Complete E material on Fundamentals of Biochemistry [2+1]; (32 Lectures)

Fundamentals of Biochemistry Pradipta Banerjee, Ph. D.
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FUNDAMENTALS OF BIOCHEMISTRY (ASBC211)
Pradipta Banerjee, Ph. D.
Assistant Professor,
Dept. of Biochemistry & Plant Physiology,
CUTM, Parlakhemundi, Odisha
LESSION 1: IMPORTANCE OF BIOCHEMISTRY
The term biochemistry derived from the Greek word 'Bios' meaning ‘life’ may be defined as
the science that deals with the chemical basis of life. Biochemistry, as the name implies, is the
chemistry of living organisms. Living organisms, whether they are microorganisms, plants or
animals are basically made up of the same chemical components. Biochemistry is the study of
the way in which these components are synthesized and utilized by the organisms in their life
processes. It bridges the gap between the conventional chemistry and biology.
In other words, life is nothing but thousands of ordered chemical reactions or chemistry is the
logic of all biological phenomena.
Landmarks in Biochemistry
During 17th and 18th centuries, important foundations were laid in many fields of biology. The
19th century observed the development of concepts - the cell theory by Schleiden and
Schwann, Mendel’s study of inheritance and Darwin’s theory of evolution. The real push
to biochemistry was given in 1828 when total synthesis of urea from lead cyanate and ammonia
was achieved by Wohler who thus initiated the synthesis of organic compound from inorganic
compound. Louis Pasteur, during 1857, did a great deal of work on fermentations and pointed
out the central importance of enzymes in this process. The breakthrough in enzyme research
and hence, biochemistry was made in
1897 by Edward Buchner when he extracted enzyme from yeast cells in crude form which
could ferment a sugar molecule into alcohol. Neuberg introduced the term biochemistry in
1903. The early part of 20th century witnessed a sudden outburst of knowledge in chemical
analysis, separation methods, electronic instrumentation for biological studies (Xray
diffraction, electron microscope, etc) which ultimately resulted in understanding the structure
and function of several key molecules involved in life processes such as proteins, enzymes,
DNA and RNA. In 1926, James Sumner established the protein nature of enzyme. He was
responsible for the isolation and crystallization of urease, which provided a breakthrough in
studying of the properties of specific enzymes. The first metabolic pathway elucidated was the
glycolytic pathway during the first half of the 20th century by Embden and Meyerhof. Otto

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Warburg, Cori and Parnas also made very important contributions relating to glycolytic
pathway. Krebs established the citric acid and urea cycles during 1930-40. In 1940, Lipmann
described the central role of ATP in biological systems. The biochemistry of nucleic acids
entered into a phase of exponential growth after the establishment of the structure of DNA in
1953 by Watson and Crick followed by the discovery of DNA polymerase by Kornberg in
1956.
From 1960 onwards, biochemistry plunged into an interdisciplinary phase sharing much in
common with biology and molecular genetics. Frederick Sanger’s contributions in the
sequencing of protein in 1953 and nucleic acid in 1977 were responsible for further
developments in the field of protein and nucleic acid research. The growth of biochemistry and
molecular biology was phenomenal during the past two decades. The development of
recombinant DNA research by Snell and coworkers during 1980 allowed for further growth
and emergence of a new field, the genetic engineering.
Biochemistry includes various aspects of organic chemistry in organic chemistry, physical
chemistry, physics, biology and other basic disciplines. It is also interrelated with physiology,
microbiology, medicine and agriculture. Thus there was progressive evolution of biology to
biochemistry and then to molecular biology, genetic engineering and biotechnology.
Scope of Biochemistry
During the early part of the twentieth century, the central theme of biochemistry was the
development of the field of intermediary metabolism that is the elucidation of the pathways for
the synthesis and degradation of the constituents of living organisms. Although studies
concerned with intermediary metabolism continue to be important, at the present, biochemical
research may be classified into the following major areas:
1. Composition and characteristics of chemical compounds of living organisms.
2. Cell ultrastructure.
3. Cellular control mechanisms.
4. Physical chemistry of bio-macromolecules.
5. Structure-function, kinetics, regulation and mode of action of enzymes.
6. Intermediary metabolism.
7. Bioenergetics particularly the mechanisms of formation of adenosine triphosphate
(ATP) in the process of oxidative phosphorylation.
8. The molecular basis for genetic and developmental phenomena.
9. The molecular basis for physiological phenomena including nerve conduction, muscle
contraction, vision and transport across membrane

