Metabolism

By Ogoti
Def:
What is Metabolism?
The entire chemical reaction network of the body that are
catalysed by enzymes.
• The intermediates, substrates or products of these enzyme
catalysed reactions are referred to as Metabolites.
• The sequence of enzymatic reactions that lead to specifc
products are referred to as Metabolic Pathways. They are of
three types:
i. Catabolic pathways (Catabolism): Breakdown of complex
molecules (Carbohydrates, Proteins & Lipids) to small
molecules. Energy is generated.
ii. Anabolic pathways (Anabolism): Synthesis of complex
molecules from small molecules. Energy is consumed.
iii. Amphibolic pathways: Act as links between catabolic
pathways and anabolic pathways.
Note: These pathways do not occur in isolation.
 Carbohydrate Metabolism
• Glucose occupies a central position in the metabolism
of plants, animals and many microorganisms.
a. It is relatively rich in potential energy, thus a good fuel.(Complete
oxidation to CO2 and H2O proceeds with a ∆G value of -2,840
KJ/mol).
b. It is a versatile precursor, capable of supplying a huge array of
metabolic intermediates for biosynthetic reactions. e.g. E. coli can
obtain carbon skeleton for Amino acids, nucleotides, coenzymes,
fatty acids or other metabolic intermediates it needs for growth.

• In animals and vascular plants, glucose has three

major fates: How is glucose synthesized?
Photosynthetic organisms make
glucose by fxing CO2 to trioses,
then convert the trioses to glucose.

Non-photosynthetic cells make

glucose from simpler three and four-
carbon compounds by the process of
gluconeogenesis
 Content:
• Glycolysis.
• Fructose metabolism
• Galactose metabolism
• Gluconeogenesis.
• Pentose Phosphate Pathway.
• Tricarboxylic acid cycle (TCA cycle).
• Electron Transport Chain (Oxidative phosphorylation).
 Glycolysis

In glycolysis a molecule of glucose is degraded in a series of

enzyme-catalyzed reactions to yield two molecules of the three-
carbon compound (pyruvate).

The sequential reactions of glycolysis, conserve the free energy

released from glucose in form of ATP and NADH.
Glycolysis is the central pathway of glucose catabolism in most
cells some of which solely depend on it as a source of metabolic
energy .e.g.
i. Mammalian tissues and cell types (erythrocytes, renal
medulla, brain and sperm cell .
ii. Plants tissues that are modifed to store starch (potato
tubers)
iii. Most anaerobic microorganisms.

The breakdown of the six-carbon (glucose) into two molecules of

the three-carbon pyruvate occurs in ten steps, which are divided
in to two phases:
i. Preparatory phase
Glycolysis cont….
Preparatory phase
The frst fve reactions of
glycolysis which constitute the
preparatory phase the energy of
ATP is invested, raising the free-
energy content of the
intermediates, and the carbon
chain of all hexoses are
converted into a common
product;
Payof phasetwo molecules of
glyceraldehyde 3- phosphate
In the payof phase of each
(G3P).
molecule of G3P is oxidized and
phosphorylated by inorganic
phosphate (not by ATP) to form
1,3-bisphosphoglycerate.
Energy is then released as the
two
molecules of 1,3-
bisphosphoglycerate are
converted to two molecules of
pyruvate.

 Fates of Pyruvate
The pyruvate formed in glycolysis
is further metabolized in three
major catabolic routes.
I. Complete oxidation to CO2 and
water in the citric acid cycle
and electron transport chain in
the mitochondria. The energy
from electron-transfer reactions
drive ATP synthesis ( e.g.
aerobic organisms or tissues).
II. Pyruvate reduction to lactate
via lactic acid fermantation for
regeneration of NAD+ required
as an electron acceptor for
further oxidation of glucose to
pyruvate ( e.g. contracting
muscle, erythrocytes, retina and
anaerobic microorganisms)
III. Decarboxylation of pyruvate to
ethanol in ethanol (alcohol)
fermentation under hypoxic or
Feeder pathways for Glycolysis
 Regulation of Glycolysis

• metabolic pathways are regulated by altering activities of few enzymes of that

pathway.
• Hexokinase, phosphofructo kinase and pyruvate kinase are the regulatory
enzymes of glycolysis.
• Their activities are under allosteric and hormonal control.
Allosteric regulation of glycolysis
Phosphofructokinase-1:
• is the major regulatory enzymes of glycolysis.
• It is an allosteric enzyme and catalyzes rate limiting reaction of glycolysis.
• It is inhibited by ATP and citrate, while AMP and fructose-6-phosphate are
activators of this enzyme.
Pyruvate kinase:
• is the second regulatory enzyme.
• It is inhibited by ATP.
Hexokinase:
• Is inhibited bt Glucose-6-phosphate.
 So, when ATP (energy) concentration is highglycolysis is inhibited.
 and decrease in ATP level increases rate of glycolysis.
 Regulation of Glycolysis cont…

Hormonal regulation of glycolysis

• Insulin increases rate of glycolysis by increasing concentration of
glucokinase, phosphofructokinase-1 and pyruvate kinase.

Mechanism of Insulin Action

 FRUCTOSE METABOLISM
• Fructolysis is the term used to refer to the
metabolism of fructose from dietary sources.
• Though the metabolism of glucose through
glycolysis uses many of the same enzymes and
intermediate structures as those in fructolysis, the
two sugars have very diferent metabolic fates in
human metabolism.
• Unlike glucose, which is directly metabolized widely
in the body, fructose is almost entirely metabolized in
the liver in humans, where it is directed toward
replenishment of liver glycogen and triglyceride
synthesis.
• Under 1% of ingested fructose is directly converted
to plasma triglyceride.
• Approx. 29% - 54% of fructose is converted in liver to
glucose, and about a quarter of fructose is converted
 Reactions of Fructose Metabolism.
• Fructose is phosphorylated to fructose-1-phosphate by
fructokinase in the liver. This enzyme will not phosphorylate
glucose and unlike glucokinase its activity is not afected by
fasting or by insulin and that is why fructose disappears from the
blood of diabetic patients at a normal rate.
• •Fructose-1-phosphosphate is cleaved by liver aldolase(aldolase
B)to dihydroxy acetone phosphate(DHAP) and D-glyceraldehyde.
Aldolase-B also functions in glycolysis in the liver attacking
fructose-1,6-bisphosphate.
• •D-glyceraldehyde is phosphorylated by glyceraldehyde kinase to
D-glyceraldehyde-3-phophate.These two triose phosphates(DHAP
and glyceraldehyde-3-phosphate) then may enter the glycolytic
pathway, gluconeogenesis or glycogenesis according to the
metabolic status of the tissue.
• •In extra hepatic tissues, especially the adipose and muscle
tissues, fructose can be phosphorylated by hexokinase to fructose-
6-phosphate.This however is a slow reaction and occurs only in the
presence of high concentration of fructose because when fructose
is present with glucose, its phosphorylation is inhibited by glucose.
 Continuation.
• about a quarter of fructose is converted to lactate.
15% - 18% is converted to glycogen.
• Glucose and lactate are then used normally as
energy to fuel cells all over the body.
• Fructose is a dietary monosaccharide present
naturally in fruits and vegetables, either as free
fructose or as part of the disaccharide sucrose, and
as its polymer inulin.
• It is also present in the form of refned sugars
including granulated sugars (white crystalline table
sugar, brown sugar, confectioner's sugar, and
turbinado sugar), refned crystalline fructose , as
high fructose corn syrups as well as in honey.
Illustration of the fructose metabolism pathway
Sorbitol or Polyol Pathway for Formation of Fructose

