BCHEM 254: METABOLISM IN

HEALTH AND DISEASES II

 Course Content
1. DIGESTION

Digestion of carbohydrates

Digestion of proteins

Digestion of lipids

2. GLYCOLYSIS

Role of phosphorylated intermediates in Glycolysis

First phase or energy investment stage

Second phase or energy generation stage

Metabolic fate of pyruvate

Regulation of Glycolysis
 Course Content
3. ENTRY OF OTHER SUGARS INTO GLYCOLYTIC PATHWAY
Monosaccharide, disaccharide and polysaccharide metabolism
Regulation of glucose breakdown
Glycogenolytic Cascade
Non hormonal control of glycogenolysis

4. ENERGY CHARGE
5. CITRIC ACID CYCLE
Pyruvate oxidation
Lipoic acid
Tricarboxylic acid cycle (TCA cycle)
 Course Content
6. ANAPLEROTIC PATHWAYS OF THE CITRIC ACID CYCLE
7. GLYOXYLATE CYCLE
Reactions of the glyoxylate cycle
8. PENTOSE PHOSPHATE PATHWAY OR PHOSPHOGLUCONATE PATHWAY
Metabolic difference between NADP+ and NAD+
Human genetic Disorders of PPP
Glucose-6-phosphate dehydrogenase deficiency

9. ELECTRON TRANSPORT
10. OXIDATIVE PHOSPHORYLATION
 Course Content
11. FATTY ACID OXIDATION
Mobilization and Utilization of TAGs in mammals
Utilization of lipoproteins
Fatty acid oxidation reactions
The B-oxidation pathway
Oxidation of unsaturated fatty acids
Oxidation of fatty acids with odd number of carbon atoms

12. KETONE BODIES

Regulation of FA oxidation and ketone body formation
 Course Content
13. AMINO ACID OXIDATION OR DEGRADATION
Transamination reactions
Mechanism of transaminases
Diagnostic uses of transaminases
Fate of glutamate
Excretion of Ammonia
Energetics of urea synthesis
Genetic defects in the urea cycle
Degradation of amino acid carbon chain
Genetic abnormalities of phenylalanine catabolism
Succinyl CoA yielding amino acids
Glucogenic and ketogenic amino acids
 DIGESTION
1. Digestion of carbohydrates
It is mainly polysaccharides which begins in the mouth.
Salivary amylase secreted by the salivary glands hydrolyse α(1,4) glycosidic linkages of
starch and glycogen to produce a mixture of maltose, glucose and oligosaccharides.
The process is completed in the small intestines by the action of pancreatic amylase.
All digestible carbohydrates are converted to glucose and other monosaccharides.
Cellulose is not digested by most mammals.
But undigested cellulose residue provides fiber or roughage essential for mobility of
the intestines.
Ruminants have bacteria in their rumen which hydrolyze cellulose to glucose.
This glucose is fermented to yield lactate, acetate and propionate.
Lactate and propionate are converted to blood sugar by the liver.
Enzymes located in the outer borders of the epithelial cell lining of the small
intestines hydrolyze disaccharides.
Examples: Sucrase or Invertase hydrolyses sucrose to glucose and fructose, lactose to
glucose and galactose by Lactase and maltose to glucose units by Maltase.
Lack of lactase activity leads to lactose intolerance .
 The undigested lactose remains in the intestine, where some of it undergoes
fermentation by intestinal microorganisms.
Diarrhoea and formation of intestinal gases are the consequences
 DIGESTION OF PROTEINS
Proteins are hydrolyzed to their component amino acids which begins with the action of
pepsin in the stomach.
In the stomach, proteins stimulate the secretion of the hormone gastrin, which in turn
stimulates the secretion of HCl by the parietal cells of the gastric gland.
The acidity of gastric juice acts as an antiseptic and kills most bacteria.
It also converts pepsinogen into the active form pepsin.
Pepsin hydrolyses peptide bonds of ingested proteins to produce a mixture of small
peptides.
The low pH of the stomach contents which enter the small intestines stimulates the
pancreas to secrete bicarbonate, to neutralize gastric HCl.
The hormone cholecystokinin stimulates the secretion of trypsin, chymotrypsin
and carboxypeptidase made by the exocrine cells of the pancreas.
These hormones are released in their inactive forms and later converted to their
active forms.
Trypsin hydrolyses peptide bonds whose C=O are contributed by lysine and
arginine residues.
Chymotrypsin hydrolyses peptide bonds involving phenylalanine, tyrosine and
tryptophan residues.
Carboxypeptidase which contains zinc, splits carboxy terminal residues from
peptides.
The small intestines also secretes an aminopeptidase which cleaves off NH2
terminal residues from short peptides.
• Fibrous proteins such as keratin are only partially digested.

• Plant proteins such as those in cereal grains are also partially hydrolyzed because
they are surrounded by cellulose coat.

• Celiac disease is a rare condition in which the intestinal enzymes are unable to
digest certain water insoluble proteins of wheat, particularly gliadin which is
injurious to the cell lining of the small intestine.

• In acute pancreatitis, there is destruction of the normal pathway of secretion of

pancreatic juice into the intestines. The zymogens are converted into their active
forms prematurely inside the pancreas.
 DIGESTION OF LIPIDS
This begins in the small intestine in which the enzyme prolipase is secreted by the
pancreas.
The prolipase is converted to the active lipase and in the presence of bile salts acts on a
specific protein called colipase.
The lipase binds to droplets of Triacylglycerol and catalyzes the removal of one or both
external fatty acids residues to yield a mixture of free fatty acids and 2-mono acyl glycerols.
Bile salts are extremely important in the absorption of fat soluble nutrients.
When the formation or secretion of bile salts is deficient, undigested and undissolved fats
appear in the stool.
Also fat soluble vitamins A,D,E and K are not completely absorbed and nutritional
deficiency of vitamin A can result.
 GLYCOLYSIS

The process by which glucose molecule is enzymatically degraded to yield two

molecules of pyruvate.

It is a universal central pathway of catabolism, not only in animals and plants but
also in microorganisms.

The sequence of reactions is the same in all except its regulation which differs from
one organism to another.

Another important difference is the fate of the pyruvate formed.

 Role of phosphorylated intermediate in Glycolysis
The phosphate groups are completely ionized at pH 7, and thereby giving the
intermediates net negative charges. This prevents the product from escaping from
the cell.
Lactate, pyruvate and other charged substances can only leave the cell through
specific transport.
The intermediate forms essential components in the enzymatic conversion of
metabolic energy, especially in reactions where large amount of energy is released.
This energy is ultimately transferred to ADP to form ATP.
The phosphate group serves as recognition or binding groups required for the proper
fitting of glycolytic intermediates to the active sites of their corresponding enzymes.
First phase or energy investment stage
• Phosphorylation of glucose- phosphorylation is at position 6 at the
expense of ATP.
The reaction is irreversible under intracellular conditions, and it is catalyzed
by hexokinase, mostly found in animal, plant and microbial cells.
Hexokinase occurs in isozyme(differ only in kinetic properties) forms in
different organisms and tissues.
In muscle cells, hexokinase is strongly inhibited by its product glucose-6-
phosphate.
Glucokinase, found in the liver, catalyses the phosphorylation of glucose and
is not inhibited by glucose -6-phosphate.
It also has a higher Km for glucose than hexokinase.
 Conversion of G6P to Fructose-6-phosphate
This reversible reaction involves isomerisation of G6P to F6P.
 It is catalyzed by phosphoglucoisomerase.
 Phosphorylation of F6P
The enzyme is phosphofructokinase(PFK) and requires Mg2+ for catalysis.
The enzyme is specific for F6P.
Higher plant tissues contain a PFK that utilizes Pyrophosphate(Ppi) instead of
ATP and that reaction is reversible.
In animal tissues, this reaction is irreversible and is the second control point
in Glycolysis.
PFK is an allosteric enzyme and a major regulatory enzyme in muscle
Glycolysis.
The enzyme activity is stimulated whenever the cells ATP is depleted or
there is excess ADP or AMP.
The enzyme is however inhibited by high concentration of ATP as well as
other fuels like citrate and fatty acids.

PFK is also stimulated by F6P and fructose-1,6-bisphosphate (FBP).