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10. Role, transformation and requirement of nutrients in plants, animals and other organisms
and
11. Chemistry of inheritance: structure-function and regulation of gene expression.
LESSION 2: PROPERTIES OF WATER, pH AND BUFFER
2.1 WATER
Water is a polar molecule. The H—O bond is polarized— the H end is more positive than the
O end. This polarity is reinforced by the other H—O bond. Because of the polarity difference,
water is both a hydrogen-bond donor and a hydrogen-bond acceptor. The two hydrogens can
each enter into hydrogen bonds with an appropriate acceptor, and the two lone pairs of electrons
on oxygen can act as hydrogen-bond acceptors. Because of the multiple hydrogen-bond donor
and acceptor sites, water interacts with itself. Water does two important things: It squeezes out
oily stuff because the oily stuff interferes with the interaction of water with itself, and it
interacts favourably with anything that can enter into its hydrogen-bonding network.
The driving force for a chemical reaction is what makes it happen. It’s the interaction that
contributes the most to the decrease in free energy. For protein (and DNA) folding, it’s the
hydrophobic interaction that provides most of the driving force. As water squeezes out the
hydrophobic side chains, distant parts of the protein are brought together into a compact
structure. The hydrophobic core of most globular proteins is very compact, and the pieces of
the hydrophobic core must fit together rather precisely.
Putting a hydrophobic group into water is difficult to do (unfavorable). Normally, water forms
an extensive hydrogen-bonding network with itself. The water molecules are constantly on the
move, breaking and making new hydrogen bonds with neighboring water molecules. Water has
two hydrogen bond donors (the two H—O bonds) and two hydrogen bond acceptors (the two
lone electron pairs on oxygen), so a given water molecule can make hydrogen bonds with
neighboring water molecules in a large number of different ways and in a large number of
different directions. When a hydrophobic molecule is dissolved in water, the water molecules
next to the hydrophobic molecule can interact with other water molecules only in a direction
away from the hydrophobic molecule. The water molecules in contact with the hydrophobic
group become more organized. In this case, organization means restricting the number of ways
that the water molecules can be arranged in space. The increased organization (restricted
freedom) of water that occurs around a hydrophobic molecule represents an unfavorable

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decrease in the entropy of water. In the absence of other factors, this increased organization
(decreased entropy) of water causes hydrophobic molecules to be insoluble.
Hydrogen Bond: A hydrogen bond is an interaction between two groups in which a weakly
acidic proton is shared (not totally donated) between a group that has a proton (the donor) and
a group that can accept a proton (the acceptor). Water can be both a hydrogen-bond donor and
a hydrogen-bond acceptor. In an unfolded protein, the hydrogen-bond donors and acceptors
make hydrogen bonds with water acceptor. In an unfolded protein, the hydrogen-bond donors
and acceptors make hydrogen bonds with water.
The lonization of Water is expressed by an Equilibrium Constant
Similarly, in case of water, we can write,
[H2O] Keq = [H+
] [OH-
]
Kw = [H+
] [OH-
]
Kw is ionic product of water at 250
C.
2.2 pH Scale Designates H+ and OH- ion concentrations
When there are exactly equal concentrations of H+
and OH-
, as in pure water, the solution is
said to be at neutral pH.
Where, p denotes “ negative logarithm of”.
A cola drink (pH 3.0) or red wine fuH 3.7) has an H+ concentration approximately10, 000
times that of blood( pH 7.4).
The pH of an aqueous solution can be approximately measured with various indicator dyes,
including litmus, phenolphthalein, and phenol red, which undergo color changes as a proton
dissociates from the dye molecule. Accurate determinations of pH in the chemical or clinical
laboratory are made with a glass electrode that is selectively sensitive to H+
(pH Meter).
Measurement of pH is one of the most important and frequently used procedures in
biochemistry. The pH affects the structure and activity of biological macromolecules; for

