glycolysis
Generates ATP and NADH
Stage 2 Begins: The Oxidation of an
Aldehyde Powers the Formation of a
Compound with High Phosphoryl-
Transfer Potential
• Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
catalyzes the conversion of GAP into 1,3-
bisphosphoglycerate (1,3-BPG).
– 1,3-BPG has high phosphoryl-transfer potential.
 The Glyceraldehyde 3-Phosphate
Dehydrogenase Reaction Is the Sum
of Two Processes
• Process 1: the oxidation of the aldehyde to a
carboxylic acid by NAD+
– thermodynamically favorable (∆G°′ of approximately
−50 kJ mol−1)
 The Glyceraldehyde 3-Phosphate
Dehydrogenase Reaction Is the Sum
of Two Processes, Continued
• Process 2: the joining of the carboxylic acid and
orthophosphate to form the acyl-phosphate product
– thermodynamically unfavorable (∆G°′ of approximately
+50 kJ mol−1)
 The Glyceraldehyde 3-Phosphate
Dehydrogenase Reactions Are
Coupled
• The reactions are linked by the formation of an energy-
rich thioester intermediate in the active site of GAPDH.
– preserves much of the free energy released in the
oxidation reaction
 Mechanism: Phosphorylation Is
Coupled to the Oxidation of
Glyceraldehyde 3-Phosphate by a
Thioester Intermediate
• The active site of GAPDH includes:
– a reactive Cys residue.
– NAD+.
– a crucial His.
 The Four Steps in the GAPDH
Reaction Mechanism
• Step 1: the aldehyde substrate reacts with the sulfhydryl
group of Cys 149 on the enzyme to form a hemithioacetal
• Step 2: the hydride ion is transferred to NAD+ bound to the
enzyme to yield NADH and a thioester intermediate
– favored by the deprotonation of the hemithioacetal by His
176

• Step 3: NADH leaves and is replaced by NAD+

– NAD+ polarizes the thioester intermediate to facilitate the
attack by orthophosphate

• Step 4: orthophosphate attacks the thioester to form 1,3-

BPG and free the cysteine residue
The Active Site of GAPDH
The Catalytic Mechanism of GAPDH

• energy released by carbon

oxidation is converted into high
phosphoryl-transfer potential
ATP Is Formed by Phosphoryl Transfer
from 1,3-Bisphosphoglycerate
• 1,3-BPG has greater phosphoryl-transfer potential than
ATP.
– can be used to power the ATP synthesis

• Phosphoglycerate kinase catalyzes the transfer of the

phosphoryl group from 1,3-BPG to ADP.
– yields ATP and 3-phosphoglycerate

• ATP is formed by by substrate-level phosphorylation.

Additional ATP Is Generated with the
Formation of Pyruvate
• Phosphoglycerate mutase catalyzes the conversion of 3-
phosphoglycerate to 2-phosphoglycerate.
– requires catalytic amounts of 2,3-BPG to maintain an
active-site His residue in a phosphorylated form
 The Phosphoglycerate Mutase
Reaction
• Step 1: Phosphoryl group is transferred to 3-
phosphoglycerate to form 2,3-BPG

• Step 2: 2,3-BPG is converted to 2-phosphoglycerate

– The mutase retains the phosphoryl group to regenerate the
modified histidine.

• The net reaction is

 2-Phosphoglycerate Is Dehydrated
to Phosphenolpyruvate
• Enolase catalyzes the dehydration of 2-phosphoglycerate
to phosphoenolpyruvate (PEP).
• PEP has high phosphoryl-transfer potential because the
phosphoryl group traps the molecule in its unstable enol
form.
Phosphenolpyruvate Is Converted to
Pyruvate
• Pyruvate kinase catalyzes the transfer of a phosphoryl
group from PEP to ADP, generating pyruvate and ATP.
The Final Three Reactions in
Glycolysis
 Two ATP Molecules Are Formed in
the Conversion of Glucose into
Pyruvate
• The net reaction in glycolysis is

Glucose  2 Pi  2 ADP  2 NAD 

2 pyruvate  2 ATP  2 NADH  2 H  2 H2O

• The energy released in glycolysis is about −90 kJ mol−1.

 Reactions of Glycolysis
TABLE 16.2 Reactions of glycolysis
∆G°′ in kJ ∆G in kJ
mol−1 (kcal mol−1 (kcal
Step Reaction Enzyme Reaction type mol−1) mol−1)
1 Glucose + ATP → glucose 6-phosphate + ADP + H+ Hexokinase Phosphoryl −16.7 (−4.0) −33.5 (−8.0)
transfer
2 Phosphoglucose Isomerization +1.7 (+0.4) −2.5 (−0.6)
isomerase
3 Fructose 6-phosphate + ATP → Phosphofructokinase Phosphoryl −14.2 (−3.4) −22.2 (−5.3)
fructose 1, 6-bisphosphate + ADP + H+ transfer
4 Aldolase Aldol cleavage +23.8 (+5.7) −1.3 (−0.3)

