Amity Institute of Pharmacy

Biochemistry

BP203T

Unit 2

By

Dr. Ajay Mahor

Assistant Professor

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Carbohydrate metabolism

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Major pathways of carbohydrate metabolism

✓The important pathways of carbohydrate metabolism
are listed as per syllabus
1. Glycolysis (Embden-Meyerhof pathway)
2. Citric acid cycle (Krebs cycle or tricarboxylic acid
cycle)
3. Hexose monophosphate shunt (pentose phosphate
pathway or direct oxidative pathway)
4. Glycogenolysis
5. Gluconeogenesis

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❖ Glycolysis – Pathway, energetics and significance

✓ Glycolysis is derived from the Greek words (glycose—sweet or
sugar; lysis—dissolution)

✓ It is a universal pathway in the living cells. The complete

pathway of glycolysis was elucidated in 1940

✓ This pathway is often referred to as Embden-Meyerhof pathway

(E.M. pathway) in honour of the two biochemists who made a
major contribution to the knowledge of glycolysis

✓ Glycolysis is defined as the sequence of reactions converting

glucose (or glycogen) to pyruvate or lactate, with the
production of ATP
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1. Glycolysis takes place in all cells of the body. The

enzymes of this pathway are present in the cytosomal
fraction of the cell

2. Glycolysis occurs in the absence of oxygen (anaerobic) or

in the presence of oxygen (aerobic). Lactate is the end
product under anaerobic condition. In the aerobic
condition, pyruvate is formed, which is then oxidized to
CO2 and H2O

3. Glycolysis is a major pathway for ATP synthesis in tissues

lacking mitochondria, e.g. erythrocytes, cornea, lens etc.
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4. Glycolysis is very essential for brain which is dependent on

glucose for energy. The glucose in brain has to undergo glycolysis
before it is oxidized to CO2 and H2O

5. Glycolysis (anaerobic) may be summarized by the net reaction

Glucose + 2 ADP + 2Pi → 2 Lactate + 2ATP

6. Glycolysis is a central metabolic pathway with many of its

intermediates providing branch point to other pathways. Thus, the
intermediates of glycolysis are useful for the synthesis of amino acids
and fat

7. Reversal of glycolysis along with the alternate arrangements at the

irreversible steps, will result in the synthesis of glucose
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❖ The glycolysis pathway can be divided into three

distinct phases

A. Energy investment phase or priming stage

1. Glucose is phosphorylated to glucose 6-phosphate by hexokinase or

glucokinase (both are isoenzymes). This is an irreversible reaction,
dependent on ATP and Mg2+
2. Glucose 6-phosphate undergoes isomerization to give fructose 6-phosphate
in the presence of the enzyme phosphohexose isomerase and Mg2+

3. Fructose 6-phosphate is phosphorylated to fructose 1,6-bisphosphate by

phosphofructokinase (PFK). This is an irreversible and a regulatory step in
glycolysis
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B. Splitting phase
4. The six carbon fructose 1,6- bisphosphate is split (hence the
name glycolysis) to two three-carbon compounds, glyceraldehyde
3-phosphate and dihydroxyacetone phosphate by the enzyme
aldolase (fructose 1,6- bisphosphate aldolase)

5. The enzyme phosphotriose isomerase catalyses the reversible

interconversion of glyceraldehyde 3-phosphate and
dihydroxyacetone phosphate. Thus, two molecules of
glyceraldehyde 3-phosphate are obtained from one molecule of
glucose

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C. Energy generation phase

6. Glyceraldehyde 3-phosphate dehydrogenase converts glyceraldehyde 3-
phosphate to 1,3-bisphosphoglycerate. This step is important as it is involved in
the formation of NADH + H+ and a high energy compound 1,3-
bisphosphoglycerate. Iodoacetate and arsenate inhibit the enzyme
glyceraldehyde 3-phosphate dehydrogenase. In aerobic condition, NADH passes
through the electron transport chain and 6 ATP (2 → 3 ATP) are synthesized by
oxidative phosphorylation.

7. The enzyme phosphoglycerate kinase acts on 1,3-bisphosphoglycerate

resulting in the synthesis of ATP and formation of 3-phosphoglycerate. This step
is a good example of substrate level phosphorylation, since ATP is synthesized
from the substrate without the involvement of electron transport chain.
Phosphoglycerate kinase reaction is reversible, a rare example among the
kinase reactions.