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example, the catalytic activity of enzymes is strongly dependent on pH. Measurements of the
pH of blood and urine are commonly used in medical diagnoses.
2.3 BUFFER
Note:
pH + pOH = 14

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Buffers are solutions that contain both the acidic and the basic forms of a weak acid. Buffers
minimize changes in pH when strong acids and bases are added.
Example:
Acetic Acid-Acetate Pair Buffer System

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This equation flts the titration curve of all weak acids and enables us to deduce some important
quantitative relationships. For example, it shows why the pKa of aweak acid is equal to the pH
of the solution at the midpoint of its titration. At this point, [A-
] = [HA]
Lower pKa implies stronger acid, weaker base.
Numerical Problem

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The Bicarbonate Buffer
The CO2–bicarbonate buffer is a little different from buffers using the usual kind of acids and
bases, but it is extremely important in maintaining the acid–base balance of the blood. The acid
form of the bicarbonate buffer is actually a gas dissolved in water. Dissolved CO2 is turned
into an acid by hydration to give H2CO3. Hydrated CO2 is then much like a carboxylic acid. It
gives up a proton to a base and makes bicarbonate, HCO-3
.
When CO2 is dissolved in water, there is never very much H2CO3, so we can ignore it and
count CO2 as the acid and HCO-3
as the base.
There are two ways of dealing with the bicarbonate buffer system.
The first uses the Henderson-Hasselbalch equation and an effective pKa of 6.1. If there is more
base (HCO-3
) than acid (CO2), the pH will always be bigger than the pKa. This is usually the
case physiologically (pH 7.4; pKa 6.1) so that on a molar basis there is always more than 10-
fold more HCO-3
than CO2.
You might be wondering why the bicarbonate buffer can buffer effectively at pH 7.4 when its
pKa is 6.1. The answer is that it doesn’t buffer all that well. What makes it unique and the
major buffer system of the blood is that CO2, being a gas, can be exhaled by the lungs. Exhaling
CO2 is equivalent to exhaling protons. It’s not that a proton is exhaled; it’s just left behind and
turned into water. This gives the body control over the concentration of the CO2 by controlling
the breathing rate.

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LESSION 3: CARBOHYDRATES
Carbohydrates are polyhydroxy aldehydes or ketones of approximate composition (C.H2O)n
that are important components of biological systems.
The basic units of carbohydrates are known as monosaccharides. Many of these compounds
are synthesized from simpler substances in a process named gluconeogenesis. Others (and
ultimately nearly all biological molecules) are the products of photosynthesis, the light-
powered combination of CO2 and H2O through which plants and certain bacteria form “carbon
hydrates.” The metabolic breakdown of monosaccharides provides much of the energy used to
power biological processes. Monosaccharides are also principal components of nucleic acids,
as well as important elements of complex lipids.
Oligosaccharides consist of a few covalently linked monosaccharide units. They are often
associated with proteins (glycoproteins) and lipids (glycolipids) in which they have both
structural and regulatory functions (glycoproteins and glycolipids are collectively called
glycoconjugates).
Polysaccharides consist of many covalently linked monosaccharide units and have molecular
masses ranging well into the millions of daltons. They have indispensable structural functions
in all types of organisms but are most conspicuous in plants because cellulose, their principal
structural material, comprises up to 80% of their dry weight. Polysaccharides such as starch
in plants and glycogen in animals serve as important nutritional reservoirs
3.1 Classification
Carbohydrates are mainly classified into three broad groups depending upon the number of
sugar units:
 Monosaccharides
 Oligosaccharides
 Polysaccharides

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Occurrence and Importance
The carbohydrates comprise one of the major groups of naturally occurring biomolecules. This
is mainly because; the light energy from the sun is converted into chemical energy by plants
through primary production and is transferred to sugars and carbohydrate derivatives.
The dry substance of plants is composed of 50-80% of carbohydrates. The structural
material in plants is mainly cellulose and related hemicelluloses.
Starch is the important form of storage polysaccharide in plants.
Pectins and sugars such as sucrose and glucose are also plant constituents.
Many non-carbohydrate organic molecules are found conjugated with sugars in the
form of glycosides.
The carbohydrates in animals are mostly found in combination with proteins as
glycoproteins, as well as other compounds.