 FBP activates the enzyme at low concentration.

The FBP is produced in animal cells when glucose is abundant and Glycolysis
needs to proceed.
 Cleavage of fructose-1,6-bisphosphate
The enzyme fructose bisphosphate aldolase or aldolase convert the
substrate to DHAP and G3P.
 Interconversion of triose phosphate
It is only G3P that can take part in the next step. DHAP can be rapidly
and reversibly converted to G3P through the action of triose phosphate
isomerase.
 Second phase or energy generation stage
This phase involves the conservation of free energy in glucose molecule in
the form of ATP.
Oxidation of Gly-3-P: This step catalyzed which is by glyceraldehyde-3-
phosphate dehydrogenase involves the oxidation of the C=O group to form an
acyl phosphate, 3-phosphoglyceryl phosphate. NAD+ is the oxidizing agent.
 Transfer of phosphate from 3-phosphoglycerol phosphate
The high energy phosphate from the carboxyl group of 3-phosphoglycerol
phosphate is donated to ADP to form ATP and 3-phosphoglycerate. The
enzyme responsible is phospoglycerate kinase.
 Formation of 2-phosphoglycerate
This reaction occurs with a reversible shift of the phosphate group
within the substrate molecule. It is catalyzed by phosphoglycerate
mutase.
 Dehydration of 2-phosphoglycerate
The enzyme enolase promotes reversible removal of a molecule of water
from the substrate.
PEP is a super high energy phosphate compound.

Although 2PG and PEP have nearly the same total amount of energy,
the loss of water from the former causes rearrangement of electrons in
the molecule so that a large amount of energy is released upon
hydrolysis.

 Enolase is inhibited by flouride ion (F-) in the presence of phosphate.

This is related to the formation of magnesium-flourophosphate complex

which removes Mg2+ from the reaction mixture.
 Formation of pyruvate
• The last step in Glycolysis which involves the transfer of the high energy
phosphate from PEP to ADP is catalyzed by pyruvate kinase.

• This is an example of substrate level phosphorylation.

• Pyruvate is produced in its enol form but its rapidly and non-enzymatically
rearranged to the Keto form at pH 7.
• Pyruvate kinase is an important regulatory enzyme in Glycolysis.
• It is an allosteric enzyme inhibited by ATP, alanine and Acetyl CoA, but
activated by AMP and fructose 1,6-bisphosphate.
 Metabolic Fate of Pyruvate
• Under aerobic conditions, NADH formed from the oxidation of G3P is
re-oxidized to NAD+ by oxygen.
• Under anaerobic conditions, such as that in active skeletal muscles,
lactic acid bacteria and some plant tissues, re-oxidation of NADH is by
pyruvate with the aid of lactate dehydrogenase producing lactic acid.
Lactate found in active skeletal muscle diffuses from the tissue into blood.
It is then carried to highly aerobic tissues like heart and liver to be
catabolized or converted to glucose.
The regeneration of NAD+ allows it to be used over and over again.
However, in yeast and other microorganisms that ferment glucose to ethanol
and carbon dioxide, they make use of a different pathway for NADH oxidation
because they lack lactate dehydrogenase.
First, pyruvate is decarboxylated by the action of pyruvate decarboxylase to
acetaldehyde and carbon dioxide.
This reaction is irreversible under cellular conditions.
In the final stage, acetaldehyde is reduced to ethanol by the reducing action
of NADH from the oxidation of G3P in glycolysis.
Pyruvate decarboxylase is present in brewer' s yeast and other organisms
that promote alcohol fermentation.
But in animal tissues and other organisms that carry out lactate
fermentation, they lack pyruvate decarboxylase. They rather have the
enzyme alcohol dehydrogenase.
 Regulation of Glycolysis
The process is controlled by:
1. The amount of glucose entering the glycolytic sequence.
2. The generation of the sequence itself.

Regulation of glucose entry

1. Phosphorylation of glucose by ATP using hexokinase.
Hexokinase is an allosteric enzyme which is inhibited by its product glucose-6-
phosphate, high levels of G6P inhibits hexokinase and low levels stimulates the
enzyme.
However, in the liver G6P is continuously formed as it contains mostly glucokinase
which is not inhibited by the G6P.
The excess G6P formed from glucose is converted to G1P and eventually to glycogen.
2. Glycogen phosphorylase
This is a regulatory enzyme and in the skeletal muscle.
 It occurs in two forms, phosphorylase a and less active dephospho form
phosphorylase b.
Phosphorylase a is converted to less active form by an enzyme called
phosphorylase a phosphatase.
Phosphorylase b is converted to active form by the enzyme phosphorylase
kinase which promote ATP phosphorylation of the essential serine residues to
form phosphorylase a.
Glycogen phosphorylase of the muscle is regulated in another way.
 Regulation of the Glycolytic pathway

There are 2 major regulatory points in the pathway:

1. Phosphofructokinase
• A complex allosteric enzyme whose modulators differ with cell type. In
skeletal muscles, they are regulated by levels of ATP, fructose-6-phosphate,
citrate and ADP. Other important regulators are AMP, Mg2+, phosphate and K+
ions.
• ATP and citrate are the most important inhibitory modulators.
• AMP and fructose-2,6-bisphosphate are the most important positive
modulators.
2. Pyruvate kinase
An allosteric enzyme that controls the Glycolytic sequence.
The enzyme occurs in three isozyme forms.
Pyruvate kinase is also inhibited by acetyl CoA and by long fatty acids which
are important fuels for TCA.
When the cell has high concentration of ATP and other fuels, Glycolysis is
inhibited by the action of either PFK or pyruvate kinase.
 On the other hand, low ATP concentrations promote the affinity of pyruvate
kinase for PEP and enable the transfer of phosphate to ADP to form ATP.
 Entry of other sugars into the Glycolytic pathway-
Monosaccharide Metabolism
Galactose utilization
Galactose is derived from the hydrolysis of lactose. The main route involves
ATP –dependent conversion to gal-1-p which is catalyzed by the enzyme
galactokinase.

Transformation of gal-1-p to glu-1-p involves epimerization at C4, however gal-

1-p should be metabolically activated before epimerization can occur.

This is achieved by transferase reaction with a nucleotide-linked sugar, uridine

diphosphate glucose or UDP-Glu.
In the liver, gal-1-p reacts with UDP-Glc to yield UDP-Gal and glucose-1-p.
The reaction is catalyzed by the enzyme UDP-glucose:α-D-galactose-1-
phosphate uridylyl transferase.
The galactose residue of UDP-gal is them enzymatically epimerized at C4 to
yield UDP-Glc by the enzyme UDP-glucose-4-epimerase.
UDP-glucose is then cleaved by UDP-glucose phosphorylase to glucose-1-p
which is them converted to glu-6-p by phosphoglucomutase.
In people with the genetic disease, galactosemia, UDP-glucose:α-D-galactose-
1-phosphate uridylyl transferase is genetically defective, preventing the overall
conversion of galactose into glucose.
 As a consequence, D-galactose and galactose-1-p cannot be metabolized and
accumulates in the blood and tissues.
The liver and other organs become enlarged, vision becomes impaired because
of the formation of cataracts and there is mental retardation.
Since the major source of galactose is lactose in milk, this deficiency appears
in infants.
Galactosemia can be alleviated by withholding milk and milk products from
the diet.
The defect can also arise when either galactokinase or UDP-glucose 4-
epimerase is genetically lacking.
 Mannose utilization
Mannose, which can be obtained from various polysaccharides and
glycoproteins present in natural foods, is converted to mannose-6-p by
hexokinase .

Mannose-6-p is then isomerized to fructose-6-p by phosphomannose

isomerase. F-6-P is channeled into Glycolysis.
 Fructose Utilization
Fructose, present in free form in many fruits and also formed by hydrolysis of
sucrose in small intestine is phosphorylated by hexokinase to form Fructose-6-
phosphate.
This is the major pathway in muscle and kidney.
However, in the liver, the enzyme fructokinase catalyses the phosphorylation
at C1, to form Fru-1-P.
The Fru-1-P is then cleaved to form D-glyceraldehyde and DHAP by aldolase B.
D-glyceraldehyde is phosphorylated by ATP by triose kinase to gly-3-p.
This pathway by passes PFK regulatory point and may explain why dietary
sucrose is converted to fat.
 Glycerol catabolism
Digestion of triacylglycerols and most phospholipids generate glycerol.
In animals, glycerol enters the Glycolytic pathway by the action of
glycerokinase in the liver.
The product is then oxidized by glycerol-3-p dehydrogenase to yield DHAP, an
intermediate of Glycolysis.
 Disaccharide metabolism
The three most abundant disaccharides in foods are maltose, lactose and
sucrose, and these are hydrolyzed in cells lining the small intestines to give the
constituent sugars.