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8. 3-Phosphoglycerate is converted to 2-phosphoglycerate by phosphoglycerate

mutase. This is an isomerization reaction.

9. The high energy compound phosphoenol pyruvate is generated from 2-

phosphoglycerate by the enzyme enolase. This enzyme requires Mg2+ or Mn2+ and is
inhibited by fluoride. For blood glucose estimation in the laboratory, fluoride is
added to the blood to prevent glycolysis by the cells, so that blood glucose is
correctly estimated. (Fluoride combines with Mg2+ and phosphate to form a
complex that binds with active site of enolase and blocks access of substrate. Thus,
fluoride is an unusual competitive inhibitor).

10. The enzyme pyruvate kinase catalyses the transfer of high energy phosphate
from phosphoenol pyruvate to ADP, leading to the formation of ATP. This step also is
a substrate level phosphorylation. This reaction is irreversible.

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❖ Energetics of glycolysis

✓ The details of ATP generation in glycolysis (from glucose) are given in Table. Under
anaerobic conditions, 2 ATP are synthesized while, under aerobic conditions, 8 or 6 ATP
are synthesized—depending on the shuttle pathway that operates.

✓ When the glycolysis occurs from glycogen, one more ATP is generated. This is because
no ATP is consumed for the activation of glucose (glycogen directly produces glucose 1-
phosphate which forms glucose 6-phosphate). Thus, in anaerobic glycolysis, 3 ATP are
produced from glycogen

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❖ Significance of glycolysis

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✓ The citric acid cycle was proposed by Hans Adolf Krebs in 1937, based on the
studies of oxygen consumption in pigeon breast muscle

✓ The cycle is named in his honour (Nobel Prize for Physiology and Medicine in
1953.)

✓ The enzymes of the TCA cycle are located in the mitochondrial matrix, in close
proximity to the electron transport chain. This enables the synthesis of ATP by
oxidative phosphorylation without any hindrance

✓ Krebs cycle basically involves the combination of a two-carbon acetyl-CoA with

a four-carbon oxaloacetate to produce a six-carbon tricarboxylic acid, citrate

✓ In the reactions that follow, the two carbons are oxidized to CO2, and
oxaloacetate is regenerated and recycled

✓ Oxaloacetate is considered to play a catalytic role in the citric acid cycle

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❖ CITRIC
ACID
CYCLE
Pathway

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Significance of the Citric acid cycle

✓ The Krebs cycle (also known as the Citric Acid Cycle or TCA cycle) is a crucial metabolic
pathway with several significant roles

✓ It's the primary source of ATP production during aerobic respiration, generating high-
energy molecules like NADH and FADH2 that are used to generate more ATP in the
electron transport chain

✓ Additionally, the Krebs cycle provides carbon skeletons for the synthesis of essential
molecules like amino acids, nucleotides, and fatty acids.

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❖ HMP shunt and its significance; Glucose-6-

Phosphate dehydrogenase (G6PD) deficiency

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Pathway

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❖ Glycogen metabolism Pathways and glycogen storage diseases (GSD)

➢ GLYCOGENOLYSIS

✓ The degradation of stored glycogen in the liver and muscle constitutes

glycogenolysis

✓ The pathways for the synthesis and degradation of glycogen are not reversible

✓ An independent set of enzymes present in the cytosol carries out glycogenolysis

✓ Glycogen is degraded by breaking -1,4- and -1,6-glycosidic bonds

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Pathway

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❖ Gluconeogenesis- Pathway and its significance

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Pathway

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Significance of Gluconeogenesis
✓ Gluconeogenesis is significant because it maintains stable blood glucose levels when
dietary carbohydrates are limited, especially during fasting or prolonged periods of
exercise

✓ It ensures that tissues like the brain and red blood cells, which rely on glucose for
energy, have a continuous supply

✓ Additionally, gluconeogenesis helps clear metabolic byproducts like lactate from the
blood

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Hormonal regulation of blood glucose level

✓ Hormones play a significant role in the regulation of blood glucose concentration as
depicted below
✓ Primarily, insulin lowers blood glucose level (hypoglycemic) while the rest of the
hormones oppose the actions of insulin (hyperglycemia)

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Diabetes mellitus
✓ Diabetes mellitus is the third leading cause of death (after heart disease and
cancer) in many developed countries.