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The storage form of carbohydrates, glycogen, found in liver and muscles, the blood
group substances, mucins, ground substance between cells in the form of
mucopolysaccharides are few examples of carbohydrates playing important roles in
animals.
Chitin found in the exo-skeleton of lower animals, is a polymer of N-acetyl
glucosamine.
3.2 Structures of Monosaccharides
Structures of aldose and ketose sugars are described below.
Structure of Aldose Sugars

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3.3 Reducing and oxidizing properties of Monosaccharides
3.3.1 Molisch Test
Principle: Carbohydrates when treated with concentrated sulphuric acid undergo dehydration
to give furfural derivatives. These compounds condense with α-naphthol to form colored
products. Pentoses yield furfural while Hexoses yield 5-Hydroxy methyl furfurals.
Observation: An appearance of reddish violet or purple colored ring at the junction of two
liquids is observed in a positive Molisch test.
Interpretation: This is a sensitive but a nonspecific test and is given positive by all types of
carbohydrates. If the oligosaccharides or polysaccharides are present they are first hydrolysed
to mono saccharides which are then dehydrated to give the test positive.
3.3.2 Benedict’s Test
Principle: Carbohydrates with free aldehyde or ketone groups have the ability to reduce
solutions of various metallic ions. Reducing sugars under alkaline conditions tautomerise and
Structure of Ketose Sugars

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form enediols. Enediols are powerful reducing agents. They reduce cupric ions to cuprous form
and are themselves converted to sugar acids. The cuprous ions combine with OH- ions to form
yellow cuprous hydroxide which upon heating is converted to red cuprous oxide.
3.3.3 Seliwanoff’s Test
Principle: Keto hexoses on treatment with hydrochloric acid form 5-hydroxy methyl furfural
which on condensation with resorcinol gives a cherry red colored complex.
Interpretation
 This test is given positive by ketohexoses so it is answered by fructose, sucrose and
other fructose containing carbohydrates.
 This test distinguishes between glucose and fructose.
 Overheating of the solution should be avoided. Upon continuous boiling, aldoses get
converted to ketoses and give a positive reaction with Seliwanoff reagent.
3.4 Mutarotation
 Two anomers of D-glucose, have different physical and chemical properties.
 The values of the specific optical rotation, [α]D
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, for α-D-glucose and β-D-glucose
are112.2° and 18.7°, respectively.
 When either of these pure substances is dissolved in water, specific optical rotation of
the solution slowly changes until it reaches an equilibrium value of [α]D
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52.7°.
 This phenomenon is known as mutarotation; in glucose, it results from the formation
of an equilibrium mixture consisting of 63.6% of the β anomer and 36.4% of the α
anomer.
 The interconversion between these anomers occurs via linear form of glucose.
 Yet, since the linear forms of these monosaccharides are normally present in only
minute amounts, these carbohydrates are accurately described as cyclic polyhydroxy
hemiacetals or hemiketals.

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3.5 Structure of Disaccharides and Polysaccharides
3.5.1 Disaccharides
Disaccharides are two monosaccharides linked by an O-glycosidic bond. Important examples
are Trehalose, Maltose, Sucrose, Lactose.

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3.5.2 Polysaccharides
3.5.2.1 Cellulose
 Linear homopolysaccharide composed exclusively of D glucose units held together in
(ß14) linkages.
 A single chain of cellulose can contain 10-to-15,000 residues.
 Due to presence of ß linkages, cellulose chains fold quite differently than chains of D-
glucose in starches and glycogen.
 Cellulose molecules are insoluble in water and form tough fibers.
 Cellulose is found in the cell walls of plants, particularly in stalks, stems, trunks, and
all the woody portions of the plant body.
 Cellulose constitutes much of the mass of wood, and cotton is almost pure cellulose.
 Vertebrate animals lack the hydrolytic enzymes (cellulases) that can cleave the (ß14)
linkages between glucose units in cellulose.

