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Chapter 18
Glycolysis
Dr Khairul Ansari
Chapter 18
Living organisms, like
machines, conform to the law
of conservation of energy, and
must pay for all their activities
in the currency of catabolism.
Ernest Baldwin
Dynamic Aspects of
Biochemistry
Louie Pasteur s scientific
investigations into
fermentation of grape sugar
were pioneering studies of
glycolysis.
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What is the chemical basis and logic for glycolysis, the centralpathway of metabolism; that is, how does glycolysis work?
What are the essential features of glycolysis?
Why are coupled reactions important in glycolysis?
What are the chemical principles and features of the first phaseof glycolysis?
What are the chemical principles and features of the secondphase of glycolysis?
What are the metabolic fates of NADH and pyruvate producedin glycolysis?
How do cells regulate glycolysis?
Are substrates other than glucose used in glycolysis? How do cells respond to hypoxic stress?
Essential Question
Living organism appears in O2 free environment
Gycolysis does not need O2
Gycolysis plays significant roles in anaerobic metabolism in the
first 2 billion years of evolution
Otto Warburg, Gustav Embden and Otto Fritz Meyerhof- worked
out of the glycolysis pathway
Gycolysis is also known as Embden-Meyerhof (or Warburg)
Pathway
Brain, Kidney medulla and rapidly contracting skeleting muscle
and some cells such as erythrocytes and sperm use glucose as
only energy source.
Gycolysis
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Glycolysis
Essentially all cells carry out glycolysis
Ten reactions essentially the same in all cells but with differentrates
Two phases:
First phase converts glucose to two glyceraldehyde-3-P
Second phase produces two pyruvates
Products are pyruvate, ATP and NADH
Three possible fates for pyruvate
Under aerobic conditions pyruvate can be converted to acetyl-CoA
that enter TCA cycle
It can also be converted into glucose via gluconeogenesis.
Under anaerobic conditions, it may be converted into lactic acid. In
yeast, pyruvate is converted into ethanol instead.
The Fates of Pyruvate From Glycolysis
Figure 18.2 Pyruvate produced in glycolysis can be used by cells in
several ways. In animals, pyruvate is normally converted to acetyl-
coenzyme A, which is then oxidized in the TCA cycle to produce CO2.
When oxygen is limited, pyruvate can be converted to lactate.
Alcoholic fermentation in yeast converts pyruvate to ethanol and CO2.
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The Glycolysis Pathway
Phase I:- Five reaction break down glucose to two molecules of
Glyceraldehyde -3-Phosphate (G-3-P) consume two ATP
Phase II:- G-3-P is converted to two molecules of pyruvate-produce 4
ATP
The Glycolysis Pathway
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Why Are Coupled Reactions Important in Glycolysis?
Coupled reactions convert some, but not all of the metabolic energy
of glucose into ATPUnder cellular conditions, approximately 5% of the energy of glucose
is released in glycolysis
Coupled reactions involving ATP hydrolysis are also used to drive the
glycolytic pathway
Net gain of Glycolysis is 2 ATP (61 kj/mol) and 2 pyruvate
Glucose 2 molecules of lactate
C6H12O6 2 H3C-CHOH-COO- + 2 H+
G = -183.6 KJ/molThe excess energy is utilized in rearrangement of the molecules
Glucose + 2 ADP + 2 P i 2 lactate + 2 ATP + 2 H+ 2 H2O
G = -183.6 + 61 = - 122.6 KJ/molUnder standard condition (61/183.6)x 100 = 33% energy is stored in
ATP
What Are the Chemical Principles and Features of
the First Phase of Glycolysis?
The first reaction - phosphorylation of glucose
Hexokinase or glucokinase
This is a priming reaction - ATP is consumed here in order to getmore later
ATP makes the phosphorylation of glucose spontaneous
Be sure you can interconvert Keq and standard-state free energychange
Be sure you can use Eq. 3.13 to generate the values shown in thefar right column of Table 18.1.
-D-Glucose + ATP4- -D-Glucose -6-phosphate2- +ADP3- + H+
G = -16.7 KJ/molPhosphorylation of glucose cost 13.8 KJ/mol
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Hexokinase Primes the Pump for Glycolysis
Figure 18.3 Just as a water pump
must be primed with water to
get more water out, the glycolytic
pathway is primed with ATP in
steps 1 and 3 in order to achieve
net production of ATP in the
second phase of the pathway.
Advantages of Phosphorylation
1. Plasma membrane is impermeable to -D-Glucose -6-phosphate2-
(negatively charged)2. Conversion of glucose to -D-Glucose -6-phosphate2- , keeps the
intracellular concentration low, favoring diffusing into the cell
3. Favorable thermodynamics of the first reaction makes it an
important site for regulation
Glucose is kept in the cell by phosphorylation to
glucose-6-phosphate
Figure 18.4 Glucose-6-P cannot cross the plasma membrane.
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Reactions and Thermodynamics of Glycolysis
Rxn 1: Hexokinase1st step in glycolysis; G large, negative
Hexokinase (and glucokinase) act to phosphorylate glucose andkeep it in the cell
Km for glucose is 0.1 mM; cell has 4 mM glucose
So hexokinase is normally active!