✓ It affects about 6 to 8% of the general population. The complications of

diabetes affect the eye, kidney and nervous system

✓ Diabetes is a major cause of blindness, renal failure, amputation, heart attacks

and stroke

✓ (The term diabetes, whenever used, refers to diabetes mellitus. It should,

however, be noted that diabetes insipidus is another disorder characterized by
large volumes of urine excretion due to antidiuretic hormone deficiency)

✓ Diabetes mellitus is a clinical condition characterized by increased blood

glucose level (hyperglycemia) due to insufficient or inefficient (incompetent)
insulin. In other words, insulin is either not produced in sufficient quantity or
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✓ As a consequence, the blood glucose level is elevated which spills over into
urine in diabetes mellitus (Greek : diabetes—a siphon or running through;
mellitus—sweet)

✓ An important feature of diabetes is that the body cells are starved of glucose
despite its very high concentration around i.e. scarcity in plenty

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Long term effects of diabetes

Hyperglycemia is directly or indirectly associated with several complications. These
include atherosclerosis, retinopathy, nephropathy and neuropathy

Management of diabetes
✓ Dietary management : A diabetic patient is advised to consume low calories (i.e. low
carbohydrate and fat), high protein and fiber rich diet

✓ Hypoglycemic drugs : The oral hypoglycemic drugs are broadly of two categories
sulfonylureas and biguanides. The latter are less commonly used these days due to
side effects
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Biological oxidation

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✓ Oxidation is defined as the loss of electrons and reduction as the gain of

electrons
✓ This may be illustrated by the interconversion of ferrous ion (Fe2+) to ferric ion
(Fe3+)

✓ The electron lost in the oxidation is accepted by an acceptor which is said to be

reduced
✓ Thus oxidation-reduction is a tightly coupled process

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✓ The general principle of oxidation-reduction is applicable to biological systems

also
✓ The oxidation of NADH to NAD+ coupled with the reduction of FMN to FMNH2
is illustrated

✓ In the above illustration, there are two redox pairs NADH/NAD+ and
FMN/FMNH2
✓ The redox pairs differ in their tendency to lose or gain electrons

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Redox potential (E0)

✓ The oxidation-reduction potential or, simply, redox potential, is a quantitative
measure of the tendency of a redox pair to lose or gain electrons

✓ The redox pairs are assigned specific standard redox potential (E0 volts) at pH 7.0 and
25°C

✓ The redox potentials of some biologically important redox systems are given in Table

✓ The more negative redox potential represents a greater tendency (of reductant) to
lose electrons

✓ On the other hand, a more positive redox potential indicates a greater tendency (of
oxidant) to accept electrons

✓ The electrons flow from a redox pair with more negative E0 to another redox pair
with more positive E0

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✓The redox potential (E0) is directly related to the change

in the free energy (G°)

✓Redox potential and Gibbs free energy are inversely

related: a positive redox potential (E0) corresponds to a
negative Gibbs free energy change (ΔG°), indicating a
spontaneous reaction, and vice versa

✓This relationship is expressed as ΔG° = -nFE0, where 'n'

is the number of moles of electrons transferred and 'F'
is Faraday's constant

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ELECTRON TRANSPORT CHAIN

✓ The energy-rich carbohydrates (particularly glucose), fatty acids, and
amino acids undergo a series of metabolic reactions and, finally, get
oxidized to CO2 and H2O
✓ The reducing equivalents from various metabolic intermediates are
transferred to coenzymes NAD+ and FAD to produce, respectively,
NADH and FADH2
✓ The latter two reduced coenzymes pass through the electron
transport chain (ETC) or respiratory chain and, finally, reduce oxygen
to water
✓ The passage of electrons through the ETC is associated with the loss
of free energy
✓ A part of this free energy is utilized to generate ATP from ADP and Pi

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✓ An overview of the biological oxidation is depicted below.

✓ An overview of the ETC is depicted below.