In some humans, lactase disappears from the intestinal mucosal cells after
ages 4-6 years, when milk drinking usually decreases.

Plants and microorganisms have different pathways for sucrose metabolism.

Bacteria metabolize sucrose through the action of sucrose phosphorylase.
 Polysaccharide catabolism
The most important storage polysaccharides are glycogen and starch. The
glucose units of the outer branches of glycogen and starch gain entry into the
Glycolytic pathway through the action of glycogen phosphorylase (starch
phosphorylase in plants) and phosphoglucomutase.

(Glucose)n + Pi Glucose-1-p + (Glucose)n

In this reaction, the terminal α (1,4) glycosidic linkage at the non-reducing end
of the glycogen branch undergoes phosphorolysis, the removal of the terminal
glucose residues by attack of a phosphate.

Glycogen phosphorylase acts repetitively on the non-reducing end of glycogen

until it reaches a point 4 glucose residues next to α (1,6) branch linkage. Here,
its action stops or its inactive.
Further degradation of glycogen by phosphorylase can take place only after
the action of the second enzyme oligo (α 1,4-α 1,6) glucan transferase which
catalyzes two reactions.

Glucose-1-p, the end product of glycogen phosphorylase (and starch

phosphorylase), is converted to glu-6-p by phosphoglucomutase.

 Gluse-1-p ---------- Glucose-6-p

 Regulation of glycogen breakdown
Most vertebrate glycogen is stored as granules in cells of the liver and skeletal
muscle.
 The liver provides glucose for metabolism by other tissues and this is achieved
through glycogen mobilization and gluconeogenesis.
These processes produce phosphorylated forms of glucose which cannot leave
the liver.
Free glucose is therefore obtained through the action of G-6-phosphatase
which hydrolyses G-6-P to glucose and serves as the source of glucose for other
tissues.
However, muscle glycogen essentially serves as source of G-6-P for catabolism
within the muscle cells and therefore G-6-phosphatase is absent.
This ensures that G-6-P formed in the muscle is primarily used as energy
source.
 Glycogenolytic cascade
Glycogenolysis or glycogen breakdown involves a process in which an initial
regulatory signal is amplified so many times through a series of enzyme activations.

This is particularly in situations of fight or flight when there is instantaneous

requirement for increased energy generation and utilization.

Glycogen therefore represents the most immediately available large-scale source of

metabolic energy and hence it is essential that animals be able to activate glycogen
mobilization very rapidly.

Glycogen phosphorylase is a dimer containing 2 identical polypeptide chains.

The serine residue is at position 14 of each of the chain (N-terminal)

Inactive phosphorylase b is activated by a specific phosphorylase b kinase which
transfers phosphate group from ATP to the two serine residues.

Deactivation is effected by a specific phosphorylase phosphatase.

Phosphorylase b kinase is also activated by phosphorylation of the enzyme from an

inactive to an active form through the action of the enzyme cyclic AMP-dependent
protein kinase and ATP.

This enzyme's activity is in turn stimulated by adenosine-3',5'-monophosphate or

cyclic AMP.

Cyclic AMP dependent protein kinase is a tetramer consisting of two catalytic

subunits C2 and two regulatory subunits R2 .

The tetramer, C2R2 is catalytically inactive

The cAMP binds to the tetramer to dissociate.
The protein kinase catalyzes the phosphorylation of phosphorylase b kinase.
 R2C2 + 2cyclic AMP ----------- R2(cAMP)2 + C2
Phosphorylase b kinase is a complex enzyme made up of 4 subunits "a,b,y,d".

It is the 'a' and 'b' subunits that are phosphorylated to form the active enzyme.

cAMP, also called a second messenger, receives messages from outside the cell in the
form of hormonal stimuli and transmits them within the cell.

 The primary hormone that promotes glycogenolysis in muscle is epinephrine, whereas in

the liver it is glucagon.

Epinephrine binds to specific receptors on muscle cell membranes.

Secretion of few molecules (hormones) trigger a massive conversion of glycogen to

glucose-1-p molecules
 Non hormonal control of glycogenolysis

Phosphorylase b is allosterically and non-covalently activated by AMP (or 5-AMP).

 Under normal cellular conditions, this does not occur because ATP (which does not
stimulate phosphorylase b) is much more abundant and competes with AMP for
binding to the enzyme.

However, under deprived conditions, AMP may accumulate during ATP breakdown
which may result in phosphorylase b activation and then to glycogen breakdown.
 Energy Charge
It involves energy regulation in which 2 or more levels of control are overlaid
on a particular enzyme, to allow appropriate response to complicate
situations.
The hormones, epinephrine and glucagon, are secreted and circulated to cells
when the organism as a whole needs a burst of energy.
They affect a preparation for glycogen breakdown in all muscle cells via
phosphorylase b to phosphorylase a conversion.
Cells well supplied with instant available energy (high glucose or high
ATP/AMP ratio) need not respond by breaking down glycogen at high rates
until these energy reserves are depleted.
Conversely, individual cells with low energy supplies can draw on glycogen by
release of inhibition on the phosphorylase b form, even in the absence of
extra cellular signal.
CITRIC ACID CYCLE
The central oxidative pathway in respiration in which all metabolic fuels namely
carbohydrate, lipids and proteins are catabolized in aerobic organisms and tissues.

Reactions of the TCA cycle take place within the interior, gel matrix of the
mitochondria.

This arrangement ensures that the electrons are removed during oxidative
interaction with the electron transport chain.

Essentially, a 2C compound enters the cycle as acetyl CoA which reacts with a 4C
organic acid, oxaloacetate, to yield a 6C tricarboxylic acid, citrate.

The citrate enters a series of 7 reactions which lead to the release of 2C as CO2
and the remaining 4Cs regenerated as oxaloacetate which begins the process again.
 Pyruvate oxidation
The primary chemical input for the citric acid cycle (CAC) is acetyl CoA.
For carbohydrate metabolism, the end product of Glycolysis (pyruvate)
undergoes oxidative decarboxylation to yield acetyl CoA.
It is a complex reaction catalyzed by the pyruvate dehydrogenase complex.
The reaction is highly exergonic and is essentially irreversible in vivo.

Three enzymes and 5 different coenzymes are involved which constitute a highly
organized multi-enzyme complex, found in prokaryotic and eukaryotic cell
mitochondria.

The enzymes are pyruvate decarboxylase (E1), dihydrolipoyl transacetylase (E2) and
dihydrolipoyl dehydrogenase (E3).

E1 is linked to the coenzyme TPP, E2 contains tightly bound lipoic acid and E3 contains
FAD.

In eukaryotic cells, small amounts of 2 regulatory enzymes are present, a kinase that
phosphorylate 3 serine residues of E1 and a phosphatase that removes them.
 Steps in TCA cycle

All reactions of the TCA cycle takes place in the mitochondria of animal cells and
indeed all the enzymes and coenzymes have been located in the mitochondria.
Distribution of enzymes is as follows:

Inner membrane- Aconitase and succinate dehydrogenase

Matrix space- Citrate synthase, isocitrate dehydrogenase complex, succinyl CoA

synthase, fumarase, malate dehydrogenase complex and alpha ketoglutarate
dehydrogenase
The TCA cycle
Step 1
This is the condensation of acetyl CoA with OAA to form citrate, catalyzed by
citrate synthase.

 The methyl C of the acetyl group of the acetyl CoA loses a proton, and there is
a nucleophilic attack of the resultant carbanion on the carbonyl C of OAA.