Sorbitol
• Sorbitol less commonly known as glucitol, is a sugar
alcohol with a sweet taste which the human body
metabolizes slowly.
• It can be obtained by reduction of glucose, which
changes the converted aldehyde group (−CHO) to a
primary alcohol group (−C(OH)H2).
• Most sorbitol is made from potato starch, but it is
also found in nature, for example in apples, pears,
peaches, and prunes.
• It is converted to fructose by sorbitol
dehydrogenase.
Continuation
 Continuation.
• The sorbitol (polyol)pathway is for formation of fructose
from glucose.
• Aldose reductase reduces glucose to sorbitol(glucitol).
• It’s found in many tissues such as lens, retina,
peripheral nerve cells, kidney, placenta,red blood cells,
ovaries and seminal vesicles. But it is not present in
liver.
• A second enzyme, sorbitol dehydrogenase that can
oxidize the sorbitol to fructose is present in liver, ovaries
and sperm cell.
• In liver, ovaries and sperm cells, the same enzyme,
sorbitol dehydrogenase oxidizes the sorbitol to fructose.
• The pathway from sorbitol to fructose in the liver
provides a mechanism by which dietary sorbitol is
converted into fructose which can enter glycolysis.
Continuation
 Inborn errors in fructose metabolism
• 1.Essential fructosuria
• The absence of fructokinase results in the inability to
phosphorylate fructose to fructose-1-phosphate within the
cell.
• As a result, fructose is neither trapped within the cell nor
directed toward its metabolism.
• Free fructose concentrations in the liver increase and
fructose is free to leave the cell and enter plasma.
• This results in an increase in plasma concentration of
fructose, eventually exceeding the kidneys' threshold for
fructose reabsorption resulting in the appearance of
fructose in the urine.
• Essential fructosuria is a benign asymptomatic condition.
 2.Hereditary fructose intolerance

• The absence of fructose-1-phosphate aldolase

(aldolase B) results in the accumulation of fructose 1
phosphate in hepatocytes, kidney and small
intestines.
• An accumulation of fructose-1-phosphate following
fructose ingestion inhibits glycogenolysis (breakdown
of glycogen) and gluconeogenesis, resulting in severe
hypoglycemia.
• It is symptomatic resulting in severe hypoglycemia,
abdominal pain, vomiting, hemorrhage, jaundice,
hepatomegaly, and hyperuricemia eventually leading
to liver and/or kidney failure and death.
 3.Reduced phosphorylation potential

• Intravenous (i.v) infusion of fructose has been shown to lower

phosphorylation potential in liver cells by trapping Pi(inorganic
phosphate) as fructose 1-phosphate.
• The fructokinase reaction occurs quite rapidly in hepatocytes
trapping fructose in cells by phosphorylation. On the other hand,
the splitting of fructose-1-phosphate to DHAP and glyceraldehyde
by Aldolase B is relatively slow.
• Therefore, fructose-1-phosphate accumulates with the
corresponding reduction of intracellular Pi available for
phosphorylation reactions in the cell.
• This is why fructose is contraindicated for total parenteral nutrition
(TPN) solutions and is never given intravenously as a source of
carbohydrate.
• It has been suggested that excessive dietary intake of fructose may
also result in reduced phosphorylation potential. However, this is
still a contentious issue. Dietary fructose is not well absorbed and
increased dietary intake often results in malabsorption.
 GALACTOSE METABOLISM

• Galactose, sometimes abbreviated Gal, is a

monosaccharide and the C4 epimer of glucose, that
is, they difer only for the position of the -OH group
on C4 .It has the molecular formula C6H12O6 and
molar mass 180.156 g/mol. It has a sweetness equal
to 33% of sucrose.
Structure of galactose
 Food sources of galactose
• In human nutrition the most part comes from the hydrolysis of the
disaccharide lactose(Galactose + Glucose),the milk sugar, including that
of the human milk.
• As mother’s milk is the only source of energy and carbohydrates for
newborn, galactose has a crucial role in human nutrition.
• The monosaccharide is also bound to caseins, and therefore it is found in
all dairy products.
• Findings from studies conducted since the 50s of last century have shown
that galactose is present not only in milk and dairy products, but also in
plant products such as legumes, grains, nuts, tubers and vegetables. It
seems that in these foods it is often engaged in bonds resistant to the
attack of human digestive enzymes, and therefore not metabolizable.
However, diferent fruits and vegetables contain it also in free form, in
variable amounts;
• Less than 0.1 mg/100 g of edible portion: artichokes, mushrooms, olives,
and peanuts; more than 10 mg/100 g of edible portion: bell peppers, date,
papaya, watermelon, tomato;
• up to 35.4 mg/100 g of edible portion in persimmon.
• These values are very low, but to take into account in case of
galactosemia
Process of Galactose Metabolism.
• Galactose, which is metabolized from the milk sugar,
lactose (a disaccharide of glucose and galactose),enters
glycolysis by its conversion to glucose-1-phosphate (G-1-
P). This occurs through a series of steps that is referred
to as the Leloir pathway, named after Luis Federico
Leloir who determined the overall process of galactose
utilization.
• Galactose can exist in two diferent stereoisomeric
forms; α-D-galactose and β-D-galactose. The α-D-form is
that which is metabolized in the Leloir pathway.
• Conversion of the β-form of galactose to the α-form
requires the enzyme galactose mutarotase encoded by
the GALM gene (also known as aldose 1-epimerase)
Continuation..
 Continuation…
• The frst reaction of the Leloir pathway is the phosphorylation
of α-D-galactose by galactokinase to yield galactose-1-
phosphate.
• Epimerization of galactose-1-phosphate to glucose-1-phosphate
(G-1-P) requires the transfer of UDP from UDP-glucose
catalyzed by galactose-1-phosphate uridyltransferase
(GALT).The galactose-1-phosphate uridyltransferase catalyzed
reaction generates UDP-galactose and glucose-1-phosphate (G-
1-P).
• The UDP-galactose is epimerized to UDP-glucose by UDP-
galactose-4 epimerase (GALE). The UDP portion is exchanged
for phosphate generating glucose-1-phosphate which then is
converted to glucose-6-phosphate (G-6-P) by
phosphoglucosemutase. The glucose-6-phosphate then joins
glycolytic pathway where it is metabolised futher to pyruvate.
 Illustration of Leloir
pathway(Galactose pathway)
 Metabolic fate of UDP-Galactose