5 Triose phosphate Isomerization +7.5 (+1.8) +2.5 (+0.6)

isomerase
6 Glyceraldehyde 3- Phosphorylation +6.3 (+1.5) −1.7 (−0.4)
phosphate coupled to
dehydrogenase oxidation
7 Phosphoglycerate Phosphoryl −18.8 (−4.5) +1.3 (+0.3)
kinase transfer

8 Phosphoglycerate Phosphoryl shift +4.6 (+1.1) +0.8 (+0.2)

mutase
9 Enolase Dehydration +1.7 (+0.4) −3.3 (−0.8)

10 Phosphoenolpyruvate + ADP + H+ → pyruvate + ATP Pyruvate kinase Phosphoryl −31.4 (−7.5) −16.7 (−4.0)
transfer

Note: ∆G, the actual free-energy change, has been calculated from ∆G°′ and known concentrations of reactants under typical physiological
conditions. Glycolysis can proceed only if the ∆G values of all reactions are negative. The small positive ∆G values of three of the above
reactions indicate that the concentrations of metabolites in vivo in cells undergoing glycolysis are not precisely known.
What step of glycolysis is this
reaction from? (1 of 2)
What step of glycolysis is this
reaction from? (2 of 2)

Step 6
 NAD+ Is Regenerated from the
Metabolism of Pyruvate
• NAD+ is derived from the vitamin niacin (B3).
• GAPDH reduces NAD+ to NADH.
• NAD+ must be regenerated for glycolysis to proceed.
• NAD+ can be regenerated by:
– oxidation of pyruvate to CO2 in the presence of O2.
– fermentation of pyruvate to ethanol or lactate in the
absence of O2.

• fermentation = an ATP-generating process in which

organic compounds act as both electron donors and
acceptors
Pyruvate Has Many Possible Fates
 Ethanol Fermentation

• ethanol fermentation = the conversion of glucose into

ethanol in anerobic conditions
– occurs in yeast and other microorganisms
– used for brewing and winemaking

• Step 1: pyruvate decarboxylase catalyzes the

decarboxylation of pyruvate
– requires the coenzyme thiamine pyrophosphate that is
derived from the vitamin thiamine (B1)

• Step 2: alcohol dehydrogenase catalyzes the reduction

of acetaldehyde to ethanol by NADH
– regenerates NAD+
The Reactions of Ethanol
Fermentation
 The Active Site of Alcohol
Dehydrogenase
• The active site contains a
zinc ion coordinated to the
sulfur atoms of two Cys
residues and a nitrogen
atom of His.
• Zinc polarizes the carbonyl
group of the substrate to
favor the transfer of a
hydride from NADH.
Two ATP Molecules Are Formed in the
Conversion of Glucose to Ethanol
• The net reaction in this anaerobic process is
Glucose  2 Pi  2 ADP  2 H 
2 ethanol  2 CO2  2 ATP  2 H2O

• NADH generated by the oxidation of GAP is consumed

in the reduction of acetaldehyde to ethanol.
The NADH Produced in Glycolysis
Must Be Reoxidized to NAD+ for the
Glycolytic Pathway to Continue
 Lactic Acid Fermentation
• lactic acid fermentation = the conversion of glucose into
lactate in anerobic conditions
– occurs in microorganisms and animal tissues, including
skeletal muscles

• Lactase dehydrogenase catalyzes the reduction of

pyruvate by NADH to lactase.
Two ATP Molecules Are Formed in the
Conversion of Glucose to Lactate
• The net reaction in this anaerobic process is
Glucose  2 Pi  2 ADP  2 lactate  2 ATP  2 H2O

• NADH generated by the oxidation of GAP is consumed

in the reduction of pyruvate.
 NAD+ Is Regenerated by Lactate
Dehydrogenase During Lactic Acid
Fermentation
Oxidation by the Citric Acid Cycle and
the Electron Transport Chain
• Pyruvate can be metabolized under aerobic conditions
through the citric acid cycle and the electron-transport
chain.
– forms CO2, H2O, and a great deal of energy
– the transfer of electrons to O2 through the electron-transport
chain regenerates NAD+

• Pyruvate dehydrogenase complex catalyzes the oxidative

decarboxylation of pyruvate to acetyl coenzyme A (acetyl
CoA) in mitochondria
Pyruvate  NAD  CoA  acetyl CoA  CO2  NADH  H
 Fermentations Provide Usable
Energy in the Absence of Oxygen
• obligate anaerobes = organisms that cannot survive in
the presence of O2
– example: the bacterium Clostridium perfringens, the cause
of gangrene