Glucokinase (Kmglucose = 10 mM) only turns on when cell is rich in
glucose
Hexokinase is regulated - allosterically inhibited by (product)glucose-6-P - but is not the most important site of regulation ofglycolysis - Why?
Hexokinase reaction is one of the three points in glycolysispathway that are regulated
Mg2+ is required (the true substrate is MgATP2-)
There are two isozymes of Hexokinase (type I and type II)
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Km for glucose is 0.03 mM for type I hexokinase and 0.3 mM fortype II hexokinase
Type I is primary hexokinase in brain
Type IV (glucokinase) is predominant in liver- doest not get
inhibited by the product (Km is approximately 10 mM)
When glucose level is high the liver hexokinase (glucokinase)
phosphorylates glucose and eventually converts to glycogen
Glucokinase is inducible (by insulin) and acts when level of
glucose is very high.
In diabetes mellitus (patient producing insufficient insulin) level
of glucokinase is less.
Rxn 1: Hexokinase
Steady-State Concentrations of Glycolytic Intermediates
These steady-state concentrations are
used to obtain the cellular values ofG
found in Table 18.1 and Figure 18.22.
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Glucose-6-P is common to several metabolic pathways
Figure 18.5 Glucose-6-phosphate is the branch point for several
metabolic pathways.
Reaction 1: Hexokinase
Figure 18.6 The (a) open and (b) closed
states of yeast hexokinase. Binding of
glucose (green) induces a conformation
change that closes the active site, as
predicted by Daniel Koshland. The induced
fit model for enzymes is discussed on page
409 of the text.
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Hexokinase is the paradigm of induced fit
Figure 18.7 (a) Mammalian
hexokinase I contains an N-
terminal domain (top) and a C-
terminal domain (bottom) joined
by a long -helix. Each of these
domains is similar in sequence
and structure to yeast hexokinase.
(b) Human glucokinase undergoes
induced fit upon binding glucose
(green).
Reaction 2: Phosphoglucoisomerase
Glucose-6-P to Fructose-6-P Why does this reaction occur?
next step (phosphorylation at C-1) would be tough for
hemiacetal -OH, but easy for primary -OH
isomerization activates C-3 for cleavage in aldolase reaction
Ene-diol intermediate in this reaction
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A mechanism for phosphoglucoisomerase
Figure 18.8 The phosphoglucoisomerase mechanisminvolves opening of the pyranose ring (step 1), proton
abstraction leading to enediol formation (step 2), and
proton addition to the double bond, followed by ring
closure (step 3)
Reaction 3: Phosphofructokinase
PFK is the committed step in glycolysis!
The second priming reaction of glycolysis
Committed step and large, negative G - means
PFK is highly regulated
ATP inhibits, AMP reverses inhibition
Citrate is also an allosteric inhibitor
Fructose-2,6-bisphosphate is allosteric activator
PFK increases activity when energy status is low
PFK decreases activity when energy status is high
The reaction is endegonic by itself (G = 16.6 KJ/mol), butexergonic when coupled with ATP hydrolysis (G = -14.2 KJ/mol
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Phosphofructokinase a Second Phosphorylation
Driven by ATP
Phosphofructokinase is the second priming reaction of glycolysis.
ATP is consumed in this priming reaction, so that more ATP can be
produced further along the pathway.
PhosphofructokinasePhosphofructokinase is the valve controlling rate of glycolysis
ATP is both substrate and allosteric regulator of PFK
High ATP turn off glycolysis in cytosol.
ATP level does not fluctuate to greater extend in most tissue
ADP + ADP ATP + AMP
Small drop of ATP level in cell results a large increase in AMP
AMP reverse the PFK inhibition by ATP
PFK activity is high when energy status of cell is low, and PFK
activity is low when energy status is high.
Fructose-2,6-bisphosphate also regulatesPFK
Citrate an intermediate of TCA cycle is also an allosteric inhibitor
of PFK (PFK activity coupled with TCA cycle)
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Phosphofructokinase behaves cooperatively at high [ATP]
Figure 18.9 At high ATP, phosphofructokinase (PFK) behaves
cooperatively and the activity plot is sigmoid.
F-2,6-BP regulates Phosphofructokinase
Phosphofructokinase is regulated by fructose-2,6-bisphosphate, a
potent allosteric activator that increases the affinity of
phosphofructokinase for the substrate fructose-6-P.
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F-2,6-BP reverses the inhibition of PFK by ATP
Figure 18.11 F-2,6-BP
stimulates PFK by
decreasing the inhibitory
effects of ATP.
Rxn 4: Aldolase (Fructose bisphosphate aldolase
A C6
intermediate is cleaved to 2 C3
s (Dihydroxyacetone phosphate
and and Glyceraldehyde-3-phosphate)
The reaction is enderonic (cellularG, however, is close to zero.)
The reaction rate (equilibrium ) is influenced by concentration and
may become exergonic
Class I and II aldolases present in animal and bacteria respectively
Class II aldolase contain Zn2- in the active site
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Reaction 5: Triose Phosphate Isomerase
Triose phosphate isomerase completes the first phase of glycolysis.