There is the formation of a highly unstable citroyl CoA which simultaneously

hydrolyses to yield the product.
The CoASH formed in this reaction is now free to participate in oxidative
decarboxylation of another molecule of pyruvate.
Citrate synthase is a regulatory enzyme and the reaction it catalyzes is the rate
limiting step of the TCA cycle.
 Step 2 -Isomerization of citrate
The tertiary alcohol functional group of citrate is difficult to oxidize.
The isomerisation reaction by aconitase generates the secondary alcohol
compound, isocitrate which is readily oxidized.

The enzyme contains non-heme iron and acid-labile sulfur in cluster called an
Fe-S center.
It is also the target site for the toxic action of flouroacetate, a plant product
that has been used as rat poison.
Step 3-Dehydrogenation of isocitrate
This reaction is catalyzed by isocitrate dehydrogenase.
There are two different forms of the enzyme; one requires NAD+ as the acceptor and
the other NADP+ .

The reaction involves dehydrogenation of oxalosuccinate, an unstable enzyme-bound

intermediate that spontaneously decarboxylate to release the product.

The NAD+ linked enzyme is found only in the mitochondrion while the NADP+ -linked
one is found in the cytosol and mitochondria
 Step 4-Oxidation of α-ketoglutarate to succinate
This reaction is comparable to the pyruvate dehydrogenase complex reaction.
The keto acid undergoes oxidative decarboxylation with the formation of acyl
CoA, succinyl CoA.
This reaction is catalyzed by α-ketoglutarate dehydrogenase complex, an enzyme
complex similar to the pyruvate dehydrogenase complex.
There are three analogous enzymes; α-KG decarboxylase, dihydrolipoyl trans
succinylase and dihydrolipoyl dehydrogenase and five coenzymes; NAD+,FAD,
CoASH, lipoic acid and TPP.
The first reaction is catalyzed by α-KG decarboxylase.
There is then the transfer of a 4C unit to lipoic acid and subsequent oxidation by
NAD+ and FAD.
The important difference between the two multienzyme complexes is that the
regulatory activities associated with the pyruvate dehydrogenase complex is absent
from the α-KG dehydrogenase complex.
 Step 5- Substrate level phosphorylation
Succinyl CoA is an energy rich compound and its hydrolysis to succinate yields
large free energy.
This energy is used to drive the formation of an energy rich phosphate bond.
This substrate level phosphorylation reaction is catalyzed by succinyl CoA
synthase.
 In animal cells, the product is GTP and not ATP.
This energy conserving step has an intermediate in which the enzyme
molecule itself becomes phosphorylated at a histidine residue in the active site.
Much of the GTP formed drives the ultimate synthesis of ATP through the
action of nucleoside dephospho kinase.

 GTP + ADP -------- ATP + GDP

In plants and bacteria, however, ATP is formed directly.

Step 6 – dehydrogenation of succinate to fumarate
This oxidation is achieved through the flavoprotein succinate dehydrogenase
which contains covalently bound FAD.
The enzyme is tightly bound to the mitochondria inner membrane.
Succinate dehydrogenase is competitively inhibited by malonate and
oxaloacetate.

Step 7- Hydration of fumarate to malate

This reaction is catalyzed by fumarate hydratase, also called fumarase.
 It is highly specific and does not hydrolyse malate, the cis former of fumarate.
Step 8- Dehydrogenation of malate

This reaction is catalyzed by NAD-linked malate dehydrogenase which is

found in the mitochondrial matrix.

 In intact cells, the reaction proceeds to the right because OAA , the reaction
product, is rapidly removed by the citrate synthase reaction, so that the
concentration of OAA is extremely low.
Reactions of Steps 6 to 8
 CONTROL OF CAC
As in glycolysis, the rate of the CAC is initially regulated by the rate of formation of its
fuel acetyl CoA which is produced from pyruvate oxidation reaction and from the
oxidation of fatty acids.
Therefore, regulation of the cycle occurs both at the point of entry and also at key
reactions in the pathway.

Regulation of pyruvate oxidation

The pyruvate dehydrogenase complex is regulated through allosteric modulation of
its component enzymes.
The transacetylase enzyme (E2) is inhibited by acetyl CoA and activated by CoASH.
The dihydrolipoyl dehydrogenase (E3) is inhibited by NADH and activated by NAD+.
ATP is an allosteric inhibitor of the complex and AMP or ADP is an activator.
Therefore, the rate of this reaction is coordinated with the energy charge,
NAD+/NADH ratio and the ratio of acetylated to free CoASH.
The multi enzyme complex in mammals is also controlled by covalent
modification of the pyruvate dehydrogenase component of the complex.

When the ATP concentration of the mitochondrion is relatively high, it serves

as a stimulator of the auxiliary enzyme pyruvate dehydrogenase kinase.

In the presence of ATP, it phosphorylate 3 specific serine residues of pyruvate

dehydrogenase (E1), resulting in loss of activity.

Ample acetyl CoA and NADH also stimulate the activity of the auxiliary
enzyme.
A specific pyruvate dehydrogenase phosphatase hydrolytically removes the
bound P and this reactivates the E1.
This happens when the demand for ATP increases causing its levels to decline.
The phosphatase is stimulated by high concentration of free Ca+ and Mg2+ ions.
Regulation of CAC
The flux through the CAC is regulated by allosteric interactions as well as the
concentrations of substrates and the enzyme involved.

The most important factor controlling the CAC activity is the intra mitochondrial
concentrations of NAD+/NADH.

NAD+ is a substrate for three cycle enzymes namely isocitrate dehydrogenase,

α-KG dehydrogenase and maleate dehydrogenase as well as the pyruvate
dehydrogenase complex.

Under conditions that decrease the NAD+/NADH ratio such as the inhibition of
the ETS (as in cases of high ATP concentration) or substantial consumption of
ethanol (reversal of the alcohol dehydrogenase reaction), the concentration of
NAD+ can limit the activities of these dehydrogenases.
Important sites for allosteric regulation are the isocitrate dehydrogenase and the aKG
dehydrogenase reactions.
In many cells , isocitrate dehydrogenase is activated by ADP and inhibited directly by
NADH (in addition to the reduced activity caused by low NAD+/NADH ratio).
α-KG dehydrogenase activity is inhibited by succinyl CoA and by NADH.
In many tissues, the first reaction of the cycle sets its pace.
The rate of the citrate synthase reaction is controlled by the concentration of the
acetyl CoA which is in turn controlled by the activity of pyruvate dehydrogenase
complex.
The synthase activity is determined by the concentration of OAA.
The enzyme is subject to inhibition by NADH, NADPH or succinyl CoA.
Under normal conditions, the rate of Glycolysis and the rate of the CAC are coordinated so that
the glucose breakdown to form pyruvate is just what is needed to supply the CAC with its main
input, acetyl CoA.

The rate of Glycolysis is related to the rate of CAC by their inhibition by high levels of ATP and
NADH which are common intermediates and also by the concentration of citrate.

Citrate, the product of the first step of the CAC serves as an important allosteric inhibitor of
phosphofructokinase in the Glycolytic sequence.

However, some tissues such as the heart cannot transport citrate out of the mitochondria, so
interaction with cytosolic PFK may not occur to any significant extent, but citrate levels can still
control CAC in the heart.
 ANAPLEROTIC PATHWAYS OF THE CITRIC ACID CYCLE
Certain metabolic reactions tend to deplete CAC intermediates by using them as
precursors for biosynthesis.
If these intermediates are not replenished, the rate of CAC would decline because of
the low concentrations of the intermediates in the mitochondria.
Anaplerotic reactions or sequence or filling up pathways replace these intermediates
in mitochondria which usually remain relatively constant .
Some reactions that use intermediates:
Succinyl CoA is used in the synthesis of heme and other porphyrins like chlorophyll.
OAA and α-KG are the keto ACID derivatives of the amino acids aspartate and
glutamate respectively.
 In some tissues, citrate is transported out of the mitochondria to the cytosolic where it is
cleaved to provide acetyl CoA for fatty acid biosynthesis.
OAA can also be used for carbohydrate synthesis via gluconeogenesis.
 In the cytosol it is acted upon by PEP carboxykinase to provide PEP.
 Anaplerotic reactions
Conversion of pyruvate to OAA.
This occurs particularly in the liver and kidney.
The reaction uses biotin as a cofactor as is used in most carboxylation
reactions.