• facultative anaerobes = organisms that metabolize

glucose aerobically when O2 is present and perform
fermentation when O2 is absent
• Microorganisms are capable of generating a wide array
of molecules as end points of fermentation.
 Examples of Pathogenic Obligate
Anaerobes
TABLE 16.3 Examples of pathogenic obligate anaerobes
Bacterium Result of infection
Clostridium tetani Tetanus (lockjaw)
Clostridium botulinum Botulism (an especially severe type of food poisoning)
Clostridium perfringens Gas gangrene (gas is produced as an end point of the fermentation,
distorting and destroying the tissue)
Bartonella hensela Cat scratch fever (flu-like symptoms)
Bacteroides fragilis Abdominal, pelvic, pulmonary, and blood infections
Starting and Ending Points of
Various Fermentations
TABLE 16.4 Starting and ending points
of various fermentations
Glucose → Lactate
Lactate → Acetate
Glucose → Ethanol
Ethanol → Acetate
Arginine → Carbon dioxide
Pyrimidines → Carbon dioxide
Purines → Formate
Ethylene glycol → Acetate
Threonine → Propionate
Leucine → 2-Alkylacetate
Phenylalanine → Propionate

Note: The products of some fermentations

are the substrates for others.
 Identify the bond that polarizes the
substrate so that it more easily accepts a
hydride from NADH. (1 of 2)
 Identify the bond that polarizes the
substrate so that it more easily accepts a
hydride from NADH. (2 of 2)
Fructose is Converted into Glycolytic
Intermediates by Fructokinase
• Fructose is found in table sugar and high fructose corn
syrup.
• To be metabolized, fructose must be converted into a
metabolite of glucose.
• The fructose 1-phosphate pathway metabolizes fructose
in the liver.
 Additional Sugars Can Enter
Glycolysis at a Variety of Points
Fructose Metabolism in the Liver
 Outcomes of Excessive Fructose
Consumption
• Epidemiological and clinical studies have previously
linked excess consumption of fructose to fatty liver,
insulin insensitivity, obesity, and type 2 diabetes.
– not supported by recent meta-analyses

• Overconsumption of sugars and fats is still strongly

associated with these clinical outcomes.
• Excess pyruvate is converted into acetyl CoA and then
into fatty acids.
Galactose Is Converted into Glucose
6-Phosphate
• The galactose–glucose interconversion pathway, a four-
step pathway, converts galactose to glucose 6-
phosphate.

• Step 1 is the phosphorylation of galactose by

galactokinase.
 Glucose Is Converted into UDP-
Glucose
• Uridine diphosphate
glucose (UDP-glucose)
is an activated
intermediate in the
synthesis of
carbohydrates.
 The Galactose–Glucose
Interconversion Pathway
• The sum of the reactions of the galactose–glucose
conversion pathway is
Galactose  ATP  glucose 1-phosphate  ADP  H

• UDP-glucose is not consumed in the conversion of

galactose into glucose.
• Phosphoglucomutase isomerizes glucose 1-phosphate
to glucose 6-phosphate.
Galactose Can Be Highly Toxic with a
Defective Metabolic Pathway
• galactosemias = rare disorders that interfere with the
metabolism of galactose
– causes vomiting and diarrhea after milk consumption,
enlargement of the liver, jaundice, cataracts, lethargy,
and delayed neurological development
– often results from an inherited deficiency in galactose 1-
phosphate uridyl transferase activity

• Treatment is to remove galactose and lactose from the

diet.
 Cataracts Are Evident as the
Clouding of the Lens
• A cataract is the clouding
of the lens of the eye due
to pathological protein
aggregation.
 Cataract Formation

• Aldose reductase catalyzes

the reduction of galactose
to galactitol in the lens
when the transferase is not
active.
• Galactitol is poorly
metabolized and
accumulates.
• Water diffuses into the lens
to maintain osmotic
balance, triggering the
formation of cataracts.
 Many Adults Worldwide Are
Intolerant of Milk Because They Are
Deficient in Lactase
• lactase = an enzyme that cleaves lactose into glucose
and galactose

• Lactose intolerance is most commonly caused by a

deficiency of lactase.
• treatment involves avoiding lactose products or ingesting
lactase with milk products
 Lactobacillus Is One Example of an
Industrially Useful Anaerobic
Bacterium
• Lactose is a good energy
source for microorganisms in
the colon which ferment it to
lactic acid while generating
methane (CH4) and hydrogen
gas (H2)
– causes gut distension and
flatulence
– lactate and undigested lactose
are osmotically active and
draw water into the intestine,
causing diarrhea
 Which is a means by which
additional sugars enter glycolysis?
(1 of 2)
a. Galactose is converted to fructose 6-phophsophate.
b. Fructose in the adipose tissue is converted to glucose 6-
phosphate.
c. Fructose in the liver is converted to dihydroxyacetone
phosphate or glyceraldehyde 3-phosphate.
d. Galactose is converted to fructose 1,6-bisphosphate.
e. Lactose is converted to two molecules of fructose 6-
phosphate.