Each glucose has been converted to two molecules of glyceraldehyde-
3-phosphate.
Reaction 5: Triose Phosphate Isomerase
DHAP is converted to G-3-P
This reaction makes it possible for both products of the aldolase
reaction to continue in glycolysis
The reaction involves an ene-diol mechanism
Glu165 in the active site acts as a general base
Triose phosphate isomerase is a near-perfect enzyme
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What Are the Chemical Principles and Features of
the Second Phase of Glycolysis?
Metabolic energy of glucose produces 4 ATP
Net ATP yield for glycolysis is two ATP
The second phase of glycolysis involves two very high-energy
phosphate intermediates
1,3-bisphosphoglycerate (1,3-BPG)
Phosphoenolpyruvate (PEP)
Reaction 6: Glyceraldehyde-3-P DehydrogenaseG-3-P is oxidized to 1,3-BPG
Oxidation of aldehyde to carboxylic acid is highly exergonic
The overall reaction involves formation of carboxylic phosphoricanhydride and the reduction of NAD+ to NADH
The overall reaction is slightly endergonic
Energy yield from converting an aldehyde to a carboxylic acid isused to make 1,3-BPG and NADH
The mechanism involves covalent catalysis and a nicotinamidecoenzyme, and it is good example of nicotinamide chemistry
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Rxn 6: Glyceraldehyde-3-P Dehydrogenase
Figure 18.14 A
mechanism for the
glyceraldehyde-3-
phosphate
dehydrogenase reaction.
Reaction of an enzyme
sulfhydryl with G3P forms
a thiohemiacetal, which
loses a hydride to NAD+ to
become a thioester.
Phosphorolysis releases1,3-bisphosphoglycerate.
G3P-DH
Arsenate is a substrate for the G3P-DH reaction, forming1-arseno-3-phosphoglycerate. This product breaks down to
3-phosphoglycerate, essentially bypassing the phosphoglycerate
kinase reaction. The result is that glycolysis in the presence of
arsenate produces no net ATP.
G3P-OH can be inactivated by reaction with iodoacetate, whichreacts with and block the essential cysteine sulfhydryl
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Reaction 7: Phosphoglycerate Kinase
ATP synthesis from a high-energy phosphate Phosphoglycerate kinase transfers a phosphoryl group from 1,3-
bisphosphoglycerate to ADP to form an ATP.
This is referred to as substrate-level phosphorylation
This reaction pays off the ATP debt created by the priming
reactions in the first phase
Mg2+ is required (the true substrate is MgADP-)
2,3-BPG (for hemoglobin) is made by circumventing the PGK
reaction (Figure 18.15)
Reaction 7: Phosphoglycerate Kinase
Phosphoglycerate kinase transfers a phosphoryl group from 1,3-
bisphosphoglycerate to ADP to form ATP. This type of ATP-
synthesizing reaction is referred to as a substrate-level
phosphorylation
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Reaction 7: Phosphoglycerate Kinase
The open (a) and closed (b) forms of
phosphoglycerate kinase. ATP (cyan), 3-
phosphoglycerate (purple), and Mg2+
(gold).
2,3-BPG is made by reactions that detour around the
phosphoglycerate kinase reaction
2,3-bisphosphoglycerate is an important regulator of
hemoglobin (see pages 505-506 of the text)
2,3-BPG is formed from 1,3-BPG by bisphosphoglycerate
mutase
3-phosphoglycerate is then formed by 2,3-
bisphosphoglycerate phosphatase
Most cells contain only a trace of 2,3-BPG, but erythrocytes
typically contain 4-5 mM 2,3-BPG
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2,3-BPG is made by reactions that detour around the
phosphoglycerate kinase reaction
Figure 18.15 Formation and decomposition of 2,3-
bisphosphglycerate.
2,3-BPG is made by reactions that detour around the
phosphoglycerate kinase rxn
Figure 18.16 The mutase that forms 2,3-BPG from 1,3-BPG requires
3-phosphoglycerate. The reaction is actually an intermolecular
phosphoryl transfer from C-1 of 1,3-BPG to C-2 of 3-
phosphoglycerate.
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Reaction 8: Phosphoglycerate Mutase
Phosphoglycerate mutase catalyzes a phosphoryl group transfer
from C-3 to C-2
Rationale for this reaction in glycolysis: It repositions the
phosphate to make PEP in the following reaction (enolase)
Note the phospho-histidine intermediates
Zelda Rose (wife of Nobel laureate Irwin Rose) showed that a
bit of 2,3-BPG is required to phosphorylate His
Nomenclature note: a mutasecatalyzes migration of a
functional group within a substrate
Reaction 8: Phosphoglycerate Mutase
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Reaction 9: Enolase
Conversion of 2-Phosphoglycerate to PEP
The enolase makes a high-energy phosphate in
preparation for ATP synthesis in step 10
The overall G for this reaction is 1.8 kJ/mol
How can such a reaction create a PEP?