Pyruvate carboxylase is a tetrameric protein containing 4 molecules of biotin;

each is covalently bound through an amide linkage involving the 3-amino group
of specific lysine residue.
In the first step of the reaction, there is ATP-dependent carboxylation of the
enzyme bound coenzyme to give N-carboxybiotin.
This activated derivative then transfers the carboxyl group directly to pyruvate.
Pyruvate carboxylase is allosterically activated by acetyl CoA.
This is feedforward activation with the effect that acetyl CoA accumulation
facilitates its own utilization by promoting the synthesis of OAA with which it
reacts to form citrate through the citrate synthase reaction.

PEP + CO2 + GDP ---------OAA + GDP

The reaction catalyzed by PEP carboxykinase takes place in heart and muscle.
In plants and bacteria, PEP is converted to OAA in a reaction catalyzed by PEP
carboxylase.

PEP + HCO3 ----- OAA

Another reaction converts pyruvate to Malate and is catalyzed by malic enzyme
or malate dehydrogenase (decarboxylating: NADP+)

Malic enzyme occurs in both the mitochondria and cytosol of plant and animal
cells.
The cytosolic enzyme is important because of its ability to produce NADPH for
biosynthetic purposes.
Transamination reactions can also be regarded as anaplerotic.
An amino acid transfers its amino group to a keto acid with itself being
converted to keto acid.
Glutamate and aspartate undergo transamination to generate CAC
intermediates α-KG and OAA respectively.

• Glutamate can be converted to α-KG by another route catalyzed by glutamate

dehydrogenase.
Glutamate + NAD(P)+ + H2O -----------α-KG + NAD(P)H + NH4+
GLYOXYLATE CYCLE

This series of reactions take place predominantly in plant cells and some
microorganisms.
They result in the net synthesis of carbohydrate from fat.
This is particularly important in the development of seeds which store energy
in the form of triacylglycerols (TAG).
When seed germinates, the TAGs are broken down and converted to sugars,
which provide energy and raw materials needed for the growth of the plant.
GLYOXYLATE CYCLE
The glyoxylate cycle takes place in the glyoxysomes, a specialized cytoplasmic
organelle, that carries out both β-oxidation of fatty acids to acetyl CoA and the
utilization of the acetyl CoA in the glyoxylate cycle.
Glyoxysomes appear in the cotyledons of lipid-rich seeds during germination
and disappears when the plant can photosynthesize.
Essentially, the cycle converts 2 acetyl units as acetyl CoA to succinate.
It uses some of the same enzymes as CAC but it by-passes the 2 reactions
where C is lost as CO2.
GLYOXYLATE CYCLE
 Reactions of the glyoxylate cycle
The overall reaction, which may also be regarded as anaplerotic is as follows:
2 acetyl CoA + NAD+ + 2H2O -------- Succinate + 2CoASH + NADH + H+

1. Acetyl CoA from fatty acid oxidation condenses with OAA to give citrate just as
CAC.
Alternatively, acetate can also be converted to acetyl CoA by acetate thiokinase.
CH3COO- + CoASH + ATP --------- CH3COCoA + AMP + Ppi

2. Citrate is converted to isocitrate by aconitase (as in CAC)

3. Isocitrate is cleaved by isocitrate lyase to give glyoxylate and succinate
4. Glyoxylate then accepts another molecule of acetyl CoA, in a reaction
catalyzed by malate synthase.
The succinate produced is transferred to the mitochondria where it is acted upon by
enzymes of the TCA cycle to regenerate the OAA for the glyoxylate cycle .
The OAA is then transaminated by an amino transferase to produce aspartate (OAA
cannot leave the mitochondria).
The aspartate then leaves the mitochondrion and enters the glyoxysomes where it
is converted back into OAA through transamination.
The malate produced by malate synthase can be oxidized in the cytosol to OAA and
then converted to PEP through the PEP carboxykinase reaction.
The PEP undergoes gluconeogenesis for carbohydrate synthesis.
Thus two molecules of acetyl CoA are used to synthesize carbohydrate, a reaction
which does not occur in animals.
The malate can also enter the mitochondria and be converted to OAA, aspartate
and some nitrogenous compounds like pyrimidines (from aspartate).
Still in the mitochondria, the OAA produced can react with a molecule of acetyl CoA
to provide citrate and other TCA cycle intermediates for anabolic use. Finally the
malate in the mitochondria can be used to form succinate and succinyl CoA through
the reversal of TCA reactions.
The malate dehydrogenase reaction (to produce OAA), along with the citrate
synthase and aconitase reaction, is identical to CAC reactions.
The glyoxylate reactions are catalyzed by isoenzymic forms of each enzyme that are
specialized for their role and these isoenzymes are compartmentalized in
glyoxysomes.
PENTOSE PHOSPHATASE PATHWAY OR PHOSPHOGLUCONATE PATHWAY
This pathway operates to varying extents in different cells and tissues.
The enzymes are located in the cytosol.
The PPP has three basic functions:
1. To provide NADPH for reductive biosynthesis.
2. To provide ribose-5-P for nucleotides and nucleic acid biosynthesis.
3. To metabolize dietary PENTOSE sugars derived from the digestion of nucleic acids.

Metabolic difference between NADP+ and NAD+

NAD+/NADH-linked enzymes primarily function to oxidize substrate while enzymes
using NADP+/NADPH as coenzymes catalyze reactions to provide the reducing power
required for many biosynthetic reactions .
The PPP operates in two phases- oxidative and non-oxidative.
Oxidative
1. The first reaction catalyzed by Glu-6-P dehydrogenase oxidizes Glu-6-P to 6-
phosphoglucono-δ-lactone.
2. The lactone is hydrolyzed by a specific lactonase to yield 6-phosphogluconate.
3. The 6-phosphogluconate undergoes oxidative decarboxylation to yield
ribulose-5-P (a pentose phosphate) and NADPH.
These reactions constitute the irreversible phase of PPP.
The NADP+ is competitively inhibited by NADPH.
The change in Gibbs free energy of hydrolysis of the lactone is large and
therefore the overall oxidation of Glu-6-P is irreversible.
Non-oxidative (or reversible phase)
1. Ribulose-5-P is converted to ribose-5-P, a reaction catalyzed by
phosphopentose isomerase.
2. The reaction proceeds via an enediol intermediate.
At that point, the pathway has fulfilled its two basic functions; thus, to generate
NADPH and ribose-5-P.
The significance of NADPH is to provide reducing strength for most biosynthetic
reactions.
Ribose-5-P may not be needed in large quantities and so has to be catabolized.
This is achieved through a series of reactions that converts 3 5C sugar phosphates
to 2 6C sugar phosphates and one 3C sugar phosphate.
The hexose phosphates can be broken down either by recycling through the PPP or
by Glycolysis.
The triose phosphate is glyceraldehyde-3-P, a glycolytic intermediate.
In all, 3 enzymes are involved, namely phosphopentose epimerase, transketolase
and transaldolase.
Ribulose-5-P is converted to its epimer, xylulose-5-P.

One molecule of xylulose-5-P reacts with 1 molecule of ribulose-5-P in a reaction

catalyzed by transketolase.
This enzyme transfers 2C fragment form xylulose-5-P to ribulose-5-P to give
glyceraldehyde-3-P and a 7C sugar sedoheptulose-7-P.
The transketolase reaction requires Thiamin pyrophosphate (TPP) as a
cofactor and the 2C fragment is transferred as activated glycoaldehyde
(HOCH2CHO) which becomes transiently bound to TPP.
Transaldolase transfers a 3C dihydroxyacetone unit from a 7C substrate to a 3C
substrate to produce erythrose-4-P and fructose-6-P.
Finally, transketolase acts on another molecule of xylulose-5-P and transfers
its glycoaldehyde unit to erythrocyte-4-P and fructose-6-P.