© Macmillan Learning, 2023

 Which is a means by which
additional sugars enter glycolysis?
(2 of 2)
a. Galactose is converted to fructose 6-phophsophate.
b. Fructose in the adipose tissue is converted to glucose 6-
phosphate.
*c. Fructose in the liver is converted to dihydroxyacetone
phosphate or glyceraldehyde 3-phosphate.
d. Galactose is converted to fructose 1,6-bisphosphate.
e. Lactose is converted to two molecules of fructose 6-
phosphate.

© Macmillan Learning, 2023

Section 16.3 The Glycolytic Pathway
Is Tightly Controlled
• Enzymes catalyzing irreversible reactions in metabolic
pathways are control sites.
– controlled by allosteric effectors, covalent modification, or
by regulating transcription

• In glycolysis, three enzymes catalyze irreversible

reactions:
– hexokinase
– phosphofructokinase
– pyruvate kinase
Glycolysis in Muscle Is Regulated to
Meet the Need for ATP
• Glycolysis in skeletal muscle provides ATP primarily to
power contraction.
• The primary control of muscle glycolysis is the ratio of
ATP to AMP.
 Regulation of Phosphofructokinase
by AMP and ATP in Muscle
• High levels of ATP allosterically inhibit
phosphofructokinase.
– Binding to a specific regulatory site lowers the enzyme's
affinity for F-6P.

• AMP reverses the inhibitory action of ATP.

• Enzyme activity increases when the ATP/AMP ratio is
lowered.
• committed step = the first irreversible reaction unique to
the glycolytic pathway
– example: phosphorylation of F-6P to F-l,6-BP by
phosphofructokinase
Structure of Phosphofructokinase
 A High Level of ATP Allosterically
Inhibits Phosphofructokinase
• High [ATP] converts the hyperbolic binding curve of F-6P
into a sigmoidal one.
 Regulation of Phosphofructokinase
by pH in Muscle
• A decrease in pH inhibits phosphofructokinase activity by
augmenting the inhibitory effect of ATP.
– occurs when muscle is functioning anaerobically and
producing excessive lactic acid
– prevents muscle damage from too much acid
 AMP as a Positive Regulator of
Phosphofructokinase
• ADP is not the positive regulator of phosphofructokinase
because adenylate kinase forms ATP from ADP

• AMP serves as the signal for the low-energy state.

• AMP as an allosteric regulator provides sensitive control
because in the cell [ATP] > [ADP] > [AMP], meaning
small changes in [ATP] result in larger changes in [AMP].
Regulation of Hexokinase in Muscle

• Hexokinase is inhibited by its product, G-6P

– high [G-6P] signals that the cell no longer requires glucose
for energy or synthesis of glycogen
– leads to glucose remaining in blood

• Inhibition of phosphofructokinase leads to the inhibition

of hexokinase because a rise in [F-6P] leads to a rise in
[G-6P] as they are in equilibrium.
 Regulation of Pyruvate Kinase in
Muscle
• Pyruvate kinase is allosterically inhibited by ATP.
– slows glycolysis when the energy charge is high

• feedforward stimulation (feedforward activation) = occurs

when products of a preceding irreversible step activate
the enzyme
– example: when the pace of glycolysis increases, F-l,6-BP
activates the kinase
Regulation of Glycolysis in Muscle
 During short bursts of intense
exercise, such as during a sprint, the
muscle functions anaerobically until
fatigue sets in. Does the activity of a
phosphofructokinase increase,
decrease, or stay the same? (1 of 2)
 During short bursts of intense
exercise, such as during a sprint, the
muscle functions anaerobically until
fatigue sets in. Does the activity of a
phosphofructokinase increase,
decrease, or stay the same? (2 of 2)
decrease
 The Regulation of Glycolysis in the
Liver Illustrates the Biochemical
Versatility of the Liver
• The liver maintains blood-glucose concentration,
generates reducing power for biosynthesis, and
synthesizes biochemicals.
• Regulation is more complex than in the muscle.
Regulation of Phosphofructokinase
in Liver
• Liver phosphofructokinase can be regulated by ATP.
– not an important metabolic signal as the liver does not
experience sudden ATP needs

• Liver phosphofructokinase can be regulated by pH.

– not an important metabolic signal as the liver does not
produce lactate

• Phosphofructokinase is inhibited by citrate, an early

intermediate in the citric acid cycle, by enhancing the
inhibitory effect of ATP.
– High citate means biosynthetic precursors are abundant.
 Regulation of Phosphofructokinase
in Liver, Continued
• Phosphofructokinase is
activated by fructose 2,6-
bisphosphate (F-2,6-BP).
– Binding of F-2,6-BP increases
the affinity of the enzyme for
F-6P and diminishes the
inhibitory effect of ATP.