"Energy content" of 2-PG and PEP are similar
Enolase just rearranges 2-PG to a form that releases
more energy upon hydrolysis
Reaction 9: Enolase
The enolase reaction creates a high-energy phosphate in
preparation for ATP synthesis in step 10 of glycolysis.
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Reaction 10: Pyruvate Kinase
Conversion of PEP to pyruvate makes ATP
These two ATP (from one glucose) can be viewed as the"payoff" of glycolysis
Large, negative G indicating that this reaction issubject to regulation
PK is allosterically activated by AMP, F-1,6-bisP
PK is allosterically inhibited by ATP and acetyl-CoA
Understand the keto-enol equilibrium of pyruvate; it is
the key to understanding the pyruvate kinase reaction
Reaction 10: Pyruvate Kinase
The pyruvate kinase reaction converts PEP to pyruvate, driving
synthesis of ATP. Note that two ATP are produced here, since two
PEP are formed from one glucose. These two ATP are the payoffof glycolysis.
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Reaction 10: Pyruvate Kinase
Figure 18.19 The conversion of phosphoenolpyruvate (PEP) topyruvate may be viewed as involving two steps: phosphoryl transfer,
followed by an enol-keto tautomerization. The tautomerization is
spontaneous and accounts for much of the free energy change for
PEP hydrolysis.
18.5 What Are the Metabolic Fates of NADH and Pyruvate
Produced in Glycolysis?
NADH can be recycled via aerobic or anaerobic pathways
NADH represents energy - two possible fates:
If O2 is available (aerobic conditions), NADH isoxidized in the electron transport pathway, makingATP in oxidative phosphorylation
In anaerobic conditions, NADH is oxidized bylactate dehydrogenase (LDH), providing additionalNAD+ for more glycolysis
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18.5 What Are the Metabolic Fates of NADH and Pyruvate
Produced in Glycolysis?
Figure 18.21 (a) Pyruvate reduction to ethanol in yeast provides a means for
regenerating NAD+ consumed in the glyceraldehyde-3-P dehydrogenase reaction.
(Right) Fermentation at a bourbon distillery. A mash of corn and other grains is
fermented by yeast, producing ethanol and CO2, which can be seen bubbling to the
surface.
18.5 What Are the Metabolic Fates of NADH and Pyruvate
Produced in Glycolysis?
Figure 18.21 (b) In oxygen-depleted muscle, NAD+ is regenerated in the
lactate dehydrogenase reaction. Hibernating turtles, trapped beneath
ice and lying in mud, become anoxic and convert glucose mainly to
lactate. Their shells release minerals to buffer the lactate throughout
the period of hibernation.
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18.5 What Are the Metabolic Fates of NADH and Pyruvate
Produced in Glycolysis?
Pyruvate also represents energy - two possiblefates:
Aerobic or anaerobic paths
In aerobic conditions, pyruvate proceeds throughthe tricarboxylic acid (TCA) cycle (see Chapter 19)
Anaerobic metabolism of pyruvate leads to lactate(in microorganisms and animals) or ethanol (inyeast)
These are examples offermentation theproduction of ATP energy by reaction pathways inwhich organic molecules function as donors andacceptors of electrons
The Warburg Effect and Cancer
Otto Warburg observed in 1924 that rapidlyproliferating cancer cells metabolize glucose mainly tolactate, even when O2 is plentiful
Lewis Cantley has suggested that this behavior arisesbecause cells need more than ATP they mustsynthesize large amounts of nucleotides, amino acids,and lipids
This requires lots of NADPH for biosynthesis as well asintermediates for building blocks
Cancer cells divert large amounts of glucose to thepentose phosphate pathway to produce NADPH
See page 597 for more details
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Otto Warburg
Otto Warburg, Nobel Prize in
Physiology or Medicine, 1931
for his discovery of the nature
and mode of action of the
respiratory enzyme
(cytochrome oxidase see Ch.
20)
The Warburg Effect and Cancer
Signaling proteins and
pathways regulate the
glycolytic pathway. Cancer
cells route up to 90% of
acquired glucose and
glutamine into lactate and
alanine, producing large
amounts of NADPH
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18.6 How Do Cells Regulate Glycolysis?
The elegant evidence of regulation See Figure 18.22
Standard state G values are scattered, with both plusand minus values and no apparent pattern
The plot ofG values in cells is revealing:
Most values near zero
3 of 10 reactions have large, negative G
These 3 reactions with large negative G are sites ofregulation (HK, PFK, PK)
Regulation of these three reactions can turn glycolysisoff and on
18.6 How Do Cells Regulate Glycolysis?
Figure 18.22 The free
energies of the reactions of
glycolysis under standard-
state conditions.
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18.6 How Do Cells Regulate Glycolysis?
Figure 18.22 The free
energies of the reactions of
glycolysis under actual
intracellular concentrations
of metabolites in
erythrocytes.