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❑Mitochondria – the power houses of cell

✓ The mitochondria are the centres for metabolic oxidative
reactions to generate reduced coenzymes (NADH and FADH2)
which, in turn, are utilized in ETC to liberate energy in the form
of ATP
✓ For this reason, mitochondrion is appropriately regarded as the
power house of the cell
❑Mitochondrial organization
✓ The mitochondrion consists of five distinct parts
✓ These are the outer membrane, the inner membrane, the
intermembrane space, the cristae, and the matrix, & the same is
depicted in the next slide

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A. Inner mitochondrial membrane:

✓ The electron transport chain and ATP synthesizing system are located on
the inner mitochondrial membrane which is a specialized structure, rich in
proteins
✓ It is impermeable to ions (H+, K+, Na+) and small molecules (ADP, ATP)
✓ This membrane is highly folded to form cristae
✓ The surface area of inner mitochondrial membrane is greatly increased due
to cristae
✓ The inner surface of the inner mitochondrial membrane possesses
specialized particles (that look like lollipops), the phosphorylating subunits
which are the centres for ATP production

B. Mitochondrial matrix:
✓ The interior ground substance forms the matrix of mitochondria
✓ It is rich in the enzymes responsible for the citric acid cycle, -oxidation of
fatty acids and oxidation of amino acids

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❑Structural organization of respiratory chain

✓ The inner mitochondrial membrane can be disrupted into five distinct
respiratory or enzyme complexes, denoted as complex I, II, III, IV, and V,
and the same is depicted in the image of the next slide
✓ The complexes I-IV are carriers of electrons while complex V is
responsible for ATP synthesis
✓ Besides these enzyme complexes, there are certain mobile electron
carriers in the respiratory chain and these include NADH, coenzyme Q,
cytochrome C, and oxygen
✓ The enzyme complexes (I-IV) and the mobile carriers are collectively
involved in the transport of electrons which, ultimately, combine with
oxygen to produce water
✓ The largest proportion of the oxygen supplied to the body is utilized by
the mitochondria for the operation of the electron transport chain

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❑ Components and reactions of the electron transport chain

✓ There are five distinct carriers that participate in the electron transport chain (ETC)
✓ These carriers are sequentially arranged as depicted below and are responsible for the
transfer of electrons from a given substrate to ultimately combine with proton and
oxygen to form water

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I. Nicotinamide nucleotides
✓ Of the two coenzymes NAD+ and NADP+ derived from the vitamin niacin,
NAD+ is more actively involved in the ETC

✓ NAD+ is reduced to NADH + H+ by dehydrogenases with the removal of two

hydrogen atoms from the substrate (AH2)

✓ The substrates include glyceraldehyde-3 phosphate, pyruvate, isocitrate, -

ketoglutarate and malate.

✓ NADPH + H+ produced by NADP+-dependent dehydrogenase is not usually a

substrate for ETC

✓ NADPH is more effectively utilized for anabolic reactions (e.g. fatty acid
synthesis, cholesterol synthesis).
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II. Flavoproteins
✓ The enzyme NADH dehydrogenase (NADH-coenzyme Q reductase) is a
flavoprotein with FMN as the prosthetic group

✓ The coenzyme FMN accepts two electrons and a proton to form FMNH2

✓ NADH dehydrogenase is a complex enzyme closely associated with non-heme

iron proteins (NHI) or iron-sulfur proteins (FeS)

NADH + H+ + FMN → NAD+ + FMNH2

✓ Succinate dehydrogenase (succinate-coenzyme Q reductase) is an enzyme

found in the inner mitochondrial membrane. It is also a flavoprotein with
FAD as the coenzyme. This can accept two hydrogen atoms (2H+ + 2e–) from
succinate
Succinate + FAD → Fumarate + FADH2

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III. Iron-sulfur proteins

✓ The iron-sulfur (FeS) proteins exist in the oxidized (Fe3+) or reduced
(Fe2+) state

✓ About half a dozen FeS proteins connected with respiratory chain

have been identified

✓ However, the mechanism of action of iron-sulfur proteins in the

ETC is not clearly understood

✓ One FeS participates in the transfer of electrons from FMN to

coenzyme Q

✓ Other FeS proteins associated with cytochrome b and cytochrome

c1 participate in the transport of electrons
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IV. Coenzyme Q
✓ Coenzyme Q is also known as ubiquinone since it is ubiquitous in living system. It is a
quinone derivative with a variable isoprenoid side chain

✓ The mammalian tissues possess a quinone with 10 isoprenoid units which is known as
coenzyme Q10 (CoQ10)