Recall that the pathway has consumed 3 pentose P – 2 in the first

transketolase reaction and one in second.
Considering the oxidative phase 3 molecules of glu-6-P are used:
3 Glu-6-P + 6 NADP+ + 3H2O ------- 3 Pent-5-P + 6NADPH + 6H+ + 3CO2

For the non-oxidative phase

3 Pent-5-P ------- 2 Fru-6-P + Gly-3-P
This reaction provides the pathway for the degradation of pentose sugars
from nucleic acids.
Overall balanced reaction for the pathway
3 Glu-6-P + 6NADP+ + 3H2O ---- 2 Fru-6-P + Gly-3-P + 6NADPH + 6H++ 3CO2
In the PPP, the fate of the sugar phosphates depends on the metabolic needs of the cell in which
the pathway is operating.
If it is for predominantly nucleotide synthesis, the major product is ribose-5-P and therefore most
of the rearrangement of the non-oxidative phase generates compounds that can be reconverted
to glu-6-P for subsequent passage through the oxidative phase, that is, fru-6-P can be converted
to glu-6-P.
Glu-6-P --------- Fru-6-P
Gly-3-P + DHAP ---------- Fructose bis phosphate--- Fru-6-P ---- Glu-6-P
The NADPH produced is used in the synthesis of fatty acids and in the testis and adrenal cortex
for synthesis of steroids.
In a cell with moderate needs for NADPH and for pentose phosphates, the fru-6-P and gly-3-P
produced in the non-oxidative phase can be further catabolized by Glycolysis and CAC.
Fru-6-P ------ Fructose bis phosphate ------- Gly-3-P + DHAP
|
Pyruvate
Under normal circumstances, none of the three modes of operations is exclusive in any one
cell because of the metabolic needs of the cell.
Human genetic disorders of PPP
Glucose-6-phosphate dehydrogenase deficiency
G6PD is the first enzyme of the PPP and in human erythrocytes, that's the only pathway for
the generation of NADPH.
This compound is required to maintain glutathione which is a tripeptide (δ-
glutamylcysteinylglycine) in its reduced form.
The overall reaction is
GSSH + NADPH + H+ ----- 2GSH + NADP+
which is catalyzed by glutathione reductase
Glutathione is abundant in most cells and because of its free thiol group, offers
protective mechanism against oxidative stress.
This stress is usually generated when some individuals consume a variety of
compounds like primaquine or chloroquine (or some antimalarials), some sulfur-
containing drugs such as sulfonamides or consume lava beans ( because these
legumes generate oxidants ).
Oxidative stress is manifested by the presence of H2O2 in the red cells.
Glutathione dissipates H2O2 by the reaction catalyzed by glutathione peroxidase.
2 GSH + H2O2 ------ GSSH + 2H2O
Glutathione also helps keep cysteine groups in proteins in their reduced state.
P-S-S-P + GSH ----- PSH + GSSP -----P'SH + GSSG
The most important role of GHS in erythrocytes is to maintain hemoglobin iron in its Fe2+ (or
reduced state ).
This makes erythrocytes particularly sensitive to glutathione depletion.
When individuals who are G6PD deficient consume compounds that produce oxidative
stress, available GHS become used up as NADPH levels are inadequate to convert GSSG to
GHS, through glutathione reductase reaction.
Low GSH levels are unable to maintain heme iron in the reduced state and consequently
methemoglobin accumulates at the expense of hemoglobin.
Oxidative stress can also cause oxidation of sulfydryl groups of the globin chain which will
lead to the denaturation of hemoglobin and formation of precipitates (Heinz bodies).
These precipitates of denatured hemoglobin may damage membrane sufficiently to cause
the hemolysis or hemolytic anaemia.
Several hundreds of G6PD genetic variants have been identified.
Two variants designated G6PD A- and G6PD Mediterranean lead to clinically significant
hemolysis.
The G6PD A- is predominant in some African populations while the Mediterranean is largely
found in populations in the Middle East.
The prevalence of this gene or trait is believed to have been maintained because a deficiency of
G6PD protects against Falciparum malaria.
It is believed that the plasmodium utilizes some of the products of PPP for development and in
G6PD deficient individuals, the cells may lyse before the plasmodium fully develops.
In G6PD deficient individuals, the enzyme activity may be 10-60 % in those with A- variants and
the Mediterranean may have less than 10% of the normal activity.
In both situations, the mutation does not impair enzyme synthesis but rather the stability
especially in the older cells.
ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION
In Glycolysis and Citric acid cycle, a total of 5 molecules of NADH and 1 FADH2 are
produced.
In aerobic cells and tissues, there is stepwise transfer of electrons from the reduced
nucleotides to molecular oxygen.
In respiration,NADH and FADH2 become oxidized (in eukaryotic cells) by electron
transport proteins bound to the inner membrane of the mitochondria.
Along these proteins or electron transport carriers, a series of coupled
oxidation/reduction reactions occurs with the final acceptor (oxygen) being converted
to water.
The electron transport sequence is an exergonic and forms a pair of reducing
equivalents from NADH; thus, 3 molecules of ATP could be formed from ADP and Pi.
 The Mitochondrion
The mitochondrion is the site of many metabolic processes including-oxidation of fatty acids.
It consists of three distinct parts; the outer membrane, the inner membrane, the inter-membrane
space and the matrix.
The inner membrane and the matrix are known as mitoplast. The membrane is thrown into
projections called cristae. The respiratory proteins are bound to the inner membrane and therefore
the extent of folding of the membrane depicts the respiratory activity of the cell.
For example, heart muscle cells with high respiratory rates contain mitochondria with densely
packed cristae and in contrast with liver cells whose cristae are scarcely distributed.
On the surface of cristae lies an enzyme that synthesizes ATP from ADP and orthophosphate within
the matrix.
The protein carriers, especially the cytochromes are embedded within the inner membrane and
they constitute the respiratory chain.
The Electron Transport
The carriers
Most of the carriers are organized into complexes made up of several protein components
(enzymes) and a variety of prosthetic groups.
1. NADH and NADP dehydrogenase
The NADH produced from numerous oxidative reactions is oxidized in the first reaction of the
electron transport in a reaction catalyzed by the enzyme NADH dehydrogenase complex or
complex I.
The enzyme contains flavin mononucleotide (FMN) as a tightly bound prosthetic group and
catalysis the reaction:
NADH + H+ + FMN ----- NAD+ + FMNH2
The enzyme is a large multi-subunit complex and transfers its electrons to another carrier
coenzyme Q.
NADPH can also be oxidized by the complex but less readily.
When it is necessary, NADPH transfers its 2 electrons to NAD+ via the enzyme
trans-hydrogenase.
NADPH + H+ + NAD+ ------ NADP+ + NADH + H+
The multi- enzyme complex also contains a number of iron-sulphuric centers
which transfers electrons from FMNH2 to CoQ.
The iron in these centers ( in the form of Fe2S2 or Fe4S4) undergoes cyclic
oxidoreductions between ferrous and ferric states.
Since the electrons are used to reduce CoQ, this complex is also known as
NADH-CoQ reductase.
The electron transfer is as follows:
2. CoenzymeQ
The carrier is a fat soluble quinone with a very long isoprenoid chain.
It is also called ubiquinone because it's ubiquitous in all cells.
In most mammalian tissues, it has 10 isoprene units and non-polar character
allows it to diffuse rapidly through the inner mitochondrial membrane.
CoQ accepts electrons as follows:
CoQ accepts electrons not only from NADH but also from succinate (succinyl
dehydrogenase, inner membrane protein), and intermediates in fatty acid
oxidation.

Succinate dehydrogenase flavoprotein complex or complex II transfers electrons via

FeS centers to CoQ.

The complex is also known as succinate CoQ reductase. CoQ can in fact be
regarded as a collection point for electrons from several flavoprotein
dehydrogenases and NADH dehydrogenase and pass them on to cytochromes.
3. Cytochromes
These are a group of red or brown heme proteins that act in sequence to carry
electrons to molecular oxygen. The major respiratory cytochromes are classified a, b,
and c depending on the wavelengths of their spectral absorption peaks.
Cytochrome C is the best known of the cytochromes. It is a small protein with an
iron-porphyrin group covalently attached to single polypeptide chain.
The iron-porphyrin complex is protoporphyrin IX, the heme found in haemoglobin
and myoglobin. The transfer of electrons from CoQ to cyt C involves a Fe-S protein
and is catalyzed by a cyt bc complex or complex III.
Cyt a and a3 contain a modified form of heme A or porphyrins A with two different
side chains. Cyt a and a3 have identical heme A moieties attached to the same
polypeptide chain but in different environments
Each heme iron is associated with a copper ion which undergoes cuprous/cupric
redox changes, i.e. Cu I/Cu II while the iron atoms undergo ferrous/ferric redox
reactions i.e. Fe2+/Fe3+ .