Tumor Diagnosis Using Positron Emission Tomography (PET)
Tumors show very high rates of glycolysis, as shown by OttoWarburg early in the 20th century
This observation is the basis of tumor detection by positronemission tomography (PET)
Metabolites labeled with 18F can be taken up by humancells (in the brain, for example)
Decay of18F results in positron emission
Positron-electron collisions produce gamma rays
Detection with gamma ray cameras provides 3D models of
tumor extent and location 2-[18F]fluoro-2-deoxy-glucose, used for this purpose, is a
substrate for hexokinase
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Tumor Diagnosis Using Positron Emission Tomography (PET)
2-[18F]fluoro-2-deoxy-
glucose is a substrate for
hexokinase
Decay of18F results in positron
emission.
Positron-electron collisions
produce gamma rays
Detection with gamma ray cameras provides 3D models of
tumor extent and location
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18.7 Are Substrates Other Than Glucose Used in Glycolysis?
Sugars other than glucose can be glycolytic substrates
Fructose, mannose and galactose can all be used
Fructose and mannose are routed into glycolysis by
fairly conventional means. See Figure 18.23
Galactose is more interesting - the Leloir pathway
"converts" galactose to glucose - sort of....
See Figure 18.24
18.7 Are Substrates Other Than Glucose Used in Glycolysis?
Figure 18.23
Mannose, galactose,
fructose, and other
simple metabolites
can enter the
glycolytic pathway.
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18.7 Are Substrates Other Than Glucose Used in Glycolysis?
Figure 18.24 Galactose
metabolism via the Leloir
pathway.
Galactose Enters Glycolysis Via the Leloir Pathway
Figure 18.25 The galactose-1-phosphate uridylyltransferase
reaction involves a ping-pong kinetic mechanism.
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UDP-glucose pyrophosphorylase uses Gal-1-P, reducing galactose
toxicity in adultsFigure 18.26
Glycerol Can Also Enter Glycolysis
Glycerol is produced in the decomposition of triacylglycerols. It can
be converted to glycerol-3-P by glycerol kinase. Glycerol-3-P is then
oxidized to dihydroxyacetone phosphate by the action of glycerol
phosphate dehydrogenase.
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Glycerol Can Also Enter Glycolysis
Glycerol is produced in the decomposition of triacylglycerols. It can
be converted to glycerol-3-P by glycerol kinase. Glycerol-3-P is then
oxidized to dehydroxyacetone phosphate by the action of glycerol
phosphate dehydrogenase.
Lactose From Mothers Milk to Yogurt and
Lactose Intolerance
In placental mammals, lactose is synthesize only in themammary gland, and then only during late pregnancyand lactation
The synthesis is done by lactose synthase, a dimericcomplex of galactosyl transferase and -lactalbumin
Lactose breakdown in the intestines by lactaseprovides newborns with essential galactose
Some humans are lactose intolerant, due to a
particularly low level of lactase
Lactic acid fermentation by certain bacteria is the basisfor the production ofyogurt
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Lactose From Mothers Milk to Yogurt and
Lactose Intolerance
Breakdown of lactose to
galactose and glucose by
lactase.
Many Humans are Lactose Intolerant Due to a Low
Level of Lactase
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18.8 How Do Cells Respond to Hypoxic Stress?
Glycolysis is an anaerobic pathway
The tricarboxylic acid cycle is aerobic
When oxygen is abundant, cells prefer to combine thesepathways in aerobic metabolism
When oxygen is limiting, cells adapt to carry out moreglycolysis
Hypoxia causes changes in gene expression that increaseslevels of glycolytic enzymes
A trigger for this is a DNA-binding protein called hypoxiainducible factor (HIF)
HIF is regulated at high oxygen levels by hydroxylase factor-inhibiting HIF (FIH-1)
18.8 How Do Cells Respond to Hypoxic Stress?
HIF consists of two subunits: a ubiquitous HIF-1subunit and a hypoxia-responsive HIF-1 subunit
In response to hypoxia, inactivation of the prolylhydroxylases allows HIF-1 stabilization
Dimerization with HIF-1
Binding of the dimer to the hypoxia-responsive element(HRE) of HIF target genes
Activation of transcription of these genes
VHL is the von Hippel Lindau subunit of the ubiquitinE3 ligase that targets proteins for proteasomedegradation
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18.8 How Do Cells Respond to Hypoxic Stress?
Figure 18.28
Chapter 19
The Tricarboxylic Acid Cycle
Dr Khairul Ansari
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Chapter 19
Thus times do shift, each
thing his turn does hold; New
things succeed, as former
things grow old.
Robert Herrick
Hesperides (1648)
A time-lapse photograph of a
ferris wheel at night. Aerobiccells use a metabolic wheel
the TCA cycle to generate
energy by acetyl-CoA
oxidation.
Essential Questions
How is pyruvate oxidized under aerobic conditions,
and what is the chemical logic that dictates how
this process occurs?
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Outline
What is the chemical logic of the TCA cycle? How is pyruvate oxidatively decarboxylated to acetyl-CoA?
How are two CO2 molecules produced from acetyl-CoA?
How is oxaloacetate regenerated to complete the TCA cycle?
What are the energetic consequences of the TCA cycle?
Can the TCA cycle provide intermediates for biosynthesis?
What are the anaplerotic, or filling up, reactions?
How is the TCA cycle regulated?
Can any organisms use acetate as their sole carbon source?