✓ Coenzyme Q is a lipophilic electron carrier. It can accept electrons from FMNH2

produced in the ETC by NADH dehydrogenase or FADH2 produced outside ETC (e.g.
succinate dehydrogenase, acyl CoA dehydrogenase)

✓ Coenzyme Q is not found in mycobacteria. Vitamin K performs similar function as

coenzyme Q in these organisms. Coenzyme Q has no known vitamin precursor in
animals. It is directly synthesized in the body

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V. Cytochromes
✓ The cytochromes are conjugated proteins containing heme group. The latter
consists of a porphyrin ring with iron atom. The heme group of cytochromes differ
from that found in the structure of hemoglobin and myoglobin

✓ The iron of heme in cytochromes is alternately oxidized (Fe3+) and reduced (Fe2+),
which is essential for the transport of electrons in the ETC

✓ This is in contrast to the heme iron of hemoglobin and myoglobin which remains in
the ferrous (Fe2+) state

✓ Three cytochromes were initially discovered from the mammalian mitochondria.

They were designated as cytochrome a, b and c depending on the type of heme
present and the respective absorption spectrum

✓ Additional cytochromes such as c1, b1, b2, a3 etc. were discovered later. The
electrons are transported from coenzyme Q to cytochromes (in the order) b, c1, c,
a and a3
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✓ The property of reversible oxidation-reduction of heme iron Fe2+ Fe3+ present in

cytochromes allows them to function as effective carriers of electrons in ETC

✓ Cytochrome c (mol. wt. 13,000) is a small protein containing 104 amino acids and a
heme group. It is a central member of ETC with an intermediate redox potential. It is
rather loosely bound to the inner mitochondrial membrane and can be easily extracted

✓ Cytochrome a and a3: The term cytochrome oxidase is frequently used to collectively
represent cytochrome a and a3 which is the terminal component of ETC. Cytochrome
oxidase is the only electron carrier, the heme iron of which can directly react with
molecular oxygen

✓ Besides heme (with iron), this oxidase also contains copper that undergoes oxidation-
reduction during the transport of electrons. In the final stage of ETC,
the transported electrons, the free protons and the molecular oxygen combine to
produce water

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OXIDATIVE PHOSPHORYLATION
✓ The transport of electrons through the ETC is linked with the release of free energy
✓ The process of synthesizing ATP from ADP and Pi coupled with the electron
transport chain is known as oxidative phosphorylation
✓ The complex V of the inner mitochondrial membrane is the site of oxidative
phosphorylation
P: O Ratio
✓ The P: O ratio refers to the number of inorganic phosphate molecules utilized for
ATP generation for every atom of oxygen consumed
✓ More appropriately, the P: O ratio represents the number of molecules of ATP
synthesized per pair of electrons carried through ETC
✓ The mitochondrial oxidation of NADH with a classical P: O ratio of 3 can be
represented by the following equation

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✓ Further, a P: O ratio of 2 has been assigned to the oxidation of FADH2. There is a

strong evidence now to suggest a P: O ratio of 2.5 for NADH, and 1.5 for FADH2, since
ten protons (for NADH) and six protons (for FADH2) are pumped across mitochondrial
membrane
✓ Synthesis of one ATP requires four protons
Sites of oxidative phosphorylation in ETC

✓ There are three sites in the ETC that are exergonic to result in the synthesis of 3 ATP
molecules as depicted in the image of ETC inhibitors

1. Oxidation of FMNH2 by coenzyme Q

2. Oxidation of cytochrome b by cytochrome c1
3. Cytochrome oxidase reaction

✓ Each one of the above reactions represents a coupling site for ATP production
✓ There are only two coupling sites for the oxidation of FADH2 (P: O ratio 2) since the
first site is bypassed

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MECHANISM OF OXIDATIVE PHOSPHORYLATION

✓ Several hypotheses have been put forth to explain the process of oxidative
phosphorylation

✓ The most important among them—namely, chemical coupling, and chemiosmotic—

are discussed below.