• High F-6P accelerates the

synthesis of F-2,6-BP.
Activation of Phosphofructokinase
by Fructose 2,6-Bisphosphate
 Regulation of Hexokinase and
Glucokinase in Liver
• Hexokinase is inhibited by its product, G-6P, as in
muscle.
• glucokinase = a specialized isozyme of hexokinase that
provides G-6P for glycogen synthesis and fatty acid
formation
– not inhibited by G-6P
– functions as a monomer, but displays sigmoidal kinetics
– phosphorylates glucose only when glucose is abundant
– inhibited by the liver-specific glucokinase regulatory protein
(GKRP) when [glucose] is low
 Regulation of Pyruvate Kinase in
Liver
• Several isozymic forms of pyruvate kinase are present in
mammals:
– L type = predominates in the liver
– M type = predominates in muscle and the brain

• Pyruvate kinase is allosterically inhibited by ATP and

alanine (synthesized in one step from pyruvate).
• L form is controlled by reversible phosphorylation.
– When blood-glucose level is low, the glucagon-triggered
cyclic AMP cascade leads to the phosphorylation of
pyruvate kinase, which diminishes its activity.
 Pyruvate Kinase Activity Is
Controlled by Phosphorylation and
Dephosphorylation
 In the liver, the abundance of
fructose 6-phosphate accelerates the
synthesis of fructose 2,6-
bisphosphate. Does the activity of a
phosphofructokinase increase,
decrease, or stay the same? (1 of 2)
 In the liver, the abundance of
fructose 6-phosphate accelerates the
synthesis of fructose 2,6-
bisphosphate. Does the activity of a
phosphofructokinase increase,
decrease, or stay the same? (2 of 2)
increase
 The Enzymes of Glycolysis Are
Physically Associated with One Another
• Enzymes of glycolysis in eukaryotes are organized into
complexes.
• substrate channeling = process that facilitates movement
of substrates and products between enzymes
– increases enzyme efficiency
– prevents the release of any toxic intermediates
 Aerobic Glycolysis Is a Property of
Tumor Cells and Other Rapidly
Growing Cells
• Tumors display enhanced rates of glucose uptake and
glycolysis.
• aerobic glycolysis (the Warburg effect) = process by
which rapidly growing tumor cells metabolize glucose to
lactate even in the presence of oxygen
• 2-18F-2-D-deoxyglucose (FDG) = a nonmetabolizable
glucose analog that is detectable by a combination of
positron emission tomography (PET) and computer-
aided tomography (CAT)
 Tumors Can Be Visualized by FDG
and Positron Emission Tomography
 Possible Selective Benefits of
Aerobic Glycolysis
• Aerobic glycolysis generates lactic acid.
– Acidification of the tumor environment facilitates tumor
invasion.
– Lactate impairs the activation of CD8+ T and NK immune
system cells that normally attack the tumor.

• Increased glucose uptake and G-6P formation provide

substrates for the NADPH-generating pentose
phosphate pathway.
• As solid tumors grow, [O2] in their environment falls and
the use of aerobic glycolysis reduces the dependence of
cell growth on O2.
Cancer and Endurance Training Affect
Glycolysis in a Similar Fashion
• hypoxia-inducible transcription factor (HIF-1) =
transcription factor that facilitates aerobic glycolysis
– increases the expression of glycolytic enzymes, glucose
transporters, and signal molecules
– cancer and anaerobic exercise training activate HIF-1

• vascular endothelial growth factor (VEGF) = signal

molecule that facilitates the growth of blood vessels that
will provide nutrients to the cells
Proteins in Glucose Metabolism
Encoded by Genes Regulated by
Hypoxia-Inducible Factor
TABLE 16.5 Proteins in glucose metabolism encoded by genes
regulated by hypoxia-inducible factor

GLUT1
GLUT3
Hexokinase
Phosphofructokinase
Aldolase
Glyceraldehyde 3-phosphate dehydrogenase
Phosphoglycerate kinase
Enolase
Pyruvate kinase
Hypoxia Alters Gene Expression in
Tumors
 Section 16.4 Glucose Can Be
Synthesized from Noncarbohydrate
Precursors
• gluconeogenesis = the synthesis of glucose from
noncarbohydrate precursors
– converts pyruvate into glucose
– occurs mainly in the liver, with a small amount in the kidney
and other tissues
– important during starvation and fasting as glucose is the
primary fuel for the brain and the only fuel for red blood
cells
 Gluconeogenesis Precursors

• The major precursors for gluconeogenesis are lactate,

amino acids, and glycerol.
– are converted to pyruvate or enter the pathway at later
intermediates

• Lactate dehydrogenase converts lactase into pyruvate.