Oxidation of glucose to CO2 is a
24-electron oxidation
The electrons from glucose
oxidation feed into the electron
transport pathway, driving
synthesis of ATP.
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Hans Krebs Showed That the Oxidation of Acetate is
Accomplished by a Cycle
The TCA cycle is known as the Krebs cycle and also as the citric acidcycle
Pyruvate from glycolysis is oxidatively decarboxylated to acetate
Then acetate is degraded to CO2 in the TCA cycle
Some ATP is produced
More NADH is made
NADH goes on to make more ATP in electron transport and oxidativephosphorylation
4 electrons are removed as NADH in glycolysis
2 NADH are produced during decarboxylation of two molecules of
pyruvate
For each acetyl-CoA oxidized in TCA cycle, 8 more electrons are
removed as NADH (3) and FADH2(1)
H3CCOO- + 2H20 + H
+ 2CO2 + 8H
In electron transport pathway these electrons combines with O2
to produce water
8H + 2O2 H2O
Net reaction of TCA cycle and electron transport pathway isH3CCOO
- + 2O2 + H+ 2CO2 + 2H2O
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Acetyl-CoA- entry point of new carbon into the TCA cycle
Generally (in biological system) C-C occur cleavage between a
and carbon
Or between two carbons
As acetate does not have a carbon neither of these cleavage are
possible.
Condensation of acetate with oxaloacetate facilitate cleavage
Figure 19.1 The Tricarboxylic Acid Cycle
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Figure 19.1 The Tricarboxylic Acid Cycle
Figure 19.1 The Tricarboxylic Acid Cycle
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-Cleavage of an -Hydroxyketone
This type of cleavage occurs in the transaldolase reaction (described in
Chapter 22).
But it would require hydroxylation of acetate, which is not a favorable
or facile reaction for acetate
Living things have evolved the clever chemistry of condensing acetate
with oxaloacetate and then carrying out a -cleavage TCA combines this cleavage with oxidation to form CO2, regenerating
oxaloacetate and capturing all the energy as NADH and ATP!
19.2 How Is Pyruvate Oxidatively Decarboxylated to Acetyl-
CoA?
Pyruvate must enter the mitochondria to enter the TCA cycle
Oxidative decarboxylation of pyruvate is catalyzed by thepyruvate dehydrogenase complex
Pyruvate dehydrogenase is a noncovalent assembly of threeenzymes
Five coenzymes are required
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Figure 19.4 The Reaction Mechanism of the Pyruvate
Dehydrogenase Complex
Decarboxylation of pyruvate yields hydroxyethyl-TPP. Transfer to lipoic
acid is followed by formation of acetyl-CoA.
19.3 How Are Two CO2 Molecules Produced from Acetyl-
CoA?
The citrate synthase synthase reaction initiates the TCA cycle
Citrate synthase is classic CoA chemistry
C of the acetyl group in acetyl-CoA is acidic and can bedeprotonated to form a carbanion
This carbanion is a strong nucleophile that can attack the -carbonylof oxaloacetate, yielding citryl-CoA
Thioester hydrolysis then produces citrate
NADH & succinyl-CoA are allosteric inhibitors
Note (Table 19.1) that citrate synthase has a large negative G and isa site of regulation
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The citrate synthase reaction initiates the TCA cycle
Figure 19.6 Citrate is formed in the citrate synthase reaction from
oxaloacetate and acetyl-CoA. The mechanism involves nucleophilic
attack by the carbanion of acetyl-CoA on the carbonyl carbon of
oxaloacetate, followed by thioester hydrolysis.
NADH is an allosteric inhibitor of citrate synthase
The citrate synthase synthase reaction initiates the TCA
cycle
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The citrate synthase synthase reaction initiates the TCA cycle
Figure 19.7 Citrate synthase in mammals
is a dimer of 49-kD subunits. In the
monomer shown here, citrate (blue) and
CoA (red) bind to the active site, which lies
in a cleft between two domains and is
surrounded mainly by -helical segments.
Citrate Is Isomerized by Aconitase to Form Isocitrate
Citrate is a poor substrate for oxidation
So aconitase isomerizes citrate to yield isocitrate which has asecondary-OH, which can be oxidized
Note the stereochemistry of the reaction: aconitase removes thepro-R H of the pro-R arm of citrate
Aconitase uses an iron-sulfur cluster - see Figure 19.8
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Citrate Is Isomerized by Aconitase to Form Isocitrate
Figure 19.8 The aconitase reaction converts citrate to cis-aconitate
and then to isocitrate. Aconitase is stereospecific and removes the
pro-R hydrogen from the pro-R arm of citrate.
Citrate Is Isomerized by Aconitase to Form Isocitrate
Figure 19.8 The active
site of aconitase. The
iron-sulfur cluster
(pink) is coordinated
by cysteines (orange)
and isocitrate
(purple).
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Fluoroacetate Blocks the TCA Cycle
Fluoroacetate is an extremely poisonous agent that blocks the TCA
cycle in vivo, although it has no apparent effect on any of the isolated
TCA cycle enzymes.