Chemical coupling hypothesis

✓ This hypothesis was put forth by Edward Slater (1953)

✓ According to the chemical coupling hypothesis, during the course of electron transfer
in the respiratory chain, a series of phosphorylated high-energy intermediates are first
produced which are utilized for the synthesis of ATP
✓ These reactions are believed to be analogous to the substrate-level phosphorylation
that occurs in glycolysis or citric acid cycle
✓ However, this hypothesis lacks experimental evidence, since all attempts, so far, to
isolate any one of the high-energy intermediates have not been successful
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Chemiosmotic hypothesis

✓ This mechanism, originally proposed by Peter Mitchell (1961), is now widely accepted
✓ It explains how the transport of electrons through the respiratory chain is effectively
utilized to produce ATP from ADP + Pi
✓ The concept of the chemiosmotic hypothesis is comparable with energy stored in a
battery separated by positive and negative charges

▪ Proton gradient:
✓ The inner mitochondrial membrane, as such, is impermeable to protons (H+) and
hydroxyl ions (OH–)
✓ The transport of electrons through ETC is coupled with the translocation of protons
(H+) across the inner mitochondrial membrane (coupling membrane) from the matrix
to the intermembrane space
✓ The pumping of protons results in an electrochemical or proton gradient. This is due
to the accumulation of more H+ ions (low pH) on the outer side of the inner
mitochondrial membrane than on the inner side as depicted in the next slide
✓ The proton gradient developed due to the electron flow in the respiratory chain is
sufficient to result in the synthesis of ATP from ADP and Pi
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▪ Enzyme system for ATP synthesis:

✓ ATP synthase, present in the complex V, utilizes the proton gradient for the synthesis
of ATP

✓ This enzyme is also known as ATPase since it can hydrolyze ATP to ADP and Pi

✓ ATP synthase is a complex enzyme and consists of two functional subunits, namely F1
and F0 as depicted in the next slide

✓ Its structure is comparable with ‘lollipops’

✓ The protons that accumulate on the intermembrane space re-enter the mitochondrial
matrix leading to the synthesis of ATP

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Inhibitors of electron transport chain

✓ Many site-specific inhibitors of ETC have contributed to the present knowledge of
mitochondrial respiration
✓ Three possible sites of action for the inhibitors of ETC are identified

1. NADH and coenzyme Q : Fish poison rotenone, barbituate drug amytal and antibiotic
piercidin A inhibit this site
2. Between cytochrome b & c1 : Antimycin A— an antibiotic, British antilewisite (BAL)—
an antidote used against war-gas—are the two important inhibitors of the site between
cytochrome b and c1
3. Inhibitors of cytochrome oxidase : Carbon monoxide, cyanide, hydrogen sulphide and
azide effectively inhibit cytochrome oxidase
4. Carbon monoxide reacts with reduced form of the cytochrome while cyanide and azide
react with oxidized form

Cyanide poisoning : Cyanide is probably the most potent inhibitor of ETC. It binds to Fe3+ of
cytochrome oxidase blocking mitochondrial respiration leading to cell death. Cyanide
poisoning causes death due to tissue asphyxia (mostly of central nervous system)

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INHIBITORS OF OXIDATIVE PHOSPHORYLATION

Uncouplers
✓ The mitochondiral transport of electrons is tightly coupled with oxidative
phosphorylation (ATP synthesis)

✓ In other words, oxidation and phosphorylation proceed simultaneously. There are

certain compounds that can uncouple (ordelink) the electron transport from oxidative
phosphorylation. Such compounds, known as uncouplers, increase the permeability
of inner mitochondrial membrane to protons (H+)

✓ The result is that ATP synthesis does not occur. The energy linked with the transport
of electrons is dissipated as heat. The uncouplers allow (often at accelerated rate)
oxidation of substrates (via NADH or FADH2) without ATP formation. The uncoupler,
2,4-dinitrophenol (DNP), has been extensively studied. It is a small lipophilic
molecule. DNP is a proton-carrier and can easily diffuse through the inner
mitochondrial membrane. In the people seeking to lose weight, DNP was used as a
drug. However, this is now discontinued, as it produces hyperthermia and other side
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✓ The other uncouplers include dinitrocresol, pentachlorophenol,

trifluorocarbonylcyanide phenylhydrazone (FCCP)
✓ The last compound (FCCP) is said to be 100 times more effective as an uncoupler than
dinitrophenol
✓ When administered in high doses, the drug aspirin acts as an uncoupler.

Physiological uncouplers : Certain physiological substances which act as uncouplers at

higher concentration have been identified. These include thermogenin, thyroxine and
long chain free fatty acids. The unconjugated bilirubin is also believed to act as an
uncoupler. This is, however, yet to be proved beyond doubt.

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