Cyt a and a3 form a large multi-protein complex called cytochrome oxidase or

complex IV.

All the other cytochromes except C are integral membrane proteins and are very
difficult to dissociate from the membrane.

It has been found that the cytochromes in the respiratory chain are arranged in the
following sequence: b– c1– C – aa3.

Thus, it's the reduced cyt aa3 that passes electrons to molecular oxygen.
Determination of sequence of respiratory electron carriers
Electrons flow from the more negative E0' (standard electron reduction potential of a
donor system) in the direction of the more positive system.
The standard free energy change of a reaction in which there is a transfer of
electrons is given by:
∆G0' = -nF∆E0'
∆ G0' is the standard free energy change in kJ/mol
n is the number electrons transferred in the half reactions
F is the Faraday's constant, 96500 Jmol-1 V-1
∆E0' is the difference in standard reduction potentials between the two redox couples.

∆G0' for ATP hydrolysis is -30.5 kJmol-1 .

Therefore, synthesis of ATP requires a 91.5kJ/mol under standard conditions.
Two major experiments supporting the sequence of ETC.

1. Respiratory complexes
Sonic oscillations of mitochondria or solubilization by non-ionic detergents can
fractionate mitochondria into 4 separate enzyme complexes, each of which
contains part of the entire respiratory sequence.
Each complex contains several integral membrane components and these
(complexes) accepts electrons from relatively mobile carriers i.e. those that are
not tightly bound to membrane.
The mobile carriers are CoQ10 and Cyt C.
The complexes are made up of:
a. I- NADH-CoQ reductase
b. II- Succinate-CoQ reductase
c. III- cyt bc1 reductase
d. IV- cytochrome oxidase
e. V- F0F1 ATP synthase
The presence of appropriate mobile carriers, electrons can be transferred from NADH or
succinate to oxygen.
When the mobile carriers are removed, electron flow is interrupted at those points.
In the presence of another subassembly, complex V, which is made up of ATP synthase
complex, the entire complexes can synthesize ATP from ADP and orthophosphate.
2. Inhibitors and artificial electron acceptors
Certain specific inhibitors acting at particular points in the chain have provided
information about the arrangement of protein carriers in the ETC.
The inhibitors include:
a. Rotenone, a plant product that is used as an insecticide, blocks the flow of electrons
from NADH to CoQ.
b. Amytal, a barbiturate drug, acts at the same point.
c. Antimycin A, a streptomycin antibiotic, blocks transfer of electrons from cyt b to cyt
C1.
d. Cyanide, one of the most deadly poisons known, inhibits the reduction of oxygen
catalyzed by cyt aa3. Another substance is carbon monoxide. Cyanide and azide
reacts with the oxidized form, whereas CO with the reduced form.
Considering the known sequence of the carrier proteins, inhibition of the ETC at a
given site creates a cross-over point.
At such points, the electron carriers just before the block becomes more reduced and
those after the block more oxidized.
These changes can be detected spectroscopically since oxidized and reduced carriers
have different spectra.
It has also been observed that the standard redox potentials of carriers are
successively more positive going towards oxygen.
This is expected because electrons flow from electronegative to electropositive
systems.
Furthermore, each protein carrier is specific for a given donor or acceptor.
For example, CoQ can transfer electrons directly to cyt b and not directly to cyt a.
The electron shuttling system
Reduced coenzymes generated in Glycolysis in the cytosol cannot traverse the
mitochondrial membrane to be oxidized by the respiratory chain.
For example, the NADH generated through the oxidation of gly-3-P by gly-3-P
dehydrogenase in the cytosol must be shuttled to the respiratory assemblies, in the
inner mitochondrial membrane, in the absence of physical movement of the
coenzyme.
This is achieved through the reduction of an organic molecule by NADH in the
cytoplasm, which is then transported inside the mitochondrion, and the product
oxidized and return back to the cytoplasm where it can undergo the same cycle again.
The first known shuttle which is achieved in the brain is the dihydroxyacetone
phosphate/glycerol-3-P shuttle.
Dihydroxyacetone phosphate/glycerol-3-P shuttle
In this system, DHAP is reduced by NADH in the cytosol and the resultant
glycerol-3-P via a specific transport system, passes into the mitochondrion.
It is oxidized by the flavor protein, with the help of glycerol-3-P
dehydrogenase located at the outer surface of the inner membrane.
The reduction of FAD is followed by the transfer of electron pair from FADH2
to CoQ for onward transfer to other protein carriers.
The DHAP so formed returns to the cytosol to accept another reducing
equivalent.
Malate/aspartate shuttle
Another shuttling system, especially active in the liver and heart, is the
malate/aspartate shuttle.
In this system, a cytosolic isoenzyme of malate dehydrogenase (MDH) together with
NADH reduces OAA to malate which passes into the mitochondrial matrix via specific
active transport system in the inner mitochondrion membrane.
The malate is re-oxidized by MDH of the CAC which is NAD-dependent.
OAA cannot cross the inner membrane, it is transaminated to aspartate which is then
transported out for conversion to OAA to restart the cycle.
The transamination process requires that α-KG be continuously transported out of
the mitochondria and glutamate be transported in.

L-Asp + α -KG ------- OAA + L-glu

 Oxidative phosphorylation
Site of ATP synthesis in the ETC
It has been observed that for the oxidation of NADH through the ETC, three
individual reactions of the chain are exergonic enough to drive the synthesis of ATP
molecules from ADP and Pi.
Each reaction has ∆G0 values of over 30.5 kJ/mol which is the minimum.
These reactions include the oxidation of FMN by CoQ, the oxidation of cyt b by cyt
C1 and the cyt oxidase reaction. Thus, actual sites are FMN ------- CoQ, cyt b -----c1,
a3 ----O2
This gives the P/O ratio of 3 which is the number of molecules of ATP synthesized per
pair of electrons carried through the chain.
Thus, 3 ATP coupling sites.
Evidence supporting site of ATP synthesis
1. Excess β-hydroxy butyrate was added with ADP and Pi and the P/O ratio was 3.

2. Excess succinate and ADP together with rotenone to inhibit electron transfer from
NADH. The P/O ration was 2. This means there is a coupling site before CoQ.

3. Excess tetramethyl-p-phenylamine diamine (TMPD)/ascorbate was added with

antimycin A to inhibit electron flow from FADH2 . Ascorbate is an artificial electron
donor. TMPD is an electron acceptor and can feed electrons to the respiratory
chain at cyt C. The electron carriers reduced cyt C non-enzymatically, and its
oxidation via cyt oxidase proceeded with a P/O ratio of 1. This experiment localized
a coupling site beyond cyt C.
4. Purified cyt c, when added to mitochondria can withdraw electrons from the
electron chain. Further oxidation of the cytochrome oxidase by an inhibitor
(such as cyanide) forces electrons to exit from the chain before cyt c. Under
these conditions succinate is oxidized with a P/O ratio of 1 which localized a site
between cyt b and cyt c.