• UDP-Gal is an important precursor in the synthesis of glycolipids,

such as gangliosides and galactocerebrosides, sphingolipids,
mucopolysaccharides and membrane glycoproteins.
• In the adult mammary gland, under the infuence of prolactin,
UDP-Gal can be joined to glucose to give milk sugar(Lactose).
• Regulated Reactions-No Regulation
• There is no known regulated step in the conversion of galactose
to glucose intermediates. The fate of glucose from dietary
galactose, toward either glycolysis or glycogenesis, is determined
by pathways regulating glucose metabolism in the liver.
• Unique Characteristics-UDP-Glucose Intermediate
• Although the galactose pathway forms UDP-glucose, there is no
net synthesis of this intermediate, since it is recycled. The net
output of this pathway is in the conversion of a mole of galactose
1-phosphate to glucose 1-phosphate.
 Interface with Other Pathways-Polyol
Pathway
• Free galactose can be reduced by aldose reductase
in the polyol pathway to galactitol (Figure below).
The polyol pathway is widely distributed in the
body including in the lens, where it contributes to
the formation of cataracts in both galactosemia and
diabetes.
Continuation..(sorbitol implicating diabetes)
• The pathway is implicated in diabetic complications,
especially in microvascular damage to the retina,
kidney and nerves.
• Sorbitol cannot cross cell membranes, and, when it
accumulates, it produces osmotic stresses on cells by
drawing water into the insulin-independent tissues.
• Cells use glucose for energy. This normally occurs
by phosphorylation via the enzyme hexokinase.
• However, if large amounts of glucose are present (as
in diabetes mellitus), hexokinase becomes saturated
and the excess glucose enters the polyol pathway
when aldose reductase reduces it to sorbitol
Continuation….
 Gluconeogenesis
In mammals, some tissues depend almost
completely on glucose for their metabolic energy. e.g.
Brain and the nervous system, erythrocytes, testes,
renal medulla and embryonic tissues.
Note:
i. Supply of glucose stored from stores (Glycogen) is not always
suficient (i.e. brain requires 120g of glucose/day More than
half the glucose stored as glycogen in muscle and liver.
ii. Between meals and prolonged fasts or vigorous exercise,
glycogen is depleted.
To maintain blood glucose, glucose is synthesised
from non-carbohydrate precursors through
Gluconeogenesis.
Gluconeogenic pathway convert pyruvate and
related 3 and 4 carbon cpds to glucose.
 In animals important precursors of glucose include: lactate,
pyruvate, glycerol
and some amino acids.
 In microorganisms gluconeogenesis starts from simple organic cpds
of 2 or 3 carbons such as: acetate, lactate and propionate.
An overview of
Gluconeogene
sis in Plants
and Animals
actions of the gluconeogenic pathway
Glycolysis and gluconeogenesis share several steps;
seven of the ten enzymatic reactions of
gluconeogenesis are the reverse of glycolysis
Three Glycolysis reactions have such a large
negative DG that they are essentially irreversible.
 Hexokinase (or Glucokinase)
 Phosphofructokinase
 Pyruvate Kinase.
In Gluconeogenesis the three irreversible steps are
bypassed by a separate set of enzymes that are
irreversible in the direction of glucose synthesis.
Conversion of pyruvate to phosphoenol pyruvate.
Conversion of Fructose 1,6- Bisphosphate to Fructose 6-phosphate.
Conversion of glucose 6-phosphate to glucose.
In animals, both pathways occur largely in the
cytosol, necessitating their reciprocal and coordinated
regulation.
luconeogenic pathway
st bypass: Conversion of Pyruvate to phosphoenol pyruvate
his step requires enzymes in both the Cytosol and Motochon
Pyruvate Carboxylase PEP Carboxykinase
O O-
C
O O- O O-
C ATP ADP + Pi C O GTP GDP C
C O CH2 C OPO 32-
CH3 HCO3- C CO2 CH2
-
O O
pyruvate oxaloacetate PEP

Pyruvate is transported to the mitochondria where

pyruvate carboxylase converts pyruvate to
oxaloacetate.
pyruvate + HCO3- + ATP  oxaloacetate + ADP +
Pi
Oxaloacetate formed is transported back to the
Note: The reaction requires Biotin as a carrier of activated
cytosol where it is converted
HCO3-
PEP by
phosphoenolpyruvate carboxykinase
oxaloacetate + GTP  PEP + GDP + CO2
Alternative paths
from pyruvate to
PEP
Pyruvate is transported to
the mitochondria where it is
converted to Oxaloacetate.
Oxaloacetae formed is
reduced to malate by
mitochondrial malate DH at
the expense of NADH.
Malate is transported back
to the cytosol through a
specifc transporter where it
is reoxidesed to oxaloacetate
generating cytosolic NADH.
Oxaloacetate is then
converted to PEP by a
cytosolic PEP
carboxykinase.

OR

Oxaloacetate, can directly

 N subject to O
Pyruvate carboxylation
Carboxylas C
HN NH
e uses biotin lysine
as prosthetic CH CH residue
group. O O C
H2C CH
S (CH2)4 C NH (CH2)4 CH

biotin NH

Biotin has a 5-C side chain whose

terminal carboxyl is in amide
linkage to the e-amino group of an
enzyme lysine.
The biotin & lysine side chains
form a long swinging arm that
allows the biotin ring to swing
Role of Biotin in the
pyruvate carboxylase
reaction
The reaction occurs in two
steps at diferent active sites of
pyruvate carboxylase enzyme.
nd bypass: Conversion of F 1,6- Bisphosphate to F 6-Phosp
This is catalysed by a diferent enzyme, Mg 2+- dependent
fructose 1,6-bisphosphatase (FBPase-1) which promotes the
irreversible hydrolysis of C-1 phosphate.

Phosphofructokinase 
6 CH OPO 2- 1CH2OH 6 CH OPO 2- 1CH2OPO32-
2 3 2 3
O ATP ADP O
5 H HO 2 5 H HO 2

H 4 3 OH H 4 3 OH
Pi H2O
OH H OH H
fructose-6-phosphate fructose-1,6-bisphosphate
 Fructose-1,6-biosphosphatase

Phosphofructokinase (Glycolysis) catalyzes:

fructose-6-P + ATP  fructose-1,6-bisP + ADP
Fructose-1,6-bisphosphatase (Gluconeogenesis)
catalyzes:
fructose-1,6-bisP + H2O  fructose-6-P + Pi
hird bypass: Conversion of Glc 6- phosphate to glucose.
The reaction is catalysed by glucose 6-phosphatase, without
synthesis of ATP, it is a simple hydrolysis of a phosphate ester
This Mg2+ activated enzyme is found on the lumenal side of the
ER of Hepatocytes and Renal cells.
Note: Muscle and brain tissues do not have this enzyme thus
connot carry out gluconeogenesis.
Glucose-6-phosphatase
6 CH OPO 2- CH2OH
2 3
5 O O
H H H H
H H2O H
4
OH H 1
OH H + Pi
OH OH OH OH
3 2
H OH H OH
glucose-6-phosphate glucose