• Triacylglycerol hydrolysis in fat cells yields glycerol and
fatty acids.
– Animals cannot convert fatty acids into glucose.
– Glycerol enters either the gluconeogenic or the glycolytic
pathway as dihydroxyacetone phosphate.
The Conversion of Glycerol to
Dihydroxyacetone Phosphate
Pathway of Gluconeogenesis
Which enzymes are new to
gluconeogenesis? (1 of 2)
Which enzymes are new to
gluconeogenesis? (2 of 2)
Gluconeogenesis Is Not a Reversal of
Glycolysis
• The three irreversible steps in glycolysis must be
bypassed in gluconeogenesis.
 The Conversion of Pyruvate into
Phosphoenolpyruvate Begins with
the Formation of Oxaloacetate
• Pyruvate carboxylase catalyzes the carboxylation of
pyruvate to oxaloacetate using a molecule of ATP.
– occurs in the mitochondria
– requires biotin, a covalently attached prosthetic group,
which serves as the carrier of activated CO2
 Carboxybiotin Linked to Lysine Can
Act as a Movable "Arm" Carrying
Activated CO2 Groups
• The carboxylate
group of biotin is
linked to the ε-
amino group of a
specific Lys
residue by an
amide bond.
 Carboxylation of Pyruvate

• takes place in three stages:

• Pyruvate carboxylase functions as a tetramer composed

of four identical subunits, each of which contains four
domains.
The Domains of Pyruvate Carboxylase
• biotin carboxylase domain (BC) = catalyzes the
formation of carboxyphosphate and the attachment of
CO2 to the biotin carboxyl carrier protein domain (BCCP)
• BCCP = swings to the active site of the carboxyl
transferase domain (CT)
• CT = transfers the CO2 to pyruvate to form oxaloacetate
• pyruvate carboxylase tetramerization domain (PT) =
facilitates the formation of the tetramer and is the binding
site for acetyl CoA, a required allosteric activator
A Subunit of Pyruvate Carboxylase
Shows the Complexity of the
Enzyme
 Oxaloacetate Is Shuttled into the
Cytoplasm and Converted into
Phosphoenolpyruvate
• Malate dehydrogenase catalyzes the reduction of
oxaloacetate to malate.
• Malate is transported from the mitochondria to the
cytoplasm by a cytoplasmic NAD+-linked malate
dehydrogenase and reoxidized to oxaloacetate.
– Reoxidation yields NADH.

• Phosphoenolpyruvate carboxykinase (PEPCK)

decarboxylates and phosphorylates oxaloacetate to
phosphoenolpyruvate.
– Phosphoryl donor is GTP.
Oxaloacetate Used in the Cytoplasm
for Gluconeogenesis Is Formed in
the Mitochondrial Matrix
Oxaloacetate Is Decarboxylated and
Phosphorylated to
Phosphoenolpyruvate
One ATP and One GTP Molecule Are
Expended in the Conversion of
Pyruvate into Phosphoenolpyruvate
• The sum of the reactions catalyzed by pyruvate
carboxylase and phosphoenolpyruvate carboxykinase is
Pyruvate  ATP  GTP  H2O 
phosphoenolpyruvate  ADP  GDP  Pi  2 H

• These reactions bypass the pyruvate kinase reaction in

glycolysis.
 The Conversion of Fructose 1,6-
Bisphosphate into Fructose 6-
Phosphate and Orthophosphate Is an
Irreversible Step
• Phosphoenolpyruvate is metabolized by the enzymes of
glycolysis in the reverse direction until the next irreversible
step.
• The allosteric enzyme fructose 1,6-bisphosphatase
catalyzes the hydrolysis of fructose 1,6-bisphosphate to
fructose 6-phosphate and Pi.
Fructose 1,6-bisphosphate  H2O 
Fructose 1,6-bisphosphatase

fructose 6-phosphate  Pi
 Fructose 1,6-Bisphosphatase Is a
Phosphatase
• phosphatase = an enzyme that catalyzes the hydrolysis
of a phosphate to form inorganic phosphate
• The difference between kinases and phosphatases is
whether the phosphate is transferred to water or ADP.
 The Generation of Free Glucose
Occurs Only in Some Tissues and Is
an Important Control Point
• The generation of free glucose occurs primarily in the liver.
• Glucose 6-phosphate is transported into the lumen of the
ER.
• Glucose 6-phosphatase hydrolyzes glucose 6-phosphate
to glucose.
– This enzyme is bound to the ER membrane.

• Glucose and Pi are shuttled back to the cytoplasm by

transporters.
• In most tissues, tissues, glucose 6-phosphate is converted
into glycogen for storage.
In the Liver, Free Glucose Is Generated
from Glucose 6-Phosphate by Glucose
6-Phosphatase
 Six High Transfer-Potential
Phosphoryl Groups Are Spent in
Synthesizing Glucose from Pyruvate
• The formation of glucose from pyruvate is energetically
unfavorable unless it is coupled to favorable reactions.
• The stoichiometry of gluconeogenesis is
2 Pyruvate  4 ATP  2 GTP  2 NADH  6 H2O 
glucose  4 ADP  2 GTP  6 Pi  2 NAD  2 H
G  48 kJ mol1  11kcal mol1 