These experiments demonstrate that complexes I,II and IV are each capable
of driving ATP synthesis.
 ATP synthesis enzyme system
Electron microscopy of standard mitochondrion reveals that the cristae are
covered with knot-like projections, on the matrix side, attached to the inner
membrane by a short stalk.
The knots are known as F1 spheres.
Sonic oscillation fragments the inner membrane which reveals closed vesicles
or sub-mitochondrion particles containing appropriate complexes.
These sub mitochondrial particles respire and synthesize ATP just like intact
mitochondria.
Treatment of these vesicles with trypsin or urea causes the knots to dissociate
from the vesicles.
 ATP synthesis enzyme system
Centrifugation was used to separate the knots from the stripped vesicles. It
was found that the vesicles could still oxidize substrate and reduce oxygen; but
no ATP was synthesized.
This was restored when the knots were added to the vesicles. The knots were
originally called coupling factor.
Purification of the knots and the attached stalks revealed that about 5 or 6
protons from F1 knot, 2 proteins from the stalk and then an attached complex
designated F0 which is embedded in the inner membrane.
The stalked knots have been found to have ATPase activity in vitro (i.e. ATP
hydrolysis) as does F1 alone but not synthesis of ATP.
ATP synthesis ability is restored when F1 is added to F0.
Also one of the stalk proteins is the site of binding of the ATP synthesis
inhibitor oligomycin.
From the reconstruction experiment, it is certain that the true function of the
F0F1 complexes could be referred to as ATP synthase or F0F1 coupling factor.
 Respiratory control
The reaction equation for the oxidation of NADH by mitochondria is
4NADH + 4H+ + 3ADP+ 3Pi + 2O2 ------ NAD+ + 3ATP + 4H2O
So , three important substrates are needed for electron transport to proceed,
namely oxygen, Pi and ADP.
As electron transport proceeds, Pi and ADP will be removed from the cytosol
and ATP will accumulate.
This process continues until nearly all ADP is transformed into ATP.
Concentrations of inorganic phosphates will also reduce but it is also in much
higher concentrations in the cell than in the ADP.
 Respiratory control
Therefore the rate of oxygen consumption by mitochondria or electron transport is
dependent on or limited by ADP concentration.
When the ADP concentration is nearly depleted, the rate of oxygen consumption by
mitochondria must slow down to a small fraction of the maximum rate.
This is referred to as 'idling or resting' respiration.
Respiration will be restored to its maximum rate only when ADP concentrations
increase .
Thus, when some energy requiring process breaks down ATP to ADP.
This will make ADP available to be phosphorylated.
The dependence of the rate of oxygen on ADP concentration is called acceptor
control ratio.
In most of the animal and human tissues, the acceptor control of respiration
i.e. the ratio of maximal rate to the resting state is at least 10.
In some humans, acceptor control is faulty, probably due to genetic defect and
therefore their tissues show a high rate of oxygen consumption at all times.
Mechanisms of oxidative phosphorylation
There are three mechanisms that seek to explain how energy released from
respiration of electron transport is channeled into synthesis of ATP.
Two models namely chemical coupling and conformational coupling hypothesis
have been discounted.
In chemical coupling, it is thought that the energy released during electron
transport will be used directly for the synthesis of an energy rich intermediate
which then releases its energy for ATP synthesis.
However, such an intermediate has not been isolated (oxidation of gly-3-P).
Mechanisms of oxidative phosphorylation
In conformational coupling, energy from biological oxidation leads to one or more
respiratory proteins to assume a thermodynamically unstable conformation, the
energy is used to drive the synthesis of ATP from ADP and Pi.
 Such an energy rich protein intermediate has not been identified or isolated.
The most widely accepted model is the chemiosmotic coupling proposed by Mitchell
in 1961.
The model explains that the energy released from electron transport runs an active
transport system which cause the discharge of protons from the mitochondrial matrix
into the inner membrane space.
This produces an electrochemical gradient for protons with a lower pH outside
i.e. in the inter membrane space than in the matrix.
There is the tendency for the protons to move back along the concentration
gradient to establish a 'proton balance' on both sides of the membrane.
Free energy is used to maintain the proton gradient (or imbalance).
Once substantial gradient has been established, the gradient is dissipated.
In other words, when the proton flow back into the matrix, that free energy is
released and some of it is used to drive the synthesis of ATP from ADP and
orthophosphate.
It has been proposed that the dehydrogenase activity of the ETC ( i.e. NADH,CoQ &
aa3) that target electrons as well as protons are arranged asymmetrically in the inner
membrane such that protons are always carried from the matrix to the outside.
The proton pumping converts the energy of respiration into a chemical
concentration gradient or osmotic energy that creates an electric potential.
 This does not involve an intermediate.
The F0 portion of the F0F1 coupling which is an integral protein is thought to contain
specific channels for the return of protons into the mitochondrial matrix.
The free energy released as H+ traverses this channel into the matrix is somehow
used to drive the synthesis of ATP catalyzed by the F1 component of the complex.
 Experimental Evidence
1. It has been possible to measure changes in pH and electrical potential across
mitochondrial membranes and it is clear that mitochondria can pump
protons from the matrix into the inter membrane space. The pH
measurements outside an actively respiring mitochondrion is about 1.4 units
lower than the matrix. The net movement of positively charged protons
outward across the membrane also generates an electric potential of 0.14 V.
Experimental Evidence
2. The fundamental aspects of the chemiosmotic theory is that protons are
transported from the matrix across the inner membrane. For the respiratory proteins
to serve as proton pumps, electron carriers that carry protons such as NADH
dehydrogenase should be in contact with both sides of the membrane. Also these
carriers should be asymmetrically located in the membrane so that transport could be
in one direction-outward.
Asymmetry of these proteins has been demonstrated by the use of agents that react
with respiratory proteins but cannot themselves traverse the membrane. Treatment
of intact mitochondria with such reagents like antibodies, proteolytic enzymes or
labeling reagents allows the detection of proteins located at the outer surface of the
inner membrane.
3. The mechanism of action of uncouplers supports the chemiosmotic theory of
oxidative phosphorylation.
Uncoupling agents like 2.4-dinitrophenol allow electron transport in
mitochondria but prevents phosphorylation of ADP to ADP.
At extracellular pH (nearly 7.0) the phenolic OH group is not protonated .
However, as DNP molecule approaches the inner membrane, it becomes protonated
because of the lower pH environment.
The protonated form is lipid soluble and can pass through the membrane.
Once inside the matrix, the higher pH causes the phenol OH group to deprotonate.
Therefore the uncoupler transports H+ back into the mitochondrial matrix, by passing
through the F0 proton channel and thereby preventing ATP synthesis.
Phosphorylation inhibitor oligomycin binds to the specific protein of the complex and
blocks the flow of protons through the F0 proton channel and hence inhibits oxidative
phosphorylation.
Ionophores (ion carriers) are lipid soluble substances capable of binding and
carrying specific ions through the membrane.
Unlike some uncouplers, they carry cations other than H+ ions.
The toxic antibiotic, valinomycin, which is a lipid soluble compound forms a
complex with K+ ions which readily passes through the inner mitochondrial
membrane.
The ionophore, gramicidin, facilitates diffusion of K+, Na+ and several other
monovalent cations.
Increasing the permeability of the mitochondrial membrane to those ions
reduces the membrane potential and inhibits ATP synthesis.
Energy yield from complete glucose oxidation
The chemical energy obtained in the form of ATP as glucose is oxidized to CO2 and
H2O in animal cells may be broken down as:
1. Glycolysis of one molecule of glucose under aerobic conditions yields 2 molecules of
pyruvate, 2 NADH and 2 ATPs, all in the cytosol.
 Glucose + 2Pi + 2ADP + 2NAD+ ----- 2Pyruvate + 2ATP + 2NADH + 2H+ + 2H2O
 The two cytosolic NADH from the glyceraldehyde phosphate dehydrogenase are
carried out to the mitochondria by the malate-aspartate shuttle and then enters the
ETC. 6 ATPs will be formed.
2. For the dehydrogenation of pyruvate to acetyl CoA, 2 NADH will be formed which will
provide 6 ATPs.
2 pyruvate + 2CoA + 6Pi + 6ADP + O2 ---- 2 acetyl CoA + 2 CO2 + 6ATP + 8H2O
3. Two molecules of acetyl CoA to CO2 and H2O via the CAC together with the
electron transport coupled with oxidative phosphorylation.
This is through the isocitrate, β-ketoglutarate and malate.
Each yields 3 ATPs; oxidation of succinate yields 2 ATPs, and the formation of 2
ATPs via GTP generated from succinyl CoA
2 acetyl CoA + 24Pi + 24ADP + 4O2 --- 2CoASH + 4CO2 + 24ATP + 26H2O
• The overall reaction of Glycolysis and respiration is
Glucose + 38Pi + 38ADP ----- 6CO2 + 38ATP + 44H2O
• If dG0 for glucose oxidation is 2870 kJ/mol and that for ATP hydrolysis is -
30.5kJ/mol, the efficiency of Glycolysis through oxidative phosphorylation is
(38X30.5) X 100 / 2870 = 40%
• It is higher in vivo because the concentration of ADP, ATP and Pi are unequal
and much less than 1.0M assumed in standard conditions.
• ∆G0 = -2.303RT log Keq.