The action of aconitase has been traced to aconitase
Aconitase is inhibited by fluorocitrate, which is formed from
fluoracetate in two steps, as shown here.
Isocitrate Dehydrogenase Catalyzes the First Oxidative
Decarboxylation in the Cycle
Oxidative decarboxylation of isocitrate yields -ketoglutarate
This is classic NAD+ chemistry (hydride removal) followed by adecarboxylation
Isocitrate dehydrogenase is a link to the electron transport pathwaybecause it makes NADH
The mechanism is typical of NAD+-dependent enzymes (see Figure19.10)
The reaction involves (first) oxidation of the C-2 alcohol of isocitrate
to form oxalosuccinate, then a -decarboxylation reaction thatexpels the central carboxyl group as CO2
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Isocitrate Dehydrogenase Catalyzes the First Oxidative
Decarboxylation in the Cycle
Figure 19.10 The isocitrate
dehydrogenase reaction.
Hydride removal by NAD+ is
followed by a
-decarboxylation reaction that
expels the central carboxyl
group as CO2
-Ketoglutarate Dehydrogenase Catalyzes the SecondOxidative Decarboxylation of the TCA Cycle
A second oxidative decarboxylation
This enzyme is nearly identical to pyruvate dehydrogenase -structurally and mechanistically
Five coenzymes used - TPP, CoASH, lipoic acid, NAD+, and FAD
You know the mechanism if you remember pyruvatedehydrogenase
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-Ketoglutarate Dehydrogenase Catalyzes the Second OxidativeDecarboxylation of the TCA Cycle
Like pyruvate dehydrogenase, -ketoglutarate dehydrogenase is a
multienzyme complex consisting of-ketoglutaratedehydrogenase, dihydrolipoyl transsuccinylase, and dihydrolipoyl
dehydrogenase. The complex uses five different coenzymes.
19.4 How Is Oxaloacetate Regenerated to Complete the
TCA Cycle?
Succinyl-CoA Synthetase Catalyzes
a Substrate-Level Phosphorylation
A nucleoside triphosphate is made
This is possible because succinyl-CoA is a high-energy
intermediate
Its hydrolysis (the hydrolysis of a CoA ester) drives the
phosphorylation of GDP to produce GTP
The mechanism (Figure 19.11) involves a phosphohistidine
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Succinate Dehydrogenase Is FAD-Dependent
An FAD-dependent oxidation of a single bond to a double bond The mechanism involves hydride removal by FAD and a
deprotonation
This enzyme is actually part of the electron transport pathwayin the inner mitochondrial membrane
The electrons transferred from succinate to FAD (to formFADH2) are passed directly to ubiquinone (UQ) in the electrontransport pathway
The Succinate Dehydrogenase Reaction
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Fumarase Catalyzes the trans-Hydration of Fumarate to
Form L-Malate
Hydration occurs across the newly formed double bond
Hydration involves trans-addition of the elements of water
across the double bond
Possible mechanisms are shown in Figure 19.13
The actual mechanism is not known for certain
The Fumarase Reaction
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The Malate Dehydrogenase Reaction
Steric Preferences in NAD+-Dependent Dehydrogenases
The dehydrogenases that require nicotinamidecoenzymes are stereospecific, and they transferhydride to either the pro-R or the pro-S positionsselectively
What accounts for this specificity? The enzymesinvolved are asymmetric structures.
The nicotinamide coenzyme (and substrate) fit into theactive site in only one way
The hydride transfers can only be accomplished to orfrom one side or the other of the NAD+ molecule
Dehydrogenases (and other enzymes too) are alsostereospecific with respect to the substrates as well
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The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA
Cycle
Figure 19.15 The fate of the carbon atoms of acetate in successive
TCA cycles.
The Carbon Atoms of Acetyl-CoA Have Different Fates in the
TCA Cycle
Figure 19.15
The fate of the
carbon atoms
of acetate in
successive TCAcycles.
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19.7 Can the TCA Cycle Provide Intermediates for
Biosynthesis?
The TCA cycle provides several of these
-Ketoglutarate is transaminated to make glutamate, which can beused to make purine nucleotides, as well as Arg and Pro
Succinyl-CoA can be used to make porphyrins
Fumarate and oxaloacetate can be used to make several amino acidsand also pyrimidine nucleotides
Note that mitochondrial citrate can be exported to be a cytoplasmicsource of acetyl-CoA and oxaloacetate
The TCA Cycle Can Provide Intermediates For Biosynthesis
Figure 19.16 TheTCA cycle
provides
intermediates for
biosynthesis.
Amino acids are
highlighted in
orange.
All 20 common
amino acids can
be made from
metabolites
derived from the
TCA cycle.
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19.8 What Are the Anaplerotic, or Filling Up, Reactions?
Pyruvate carboxylase - converts pyruvate to oxaloacetate
This is the most important anaplerotic reaction
PEP carboxylase - converts PEP to oxaloacetate
Malic enzyme converts pyruvate into malate
PEP carboxykinase - could have been an anaplerotic reaction, but itgoes the wrong way
CO2 binds weakly to PEP carboxykinase, but oxaloacetate bindstightly, so the reaction goes spontaneously in the oppositedirection.