• The stoichiometry for the reversal of glycolysis is

2 Pyruvate  2 ATP  NADH  2 H2O 
glucose  2 ADP  2 Pi  2 NAD  2 H
G   90 kJ mol1   22 kcal mol1 
 Reactions of Gluconeogenesis
TABLE 16.6 Reactions of gluconeogenesis
∆G°′ in
Step Reaction Enzyme Reaction type kJ mol−1
1. Pyruvate + CO2 + ATP + H2O → oxaloacetate Pyruvate carboxylase Phosphoryl –4.2
+ ADP + Pi + 2H+ transfer
2. Phosphoenolpyruvate Phosphoryl- +5.8
carboxykinase coupled oxidation
3. Enolase Hydration –3.4

4. Phosphoglycerate mutase Phosphoryl shift –9.2

5. Phosphoglycerate kinase Phosphoryl +37.6

transfer
6. Glyceraldehyde 3- Phosphoryl- –12.6
phosphate dehydrogenase coupled reduction
7. Triose phosphate Isomerization –7.6
isomerase
8. Aldolase Aldol addition –23.9

9. Fructose 1,6-bisphosphate + H2O → fructose Fructose 1,6- Phosphoryl –16.3

6-phosphate + Pi bisphosphatase transfer
10. Phosphoglucose Isomerization –1.7
isomerase
11. Glucose 6-phosphatase + H2O → glucose + Pi Glucose 6-phosphatase Phosphoryl –12.1
transfer
 Which enzyme of gluconeogenesis is
located in the mitochondria? (1 of 2)
a. glucose 6-phosphatase
b. fructose 1,6-bisphosphate
c. enolase
d. phosphoenolpyruvate carboxykinase
e. pyruvate carboxylase

© Macmillan Learning, 2023

 Which enzyme of gluconeogenesis is
located in the mitochondria? (2 of 2)
a. glucose 6-phosphatase
b. fructose 1,6-bisphosphate
c. enolase
d. phosphoenolpyruvate carboxykinase
*e. pyruvate carboxylase

© Macmillan Learning, 2023

 Section 16.4 Gluconeogenesis and
Glycolysis Are Reciprocally Regulated
• Gluconeogenesis and glycolysis are regulated within a
cell such that one pathway is relatively inactive whereas
the other is highly active.
• Glycolysis predominates when energy or glycolytic
intermediates are needed.
• Gluconeogenesis predominates when there is a surplus
of energy and glucose precursors.
Glycolysis and Gluconeogenesis Are
Regulated by Adenosine Nucleotides
and Other Metabolic Intermediates
• The interconversion of fructose 1,6-bisphosphate and
fructose 6-phosphate is a key regulation site.
• When energy is needed, AMP inhibits fructose 1,6-
bisphosphatase and stimulates phosphofructokinase.
– turns on glycolysis and inhibits gluconeogenesis

• When levels of ATP and citrate are high, ATP and citrate
inhibit phosphofructokinase, and the decrease in AMP
relieves fructose 1,6-bisphosphatase inhibition.
– turns off glycolysis and turns on gluconeogenesis
 Reciprocal Regulation at the
Interconversion of
Phosphoenolpyruvate and Pyruvate
• It occurs in the liver.
• Pyruvate kinase is inhibited by allosteric effectors ATP
and alanine.
• Pyruvate carboxylase and phosphoenolpyruvate
carboxykinase are inhibited by ADP.
• Pyruvate carboxylase is activated by acetyl CoA.
 Reciprocal Regulation of
Gluconeogenesis and Glycolysis in
the Liver
 In Mammals, Glycolysis and
Gluconeogenesis in the Liver Are
Controlled by Hormones Sensitive to
Blood-Glucose Concentration
• The rates of glycolysis and gluconeogenesis are
adjusted in the liver to maintain blood-glucose levels.
• F-2,6-BP stimulates phosphofructokinase and inhibits
fructose 1,6-bisphosphatase.
• When blood glucose is low, F-2,6-BP loses a phosphoryl
group to form F-6P.
– F-6P is not an allosteric effector of PFK.
PFK2 and FBPase2 Are Bifunctional
Enzymes
• bifunctional enzyme = contains two enzymes on a single
polypeptide chain
• A bifunctional enzyme, containing phosphofructokinase 2
(PFK2) and fructose bisphosphatase 2 (FBPase2),
determines F-2,6-BP levels.
• F-2,6-BP concentrations are regulated by two enzymes.
– PFK2 forms F-2,6-BP.
– FBPase2 degrades F-2,6-BP.
 The Bifunctional Enzyme
Phosphofructokinase 2 Has Two
Distinct Domains
• contains an
N-terminal
regulatory
domain, a
kinase
domain and a
phosphatase
domain
The Bifunctional Enzyme Is Controlled
by Reversible Phosphorylation
• PFK2 and FBPase2 activities are reciprocally controlled
by phosphorylation of a single Ser residue.
• When glucose is scarce, glucagon rises, triggering a
cyclic AMP signal cascade and leading to
phosphorylation of the bifunctional enzyme by PKA.
– activates FBPase2 and inhibits PFK2 and pyruvate kinase
– lowers F-2,6-BP levels
– gluconeogenesis predominates
 The Bifunctional Enzyme Is
Controlled by Reversible
Phosphorylation, Continued
• When glucose is high, insulin rises, triggering a signal
pathway and leading to dephosphorylation of the
bifunctional enzyme by a protein phosphatase.
– activates PFK2 and inhibits FBPase2
– increases F-2,6-BP levels
– glycolysis predominates
The Synthesis and Degradation of
Fructose 2,6-Bisphosphate Is
Hormonally Controlled
 The Bifunctional Enzyme Is Also
Under Transcriptional Control by
Glucagon and Insulin
• glucagon and insulin altering gene expression by
changing the rate of transcription
• To stimulate gluconeogenesis, glucagon:
– inhibits expression of the three regulated glycolytic
enzymes.
– stimulates the production of phosphoenolpyruvate
carboxykinase and fructose 1,6-bisphosphatase.