Hexokinase or Glucokinase (Glycolysis) catalyzes:

glucose + ATP  glucose-6-phosphate +
ADP
Glucose-6-Phosphatase (Gluconeogenesis)
catalyzes:
The source of pyruvate and oxaloacetate
for gluconeogenesis during fasting or
carbohydrate starvation is mainly amino acid
catabolism.
Some amino acids are catabolized to pyruvate,
oxaloacetate, or precursors of these.
Muscle proteins may break down to supply
amino acids. These are transported to liver
where they are deaminated and converted to
gluconeogenesis inputs. 
Glycerol, derived from hydrolysis of
triacylglycerols in fat cells, is also a signifcant
input to gluconeogenesis.
 Regulation of Gluconeogenesis
Allosteric control Glucose-6-phosphatase
1. Pyruvate glucose-6-P glucose
Carboxylase (pyruvate  Gluconeogenesis Glycolysis
pyruvate
oxaloactate) is fatty acids
allosterically activated
acetyl CoA ketone bodies
by: acetyl CoA.
[Oxaloacetate] tends to oxaloacetate citrate
be limiting for Krebs
Krebs Cycle
cycle.
2. Fructose 1,6
bisphosphatase:
When gluconeogenesis is active in liver, oxaloacetate is
AMP isto its
diverted formallosteric
glucose.
inhibitor.
Oxaloacetate depletion hinders acetyl CoA entry into Krebs
Cycle.
The increase in [acetyl CoA] activates Note: Avidin,
Pyruvate a protein
Carboxylase to
Hormonal
make regulation
oxaloacetate. in egg whites, tightly
Insulin decreases the synthesis of binds biotin.
key enzymes of gluconeogenesis, Excess consumption of
thus inhibit gluconeogenesis. raw eggs can cause
e: Glycolysis and gluconeogeesis arenutritional
regulated
biotin.
defciency of
reciprocally.
Glycolysis & Gluconeogenesis: both
spontaneous.
If both pathways were simultaneously active
in a cell, it would constitute a "futile cycle"
that would waste energy.
Glycolysis:
glucose + 2 NAD+ + 2 ADP + 2 Pi 
2 pyruvate + 2
NADH + 2 ATP
Gluconeogenesis:
2 pyruvate + 2 NADH + 4 ATP + 2 GTP

glucose + 2 NAD+ + 4 ADP
+ 2 GDP + 6 Pi
Questions:
To prevent the waste of a futile cycle, Glycolysis & Gluconeogenesis are
reciprocally regulated.
Local Control: Includes reciprocal allosteric regulation by adenine
nucleotides.
 Phosphofructokinase (Glycolysis) is inhibited by ATP and stimulated
by AMP.
Phosphofructokinase 
 Fructose-1,6-bisphosphatase
6 CH OPO 2- 1
(Gluconeogenesis)
6 CH OPO 2- is inhibited
2- by AMP.
2 3 CH2OH 1
2 3 CH2OPO3
O ATP ADP O
5 H HO 2 5 H HO 2

H 4 3 OH H 4 3 OH
Pi H2O
OH H OH H
fructose-6-phosphate fructose-1,6-bisphosphate
 Fructose-1,6-biosphosphatase

The opposite efects of adenine nucleotides on

 Phosphofructokinase (Glycolysis)
 Fructose-1,6-bisphosphatase (Gluconeogenesis)
insures that:
• When cellular ATP is high (AMP would then be low), glucose is not
degraded to make ATP.
•When ATP is low (AMP would then be high), the cell does not expend
energy in synthesizing glucose.
 Global Control:includes reciprocal efects of
a cyclic AMP cascade, triggered by the
hormone glucagon when blood glucose is low.
Activation of other enzymes
tivation of Glycogen phosphorylase
Enzymes relevant to these pathways that are
phosphorylated by Protein Kinase A include:
 Pyruvate Kinase, a glycolysis enzyme that
is inhibited when phosphorylated.
 CREB (cAMP response element binding
protein) which activates, through other
factors, transcription of the gene for PEP
Carboxykinase, leading to increased
gluconeogenesis.
 A bi-functional enzyme (PFK-2) that
makes and degrades an allosteric
regulator, fructose-2,6-bisphosphate (A
potent activator of PFK-1 and an inhibitor
of Fructose 1,6 bisphosphatase.
Reciprocal regulation by fructose-2,6-
bisphosphate:
 Fructose-2,6-bisphosphate stimulates
Glycolysis.
Fructose-2,6-bisphosphate allosterically
activates the Glycolysis enzyme
Phosphofructokinase 1.
Fructose-2,6-bisphosphate also activates
transcription of the gene for
Glucokinase, the liver variant of
Hexokinase that phosphorylates glucose
to glucose-6-phosphate, the input to
Glycolysis.
 Fructose-2,6-bisphosphate
Phosphorylation of enzymes & regulatory
proteins in liver by Protein Kinase A (cAMP
Dependent Protein Kinase) results in
 inhibition of glycolysis
 stimulation of gluconeogenesis,
making glucose available for release to the
blood.
 The Cori Cycle
The Cori Cycle operates during exercise.
For a brief burst of ATP utilization, muscle
cells utilize ~P stored as phosphocreatine.
Once phosphocreatine is exhausted, ATP is
provided mainly by Glycolysis, with the
input coming from glycogen breakdown and
from glucose uptake from the blood.
 Cori Cycle
Liver Blood Muscle

Glucose Glucose
2 NAD+ 2 NAD+
2 NADH 2 NADH
6 ~P 2 ~P

2 Pyruvate 2 Pyruvate
2 NADH 2 NADH
2 NAD+ 2 NAD+
2 Lactate 2 Lactate

Lactate produced from pyruvate passes via the

blood to the liver, where it may be converted to
glucose.
The glucose may travel back to the muscle to
fuel Glycolysis.
 Cori Cycle
Liver Blood Muscle

Glucose Glucose
2 NAD+ 2 NAD+
2 NADH 2 NADH
6 ~P 2 ~P

2 Pyruvate 2 Pyruvate
2 NADH 2 NADH
2 NAD+ 2 NAD+
2 Lactate 2 Lactate

The Cori cycle costs 6 ~P in liver for every 2 ~P

made available in muscle. The net cost is 4 ~P.
Although costly in ~P bonds, the Cori Cycle allows
the organism to accommodate to large fuctuations in
energy needs of skeletal muscle between rest and
exercise.
The equivalent of the Cori Cycle also operates
during cancer.
If blood vessel development does not keep
pace with growth of a solid tumor, decreased
O2 concentration within the tumor leads to
activation of signal processes that result in a
shift to anaerobic metabolism.
 Liver Blood Cancer Cell

Glucose Glucose
2 NAD+ 2 NAD+
2 NADH 2 NADH
6 ~P 2 ~P

2 Pyruvate 2 Pyruvate
2 NADH 2 NADH
2 NAD+ 2 NAD+
2 Lactate 2 Lactate

Energy dissipation by the Cori Cycle, which

expends 6 ~P in liver for every 2 ~P produced
via Glycolysis for utilization within the tumor, is
thought to contribute to the weight loss that
typically occurs in late-stage cancer even when
food intake remains normal.
 Pentose Phosphate pathway
Also known as:
• Pentose shunt
• Hexose monophosphate shunt
• Phosphogluconate pathway

It occurs in the cytosol.