19.8 What Are the Anaplerotic, or Filling Up, Reactions?
Figure 19.17 Pyruvate carboxylase (shown here), and also
phosphoenolpyruvate (PEP) carboxylase, and malic enzymecatalyze anaplerotic reactions, replenishing TCA cycle
intermediates.
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19.8 What Are the Anaplerotic, orFilling Up, Reactions?
Figure 19.17 Pyruvate carboxylase , phosphoenolpyruvate (PEP)
carboxylase (shown here), and malic enzyme catalyze anaplerotic
reactions, replenishing TCA cycle intermediates.
19.8 What Are the Anaplerotic, or Filling Up, Reactions?
Figure 19.17 Pyruvate carboxylase, and also phosphoenolpyruvate
(PEP) carboxylase and malic enzyme (shown here) catalyze anaplerotic
reactions, replenishing TCA cycle intermediates.
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Is PEP Carboxykinase an Anaplerotic Reaction?
This reaction *might* be an anaplerotic reaction, except for the
fact that CO2 binds weakly to PEP carboxykinase, and
oxaloacetate binds very tightly. As a result, the enzyme favors
formation of PEP from oxaloacetate.
Thus the reaction operates in the wrong direction to be an
anaplerotic reaction.
Anaplerosis Plays a Critical Role in Insulin Secretion
The pancreas releases insulin in response to an increase of
blood glucose
What cellular processes mediate this response?
It was long accepted that ATP produced by catabolism
activated K+ channels in the plasma membrane of -cells in
the pancreas
However, recent research has shown that anaplerotic
enzymes feed alternative pathways that produce cytosolic
signal molecules that also support insulin secretion
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The Reductive TCA Cycle
The TCA cycle running backward could assimilate CO2 This may have been the first metabolic pathway
This reductive TCA cycle occurs in certain extant archaea andbacteria, where it serves all their carbon needs
Energy to drive it? Maybe reaction of FeS with H2S to form FeS2(iron pyrite)
Iron pyrite, which was plentiful in ancient times, is an ancientversion ofiron-sulfur clusters found in many enzymes today!
The Reductive TCA Cycle
A reductive,
reversed TCA
cycle
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19.9 How Is the TCA Cycle Regulated?
As in TCA, 3 reactions are the key sites
Citrate synthase - ATP, NADH and succinyl-CoA inhibit
Isocitrate dehydrogenase - ATP inhibits, ADP and NAD+ activate
-Ketoglutarate dehydrogenase - NADH and succinyl-CoA inhibit,AMP activates
Also note pyruvate dehydrogenase:
ATP, NADH, acetyl-CoA inhibit
NAD+, CoA activate
Figure 19.18
Regulation of the TCA cycle.
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Pyruvate Dehydrogenase is Regulated by
Phosphorylation/Dephosphorylation
Figure 19.19 Regulation of
the pyruvate dehydrogenase
reaction.
Phosphorylation inactivates;
Dephosphorylation
activates.
19.10 Can Any Organisms Use Acetate as Their Sole
Carbon Source?
The Glyoxylate Cycle
Acetate-based growth - net synthesis of carbohydrates and otherintermediates from acetate - is not possible with TCA
The glyoxylate cycle offers a solution for plants and some bacteriaand algae
The CO2-evolving steps are bypassed and an extra acetate is utilized
Isocitrate lyase and malate synthase are the short-circuitingenzymes
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19.10 Can Any Organisms Use Acetate as Their Sole
Carbon Source?
Figure 19.20 The glyoxylate
cycle. The first two steps are
identical to TCA reactions.
Isocitrate lyase and malate
synthase short-circuit the TCA
cycle, forming malate and
succinate from isocitrate and
another acetyl-CoA.
More on the Glyoxylate Cycle
Isocitrate lyase produces glyoxylate and succinate
Malate synthase does a Claisen condensation of acetyl-CoA andthe aldehyde group of glyoxylate - classic CoA chemistry!
The glyoxylate cycle helps plants grow in the dark! Seeds are arich source of acetate (from fatty acids). Until the nascent plantsees the sun (and begins photosynthesis), it can grow using theglyoxylate cycle
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Isocitrate Lyase Short-Circuits the TCA Cycle by Producing
Glyoxylate and Succinate
Figure 19.21 The isocitrate lyase reaction. Isocitrate lyase catalyzes
an aldol cleavage and is similar to the aldolase reaction in glycolysis.
Glyoxysomes Must Borrow Three Reactions from
Mitochondria
Glyoxysomes do not contain all the enzymes needed to run
the glyoxylate cycle
Succinate dehydrogenase, fumarase, and malate
dehydrogenase are absent
Glyoxysomes borrow these three reactions from
mitochondria, so that they can convert succinate to
oxaloacetate
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Glyoxysomes Must Borrow Three Reactions from
Mitochondria
Figure 19.20
Glyoxysomes lack three
of the enzymes needed
to run the glyoxylate
cycle. Succinate
dehydrogenase,
fumarase, and malate
dehydrogenase are all
borrowed from
mitochondria.