• To stimulate glycolysis, insulin stimulates the expression

of phosphofructokinase, pyruvate kinase, and the
bifunctional enzyme that makes and degrades F-2,6-BP.
 Substrate Cycles Amplify Metabolic
Signals and Produce Heat
• substrate cycle = a set of reaction in a loop
– example: the phosphorylation of F-6P to F-1,6-BP and its
hydrolysis back to F-6P
– reciprocal regulation prevents both reactions from being
active at the same time

• futile cycles = substrate cycles with a limited degree of

cycling
– may result in pathological conditions, such as malignant
hyperthermia
– may be biologically important in enhancing metabolic
signals
 Substrate Cycles Can Have
Advantages
• A small change in the rates
of the two opposing
reactions can result in a
large change in the net flux.
 Lactate and Alanine Formed by
Contracting Muscle and Peripheral
Tissues Are Used by Other Organs
• Cori cycle = a series of reactions carried out by
cooperation between the liver and muscle
• Lactate produced by active muscle is released into the
blood and converted to glucose by the liver.
• Nitrogens from amino acids used by muscle for fuel are
transferred to pyruvate to form alanine.
– The reverse reaction takes place in the liver.
The Cori Cycle
 During a Sprint, Cooperation Between
Glycolysis and Gluconeogenesis Occurs
Within Multiple Tissues
 Deficiencies in Glycolytic or
Gluconeogenic Enzymes Are Rare
Genetic Disorders
• inborn errors of metabolism = genetically inherited
deficiencies in the activity or regulation of specific
enzymes
– examples: triose phosphate isomerase deficiency and
pyruvate carboxylase deficiency
Triose Phosphate Isomerase Deficiency
(TPID)
• TPID = a multisystem disorder resulting from
dihydroxyacetone phosphate accumulation in cells,
especially red blood cells
– presents in early childhood and may lead to death
– symptoms include congenital hemolytic anemia and
progressive neuromuscular disorder
 Triose Phosphate Isomerase
Deficiency (TPID), Continued
• When triose phosphate isomerase activity is
missing, half of the carbons of glucose cannot
be metabolized to yield ATP.
– Research suggests disruption of energy
metabolism is not the cause TPID symptoms.

• Instead, buildup of dihydroxyacetone

phosphate causes TPID symptoms, as it can
be converted into methylglyoxal.
• methylglyoxal = highly reactive molecule that
covalently binds to available amino groups on
proteins, yielding advanced glycation end
products (AGE)
– modifications inhibits protein function
 Pyruvate Carboxylase Deficiency
(PCD)
• PCD = rare disorder characterized to some extent by
hypoglycemia and lactic acidosis
– may lead to death in the first few months of life in severe
cases
– symptoms include lethargy and seizures
 Pyruvate Carboxylase Deficiency
(PCD), Continued
• Pyruvate carboxylase is a key regulatory enzyme in
gluconeogenesis, which occurs primarily in the liver.
• When pyruvate carboxylase activity is missing, the liver
cannot:
– maintain adequate blood glucose concentration.
– remove lactic acid from the blood and use it as a
gluconeogenic precursor.
Glycolysis and Gluconeogenesis Are
Evolutionarily Intertwined
• Glycolytic enzymes with similar properties do not have
similar amino acid sequences.
– most likely derived independently rather than by gene
duplication

• Glycolysis consists of the metabolism of hexoses and

the metabolism of trioses.
– Metabolism of trioses is common to glycolysis and
gluconeogenesis.
– Enzymes in the metabolism of trioses are present in all
species.
– enzymes in the metabolism of hexoses are less conserved
 After a meal, glucose is abundant
and fructose 2,6-bisphosphate is
present at higher levels. Does the
activity of a phosphofructokinase
(PFK) increase, decrease, or stay the
same? (1 of 2)
 After a meal, glucose is abundant
and fructose 2,6-bisphosphate is
present at higher levels. Does the
activity of a phosphofructokinase
(PFK) increase, decrease, or stay the
same? (2 of 2)
increase
Remember to complete your exit poll
tonight!

© Macmillan Learning, 2023