One fate of G6P is the
pentose pathway.
 What does the pentose phosphate
pathway achieve?
• The pathway yields reducing potential
in the form of NADPH to be used in
anabolic reactions requiring electrons.
• The pathway yields ribose 5-
phosphate.
– Nucleotide biosynthesis leading to:
• DNA
• RNA
• Various cofactors (CoA, FAD, NAD+/NADP+).
Tissues with active pentose phosphate pathway
Pentose phosphate pathway is especially prominent in
tissues actively synthesizing fatty acids and steroids.
These tissues use NADPH to reduce the double bonds
and carbonyl groups of intermediates in the synthetic
process.
PPP is also prominent in growing and regenerating
tissues and in tumors.
Reactions of the
Pentose Phosphate
Pathway
The pentose pathway can
be divided into two
phases:
Oxidative phase.
Oxidative reactions
Non-oxidative of the
phase.
pentose phosphate pathway
Steps
1. Dehydrogenation of glucose 6-
phosphate by glucose 6-phosphate
DH
2. The lactone is hydrolysed to the free
acid; 6-phosphogluconate by a
specifc lactonase and decarboxylation
3. Dehydrogenation
of 6-phosphogluconate by 6-
phosphogluconate dehydrogenase to
ribulose
4. 5-phosphate.
Phosphopentose isomerase
converts ribulose 5-phosphate to its
aldolase isomer ribose 5- phosphate.
Non-Oxidative reactions of the pentose
phosphate
Occurs in pathway
tissues that require primarily NADPH.
 The pentose phosphates produced in the oxidative
phase of the pathway are recycled into glucose 6-
phosphate.
Steps
1. In this non-oxidative phase,
ribulose 5-phosphate is frst
epimerized to xylulose 5-
phosphate.
In a series of rearrangements of the
carbon skeletons six fve-carbon
sugar phosphates are converted to
fve six-carbon sugar phosphates,
completing the cycle, allowing
continued production of NADPH.
Two enzymes unique to the pentose phosphate pathway
act in these interconversions of sugars: transketolase
and transaldolase.
2. Transketolase transfers C-1 and C-2 of xylulose 5-phosphate
to ribose 5-phosphate, forming the seven-carbon product
sedoheptulose 7-phosphate and glyceraldehyde 3-
phosphate.

3. Transaldolase transfers a three-carbon fragment from

sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate,
forming fructose 6-phosphate and (the tetrose) erythrose 4-
phosphate.
4. transketolase acts again,
forming fructose 6-
phosphate and
glyceraldehyde 3-
phosphate from erythrose
4-phosphate and xylulose
5-phosphate .

Note:
The frst two steps in the
oxidative pentose phosphate
pathway are essentially
irreversible in in the cell.
The reactions of the non-
oxidative part are however
readily reversible and thus
provide a means of converting
Two molecules of glyceraldehyde 3-phosphate formed by two
iterations of these reactions can be converted to a molecule of
fructose 1,6-bisphosphate as in gluconeogenesis.
In a complete cycle: six pentose phosphates are converted to
fve hexose phosphates.
 Regulation of the Pentose Pathway
Oxidative Phase
• Glucose 6-phosphate DH is the
regulatory enzyme.
• NADPH is a potent competitive inhibitor
of the enzyme.
• Usually when the ratio NADPH/NADP+
is high so the enzyme is inhibited.
• But, with increased demand for NADPH,
the ratio decreases and enzyme activity
is stimulated.
 Non-oxidative Phase
• The reactions of the non-oxidative
portion of the pentose pathway are
readily reversible.

• The concentrations of the products

and reactants can shift depending
on the metabolic needs of a
particular cell or tissue.
Diferent scenarios under which the Pentose
phosphate pathway is controlled.
1. Rapidly dividing cells require more ribose 5-
phosphate than NADPH.
2. The need for NADPH and ribose 5-phosphate is
balanced.
3. More NADPH is needed than ribose 5-phosphate;
Fatty acid synthesis in adipose cells.
4. The cell needs both NADPH and ATP
iological importance of HMP shunt
1. NADPH produced in the shunt is used for biosynthesis of
several important compounds in various organs.
(a) In the liver, NADPH is used for fatty acid synthesis,
cholesterol synthesis, bile acid synthesis, glutamate synthesis and
cytochrome P450-hydroxylase system.
(b) In the adrenal cortex and gonads, NADPH is used for
cholesterol and hormone synthesis.
(c) In the adipose tissue, NADPH is used for fatty acid synthesis.
(d) NADPH is used for formation of deoxy ribonucleotides and
In RBC, NADPH
pyrimidine
2. produced is used for the formation of
nucleotides.
reduced glutathione (GHS)from oxidized glutathione
(GSSG), catalyzed by Glutathione reductase. Reduced
glutathione is required for removal of H2O2 in RBCs by the
enzyme glutathione peroxidase for maintenance of erythrocyte
• What is glutathione?
proteins.
• Why is it important?
• How is it related to NADPH?
What is glutathione?
Glutathione is a tripeptide
composed of glutamate,
cystein, glycine.

Reduced glutathione
(GSH) maintains the
normal reduced state of
the cell.

GSH
Why is it important?
• The sulfhydryl of GSH is used to reduce peroxides
(ROS) formed during oxygen transport.
– Reactive oxygen species (ROS) damage macromolecules
(DNA, RNA, and protein) and ultimately lead to cell
death.
• The resulting oxidized form of GSH, two molecules
of glutathione are linked by a disulfde bridge
(GSSG).
• How is it related to NADPH?

The enzyme
glutathione
reductase uses
NADPH as a cofactor
to reduce GSSG back
to two moles of GSH.
Thus, the pentose
pathway is linked to
the supply of adequate
amounts of GSH.
ological importance of HMP shunt cont……

3. In neutrophils, NADPH is required for the formation of

superoxide by NADPH oxidase.
Respiratory burst of neutrophils during phagocytosis involves
superoxide formation.
4. Pentoses produced in this pathway are used for nucleic acid
synthesis and nucleotide coenzymes like NAD+, FAD and FMN
synthesis
5. Non-oxidative phase of the pathway converts pentoses of
endogenous or dietary nucleic acids into intermediates of
glycolysis where they are, further oxidized to generate energy.
Medical implications of HMP shunt
Glucose-6-phosphate dehydrogenase defciency
G6PD defciency is an inheritable X-linked recessive
disorder.
Approximately 10-14% of the male African American
population is afected.
Individules with G6PD defciency can not produce
suficient GSH to cope with the ROS.
People with the disorder are not normally anemic
and display no evidence of the disease until the red
cells are exposed to an oxidant or stress.
i.e. The ingestion of oxidative agents that generate
peroxides or reactive oxygen species (ROS).
Antimalarials - pamaquine
purine glycoside from fava beans.

The condition is often clinically seen as black

urine under certain conditions.
 Drugs that can precipitate this reaction:
• antimalarial agents
• sulfonamides (antibiotic)
• aspirin
• nonsteroidal antiinfammatory drugs (NSAIDs)
• nitrofurantoin
• quinidine
• quinine
• exposure to certain chemicals - mothballs
edical implications of HMP shunt cont…

Wernicke-korsakof encephalopathy

It is due to defective trasketolase genes.

Transketolase of afected individuals has lower
afinity for TPP.
The characteristic symptoms are abnormal walking
and standing, memory loss and partial paralysis.
The disease manifests only when there is thiamine
defciency.
The syndrome is more common among alcoholics
than in the general population.
Summary of the Pentose Phosphate Pathway
 The Citric Acid
Cycle
ellular Respiration:
ccurs in three stages;
1. Oxidation of organic fuel
molecules—glucose, fatty acids, and
some amino acids—
to yield two-carbon fragments in the
form of the acetyl group of acetyl-
coenzyme
2. A (acetyl-CoA).
The acetyl groups are fed into the
citric acid
cycle, which enzymatically oxidizes
them to CO2; conserving energy in
form of reduced electron
3. Oxidation
carriers NADH NADH
of and and
FADH 2.
FADH2 and
transfer of
electrons to O2—the fnal electron
acceptor—via a chain of electron-
carrying molecules known as the
respiratory chain; Energy is conserved
in the form of ATP (oxidative
phosphorylation)
 Enzymatic conversion of Pyruvate to acetyl CoA

• The fate of pyruvate produced during glycolysis and oxidation of other

biofuels depends on the amount of energy in the tissues.
• If tissues have adequate energy pyruvate is oxidized through
gluconeonegenesis to generate glucose which can be converted glycogen for
storage.
• However, when the energy is low pyruvate is preferentially oxidized to CO 2
and H2O in the TCA cycle, with generation of 15 molecules ATP per
pyruvate.
• The enzymatic activities of the TCA cycle (and of oxidative phosphorylation)
are located in the mitochondrion.
• When transported into the mitochondrion, pyruvate encounters two principal
metabolizing enzymes: pyruvate carboxylase (a gluconeogenic enzyme) and
pyruvate dehydrogenase (PDH), the first enzyme of the PDH complex.
• The activities of these two enzymes depend on the acylation state state of
Coenzyme A (CoA).
 CONT’
• In the presence of CoA, NAD, FAD and TPP pyruvate is converted to acetyl
CoA with release CO2.
• The acetyl CoA formed enters the TCA cycle as a substrate for energy
generation.
• This reaction is illustrated in the reaction here below.
 Citric Acid Cycle
Hans Adolf Krebs.
Biochemist; born in
Names: Germany. Worked in
The Citric Acid Cycle Britain. His discovery
in 1937 of the ‘Krebs
Tricarboxylic Acid cycle’ of chemical
Cycle reactions was critical
Krebs Cycle to the understanding of
cell metabolism and
earned him the 1953
Nobel Prize for
Physiology or
Medicine.
In eukaryotes the
reactions of the
citric acid cycle
take place inside
mitochondria
 An Overview of the Citric Acid Cycle
A four-carbon oxaloacetate condenses
with a two-carbon acetyl unit to yield a
six-carbon citrate.
An isomer of citrate is oxidatively
decarboxylated to fve-carbon -
ketoglutarate.
-ketoglutarate is oxidatively
decarboxylated to yield a four-carbon
succinate.
Oxaloacetate is then regenerated from
succinate.
Two carbon atoms (acetyl CoA) enter
the cycle and two carbon atoms leave
the cycle in the form of two molecules of
carbon dioxide. The function of the citric acid
cycle is the harvesting of
Three hydride ions (six electrons) are high-energy electrons from
transferred to three molecules of NAD+, acetyl CoA.
one pair of hydrogen atoms (two
electrons) is transferred to one molecule
of FAD.
 Functions of the Citric Acid Cycle

• Integration of metabolism. The citric acid

cycle is amphibolic (both catabolic and
anabolic).
The cycle is involved Intermediates of the
in the aerobic cycle are starting points
catabolism of for many anabolic
carbohydrates, lipids reactions.
and amino acids.
• Yields energy in the form of GTP (ATP).

• Yields reducing power in the form of NADH2

and FADH2.
Reactions of
the Citric
acid cycle
 1. Citrate Synthase
• Citrate formed from acetyl CoA and
oxaloacetate
• Only cycle reaction with C-C bond formation
• Addition of C2 unit (acetyl) to the keto double
bond of C4 acid, oxaloacetate, to produce C6
compound, citrate

citrate synthase
 2. Aconitase
• Elimination of H2O from citrate to form C=C
bond of cis-aconitate.
• Stereospecifc addition of H2O to cis-aconitate to
form isocitrate.

aconitase aconitase
 3. Isocitrate Dehydrogenase
• Oxidative decarboxylation of isocitrate to
a-ketoglutarate (a metabolically irreversible
reaction)
• One of four oxidation-reduction reactions of the cycle
• Hydride ion from the C-2 of isocitrate is transferred to
NAD+ to form NADH
• Oxalosuccinate is decarboxylated to a-
ketoglutarate
The -Ketoglutarate Dehydrogenase Compl
• Similar to pyruvate dehydrogenase complex
• Same coenzymes, identical mechanisms
E1 - a-ketoglutarate dehydrogenase (with TPP)
E2 – dihydrolipoyl succinyltransferase
(with fexible lipoamide prosthetic group)
E 3 - dihydrolipoyl
dehydrogenase (with FAD)

-ketoglutarate
dehydrogenase
 5. Succinyl-CoA Synthetase
• Free energy in thioester bond of succinyl CoA is
conserved as GTP or ATP in higher animals (or
ATP in plants, some bacteria)
• Substrate level phosphorylation reaction

+ HS-
Succinyl-CoA
Synthetase

GTP + ADP GDP + ATP

. The Succinate Dehydrogenase Complex
• Complex of several polypeptides, an FAD prosthetic
group and iron-sulfur clusters
• Embedded in the inner mitochondrial membrane
• Electrons are transferred from succinate to FAD and
then to ubiquinone (Q) in electron transport chain
• Dehydrogenation is stereospecifc; only the trans
isomer is formed

Succinate
Dehydrogenase
 7. Fumarase
• Stereospecifc trans addition of water to the
double bond of fumarate to form L-malate
• Only the L isomer of malate is formed

Fumarase
 8. Malate Dehydrogenase

Malate is oxidized to form oxaloacetate.

Malate
Dehydrogenase
 Regulation of the Citric Acid Cycle
• Pathway controlled by:
(1) Allosteric modulators
(2) Covalent modifcation of cycle enzymes
(3) Supply of acetyl CoA (pyruvate dehydrogenase complex)

Three enzymes have regulatory properties

- citrate synthase (is allosterically inhibited by NADH, ATP,
succinyl CoA, citrate – feedback inhibition)
- isocitrate dehydrogenase
(allosteric efectors: (+) ADP; (-) NADH, ATP. Bacterial
ICDH can be covalently modifed by kinase/phosphatase)
--ketoglutarate dehydrogenase complex (inhibition by
ATP, succinyl CoA and NADH
Regulation of the citric acid cycle

- NADH, ATP,
succinyl CoA,
citrate
Krebs Cycle is a Source of Biosynthetic
Precursors
 Stoichiometry of the Citric Acid Cycle
 Two carbon atoms enter
the cycle in the form of
acetyl CoA.
 Two carbon atoms leave
the cycle in the form of CO2 .
 Four pairs of hydrogen
atoms leave the cycle in
four oxidation reactions
(three molecules of NAD+
one molecule of FAD are
reduced).
 One molecule of GTP,
is formed.

 9 ATP (3 ATP per NADH, and 2 ATP

per FADH2) are produced during
oxidative phosphorylation.
 1 ATP is directly formed in the
citric acid cycle.
 1 acetyl CoA generates
approximately 12 molecules of
ATP.
 Summary of Krebs Cycle
How many energy-producing molecules do we have per 1
glucose molecule?

Glycolysis: 2 ATP, 2 NADH

Intermediate step: Pyruvate oxidation

1 NADH x2= 2 NADH
 
Krebs Cycle:  3 NADHx2= 6 NADH
1 ATPx2= 2 ATP
1 FADH2x2= 2 FADH2

Total: 4 ATP, 10 NADH, 2 FADH2 --> forms 38 ATP in the

electron transport chain
 The role of vitamins in TCA cycle
• Vitamins, mainly of the B-group play important roles in TCA
cycle. Four of the B vitamins that are essential in the TCA cycle
and hence energy-yielding metabolism are:
• Riboflavin in the form of flavin adenine dinucleotide (FAD), it is
a co-factor for succinate dehydrogenase.
• Niacin in the form the form of adenine dinucleotide is the electron
acceptor for isocitrate dehydrogenase, α-ketoglutarate
dehydrogenase, and malate dehydrogenase.
• Thiamine (Vitamin B1) as thiamine diphosphate is the co-enzyme
for decarboxylation in the α-ketoglutarate dehydrogenase reaction.
• Pantothenic acid is a part of coenzyme A, the co-factor attached
to "active" carboxylic acid residues such as acetyl-CoA and
succinyl-CoA.
Electron Transfer Chain
 Oxidative phosphorylation

Mitochondria, has two menbranes:

The outer mitochondrial membrane is
readily permeable to small molecules (Mr
5,000) and ions, which move freely
through transmembrane channels formed by
a family of integral membrane proteins
called porins.
The inner membrane is impermeable to
most small molecules and ions, including
protons (H).
the only species that cross this membrane
do so through specific transporters.
The selectively permeable inner
membrane segregates the intermediates and
enzymes of cytosolic metabolic pathways
from those of metabolic processes occurring
in the matrix.
 Electron Carriers
Oxidative phosphorylation begins with the entry of
electrons into the respiratory chain.
Dehydrogenases collect electrons from catabolic pathways
and funnel them into universal electron acceptors—
nicotinamide nucleotides (NAD or NADP) or flavin
nucleotides (FMN or FAD).
FMN (Flavin MonoNucleotide) is a prosthetic group of
some flavoproteins.
It is similar in structure to FAD (Flavin Adenine
Dinucleotide), but lacking the adenine nucleotide.
FMN (like FAD) can accept 2 e- + 2 H+ to form FMNH2.
 O O O
H H H H H
C N C C N C C N C
H
3CC C C NH H
3CC C C NH H
3CC C C NH

H
3CC C C CO H
3CC C C CO H
3CC C C CO
C N N C N N C N N
H H H H
CH
2  + CH
2  +
CH
2
e+
H e+
H
H
COH H
COH H
COH

H
COH H
COH H
COH

H
COHO H
COHO H
COHO

H
2C OP O
- H
2C OP O
- H
2C OP O
-

O
- F
MNH O
-
F
MN F
MN·O
H -
2

FMN, when bound at the active site of some enzymes, can

accept 1 e to form the half-reduced semiquinone radical.
The semiquinone can accept a 2nd e to yield FMNH2.
Since it can accept/donate 1 or 2 e, FMN has an important
role mediating e transfer between carriers that transfer 2e
(e.g., NADH) & those that can accept only 1e (e.g., Fe+++).
FAD
 NAD(P)H
Nicotinamide nucleotide–linked dehydrogenases catalyze
reversible reactions of the following general types:

Neither NADH nor NADPH can cross the inner mitochondrial

membrane, but the electrons they carry can be shuttled across
indirectly.
How?
Malate-aspartate shuttle. (liver, kidney, and heart mitochondria)
glycerol 3-phosphate shuttle (Skeletal muscle and brain)
In addition to NAD and flavoproteins, three other types of electron-
carrying molecules function in the respiratory
chain:

Ubiquinone (Coenzyme Q).

Cytochromes
Iron sulfur proteins
 Coenzyme Q (Ubiquinone ,CoQ, Q)

O
CH3
C H 3O CH 3
H2C C C CH2
CH 3 H
C H 3O (CH 2 CH C CH 2 ) n H isoprene
O
coenzym e Q
 Is the only non-protein component of electron transport chain.
 Coenzyme Q (CoQ, Q, ubiquinone) is very hydrophobic.
 It dissolves in the hydrocarbon core of the inner mitochondrial
membrane.
 It includes a long isoprenoid tail, with multiple units having a
carbon skeleton comparable to that of isoprene.
In human cells, most often n = 10.
 Ubiquinone can accept one
electron to become the
semiquinone radical (∙QH) or
two electrons to form ubiquinol
(QH2) :

Coenzyme Q functions as a
mobile e carrier within the
mitochondrial inner membrane.
 Cytochromes
The cytochromes are proteins with characteristic strong
absorption of visible light, due to their iron containing heme
prosthetic groups.
Three types exists: Cytochrome a,b and c.
Are distinguished by differences in their light-absorption spectra.
The Heme prosthetic group of
cytochromescontains an iron atom in a
porphyrin ring system.

The Fe is bonded to 4 N atoms of the

porphyrin ring.

The iron participates in redox reactions

and oscillates between Fe2+ and Fe3+ states.

Hemes in the 3 classes of cytochrome (a,

b, c) differ slightly in substituents on the
porphyrin ring system
 Prosthetic groups of cytochromes
 Heme  is a prosthetic group of cytochromes
 Mitochondria has 3 classes of cytochromes,designated a, b,
and c

Found in Hb

The heme iron atom can

undergo a 1 electron
transition between ferric and
ferrous states: 
     Fe3+ + e- <--> Fe2+ 
Only heme c is covalently linked to the protein via thioether
bonds to cysteine residues.
Heme a is unique in having a long farnesyl side-chain
that includes 3 isoprenoid units.
Note: A common feature is 2 propionate side-chains.
Cytochromes are proteins with heme prosthetic groups.
They absorb light at characteristic wavelengths.
Absorbance changes upon oxidation/reduction of the
heme iron provide a basis for monitoring heme redox state.
 Some cytochromes are part of large integral membrane
complexes, each consisting of several polypeptides &
including multiple electron carriers. 
E.g., hemes a & a3 that are part of the respiratory chain
complex IV are often referred to as cytochromes a & a3.
 Cytochrome c is instead a small, water-soluble protein
with a single heme group.
 Iron-sulfur centers

Iron-sulfur centers (Fe-S) are prosthetic groups containing

2, 3 , 4 or 8 iron atoms complexed to elemental & cysteine S
Cysteine residues provide S ligands to the iron, while also
holding these prosthetic groups in place within the protein.
 Cys S
Electron transfer proteins may S Fe
Cys
S Fe S
contain multiple Fe-S centers. S Fe S
Cys Cys
S Fe
Iron-sulfur centers transfer S
only one electron, even if they Cys S S S Cys
contain two or more iron Fe Fe
atoms, because of the close
Cys S S S Cys
proximity of the iron atoms.
Iron-Sulfur C enters

E.g., a 4-Fe center might cycle between redox states:

Fe+++3, Fe++1 (oxidized) + 1 e  Fe+++2, Fe++2 (reduced)
Respiratory
Chain: matrix

inter-
cristae membrane
space

inner outer
membrane mitochondrion membrane

Most constitutents of the respiratory chain are

embedded in the inner mitochondrial membrane (or in
the cytoplasmic membrane of aerobic bacteria).
The inner mitochondrial membrane has infoldings
called cristae that increase the membrane area.
 Electrons are
transferred from
NADH  O2 via
multisubunit
inner membrane
complexes I,II
III & IV, plus
CoQ & cyt c.
Within each complex, electrons pass sequentially through
a series of electron carriers.
CoQ is located in the lipid core of the membrane. There
are also binding sites for CoQ within protein complexes
with which it interacts.
Cytochrome c resides in the intermembrane space.
It alternately binds to complex III or IV during e transfer.
Individual respiratory chain
complexes have been isolated and
their composition determined.

Components of the electron

transport chain can be
purifed from the
mitochondrial inner
membrane

There is also evidence for

the existence of stable
supramolecular
aggregates containing
multiple complexes.
 Method for determining the sequence if electron carreirs

Inhibitors at various points of the respiratory chain were used

Composition of Respiratory Chain Complexes
No. of Prosthetic
Complex Name Proteins Groups

Complex I NADH 46 FMN,

Dehydrogenase 9 Fe-S cntrs.

Complex II Succinate-CoQ 5 FAD, cyt b560,

Reductase 3 Fe-S cntrs.

Complex III CoQ-cyt c 11 cyt bH, cyt bL,

Reductase cyt c1, Fe-SRieske

Complex IV Cytochrome 13 cyt a, cyt a3,

Oxidase CuA, CuB
Complex I
catalyzes
oxidation of
NADH, with
reduction of
coenzyme Q:
NADH + H+ + Q  NAD+ + QH2
An atomic-level structure is not yet available for the entire
complex I, which in mammals includes at least 46 proteins,
along with prosthetic groups FMN & several Fe-S centers.
Complex I is
L-shaped.

The peripheral domain, containing the FMN that accepts

2e from NADH, protrudes into the mitochondrial matrix.
Iron-sulfur centers are also located in the hydrophilic
peripheral domain, where they form a pathway for e
transfer from FMN to coenzyme Q.
A binding site for coenzyme Q is thought be close to the
interface between peripheral and intra-membrane domains.
 Mechanism of e- transfer in Complex I

o
r
o

r
o
r
o

 Estimated mass of this complex 850 kD

 Involves more than 30 polypeptide chains
 One molecule of FMN
 As many as 7 Fe-S clusters (2Fe-2S & 4Fe-4S)
 Precise mechanism of this complex is unknown
The initial electron transfers are:
NADH + H+ + FMN  NAD+ + FMNH2
FMNH2 + (Fe-S)ox  FMNH· + (Fe-S)red + H+

After Fe-S is reoxidized by transfer of the electron to

the next iron-sulfur center in the pathway:
FMNH· + (Fe-S)ox  FMN + (Fe-S)red + H+

Electrons pass through a series of iron-sulfur centers,

and are eventually transferred to coenzyme Q.
Coenzyme Q accepts 2 e and picks up 2 H+ to yield the
fully reduced QH2.
 Inhibitors of complex 1

Amytal (a barbiturate drug), rotenone (a plant product commonly

used as an insecticide), and piericidin A (anantibiotic) inhibit
electron flow from the Fe-S centers of Complex I to ubiquinone and
therefore block the overall process of oxidative phosphorylation.
 Complex II
Succinate Dehydrogenase
of the Krebs Cycle is also
called complex II or
Succinate-CoQ Reductase.
FAD is the initial e
acceptor.
FAD is reduced to FADH2 during oxidation of succinate to
fumarate.
FADH2 is then reoxidized by transfer of electrons through a
series of 3 iron-sulfur centers to CoQ, yielding QH2.
The QH2 product is then reoxidized via complex III,
providing a pathway for transfer of electrons from succinate
into the respiratory chain.
 H+ transport
Complex II does not
Succinate-CoQ Reductase occur in this
or complex
Succinate dehydrogenase
(from TCA cycle!)

 Mass of 100 – 140 kD

 Composed of 4 subunits, including 2 Fe-S proteins
 Three types of Fe-S cluster: 4Fe-4S, 3Fe-4S, 2Fe-2S

Path: Succinate FADH2 2Fe2+ UQH2

 Path of electrons from NADH, succinate, fatty
acyl–CoA, and glycerol 3-phosphate to ubiquinone.
Inhibitors of complex II

2-Thenoyltrifluoroacetone &
carboxin block complex II
Complex III (Cytochrome Reductase)
Accepts electrons from coenzyme QH2 that is generated by electron
transfer in complexes I & II.
Couples the transfer of electrons from ubiquinol (QH 2) to cytochrome c
with the vectorial transport of protons from the matrix to the
intermembrane space.

Cytochrome c1, a prosthetic group within complex III, reduces

cytochrome c, which is the electron donor to complex IV.
The Q cycle.
Inhibitors of complex III

• Antimycin A:
An antibiotic blocks electron transport by
inhibiting cytochrome reductase.
• BAL (British anti lewisite):
Used as therapeutic agent in the cases of
arsenic poisoning. It inhibits activity of
cytochrome reductase.
Cytochrome oxidase (complex IV) carries out the following
irreversible reaction:

The four electrons are transferred into the complex one at a time
from cytochrome c.
Inhibitors of complex IV
• Cyanide (CN)
A powerful poison that inhibit cytochrome oxidase by
combining with cytochrome a3.
Cyanide may arise from cyanogenic substance.
• Carbon monoxide (CO)
It inhibits activity of cytochrome oxidase. (a pollutant).
• 3. Hydrogen sulfide (H2S)
It inhibits cytochrome oxidase. H2S toxicity occurs during
oil drilling operations.
It is toxic as cyanide. It is a part of natural gas.
• Azide:
Sodium azide also inhibits cytochrome oxidase activity.
The Energy of Electron Transfer Is Efficiently Conserved in a
Proton Gradient

Proton gradient
• Proton gradient created as electrons
transferred to oxygen forming water
10 H+ / NADH
6 H+ / FADH2
ATP synthesis

The Proton Gradient Drives ATP Synthesis. How??

F1F0-ATP synthase of E.Coli.
F1 portion
is composed of several subunits.
It contains three α, three β and one γ, δ and ε sub
units,designated as α3 β3 γ1 δ1 ε1.
F0 portion
is composed of one a, two b, and twelve c
subunits.
Binding-change model for ATP synthase.
Proposed by Paul Boyer.
The F1 complex has three nonequivalent adenine
nucleotide–binding sites, one for each pair of α and β
subunits.
At any given moment, one of these sites is in the β-ATP
conformation (which binds ATP tightly), a second
is in the β-ADP (loose-binding) conformation, and a third is
in the β-empty (very-loose-binding) conformation.
proton-motive force causes rotation of the central shaft—
the γ subunit comes into contact with each αβ subunit pair in
Succession, producing cooperative conformational changes.
 Generation of ATP
• Proton dependant ATP synthetase
– Uses proton gradient to make ATP
– Protons pumped through channel on enzyme
• From intermembrane space into matrix
• ~4 H+ / ATP
– Called chemiosmotic coupling theory.
Other theories include: Chemical coupling theory &
Conformational coupling theory.
Totals
NADH
10 H+ X 1 ATP = 2.5 ATP
4 H+
FADH2
6 H+ X 1 ATP = 1.5 ATP
4 H+
 Uncouplers of Oxidative phosphorylation
These compounds dissociates or uncouples oxidation in
respiratory chain from phosphorylation.
So, the oxidation takes place without ATP synthesis.
Examples:
• 2, 4-dinitrophenol
• Dinitrocresol
• Salicylanilides
• Pentachlorophenol
• CCCP (Carbonylcyanide chloromethoxy phenyl hydrazone).
• FCCP (Carbanoyl cyanide p. trifluoromethoxy phenyl
hydrazone).
 Thermogenin
 Regulation of Oxidative
phosphorylation.
Oxidative phosphorylation in the
respiratory chain is subjected to
regulation like any metabolic pathway.

The rate of respiration is generally

limited by the availability of substrates
such as: ADP, Pi, NADH, FADH2 and
O2 .
 Oxidative phosphorylation
• This is the process by which ADP is phosphorylated
with inorganic phosphate to generate ATP.
• NADH2 has a central role in this process.
• Electrons are transferred down the respiratory chain
from NADH2 to oxygen. This is because NADH2 is a
strong electron donor while oxygen is a strong
electron acceptor.