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 These enzymes are produced by many cellulolytic microorganisms. These
microorganisms, such as Trichonympha, a symbiotic protist that resides in the termite
gut, allow the host to derive energy from the glucose units stored in cellulose.
 Cellulases produced by microorganisms living in the rumens of cattle, sheep, and goats
allow these animals to obtain energy from cellulose present in soft grasses in the diet.
3.5.2.2 Starch
 Starch is a storage homopolysaccharides of D-glucose residues that is found in the
cytoplasm of plant cells.
 Starch (and glycogen) is extensively hydrated because it has many exposed hydroxyl
groups available to hydrogen-bond with water.
 Starches consist of two types of polymers called amylose and amylopectin
 Amylose is a linear polymer of D glucose residues that all are connected via (-14)
linkages.
 The molecular weights of amylose chains vary from a few thousand to more than a
million.
 Amylopectin is a branched polymer of D-glucose residues that can weigh up to 200
million Da.
 The glycosidic linkages between D-glucose residues in amylopectin chains are also (-
14); the branch point linkages between D-glucose units, however, are (-16)
linkages.
 Branch points occur about every 24 to 30 residues.
 Strands of amylopectin (black) form double-helical structures with each other or with
amylose strands (blue).
 Amylopectin has (-16) branch points (red).
 Glucose resides at the non-reducing ends of the outer branches are removed
enzymatically during the mobilization of starch for energy production.
 Glycogen has a structure that is similar to amylopectin, but is more highly branched
and more compact.
3.5.2.3 Chitin
 Chitin is a linear Homopolysaccharides composed of N-acetylglucosamine residues in
(ß14) linkage.

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 The only chemical difference from cellulose is the replacement of the hydroxyl group
at C-2 with an acetylated amino group.
 Chitin also forms extended fibers similar to those of cellulose. Like cellulose, chitin
cannot be digested by enzymes found in vertebrates.
 Chitin is the principal component of the hard exoskeletons of nearly a million species
of arthropods--insects, lobsters, and crabs, for example--and is probably the second
most abundant polysaccharide in nature.

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LESSION 4: LIPIDS
Lipids (Greek: lipos, fat) are substances of biological origin that are soluble in organic
solvents such as chloroform and methanol but are only sparingly soluble, if at all, in water.
Hence, they are easily separated from other biological materials by extraction into organic
solvents and may be further fractionated by such techniques as adsorption chromatography,
thin layer chromatography, and reverse-phase chromatography. Fats, oils, certain vitamins and
hormones, and most non-protein membrane components are lipids. In this section, we discuss
the structures and physical properties of the major classes of lipids.
Saturated and Unsaturated Lipids
Saturated fatty acids are highly flexible molecules that can assume a wide range of
conformations because there is relatively free rotation about each of their C¬C bonds.
Nevertheless, their fully extended conformation is that of minimum energy because this
conformation has the least amount of steric interference between neighboring methylene
groups.

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The melting points (mp) of saturated fatty acids, like those of most substances, increase with
molecular mass. Fatty acid double bonds almost always have the cis configuration. This puts
a rigid 30° bend in the hydrocarbon chain of unsaturated fatty acids that interferes with their
efficient packing to fill space. The consequent reduced van der Waals interactions cause fatty
acid melting points to decrease with their degree of unsaturation. Lipid fluidity likewise
increases with the degree of unsaturation of their component fatty acid residues. This
phenomenon has important consequences for membrane properties.
4.1 Importance and classification
4.1.1 Importance of Lipids
The word lipids is derived from the Greek word 'lipos' meaning fat.
Lipids are chemically heterogenous group of compounds that are insoluble in water
but soluble in non-polar solvents such as chloroform.
Lipids occur in plants and animals as storage and structural components
Structural lipids present in animals and plants are in the form of meat and vegetables
respectively.
Storage fats occur in milk and adipose tissue of farm animals and in seed oils.
Fats supply over twice as much energy per unit weight as proteins or carbohydrates.
Lipids are anhydrous due to non-polar nature and represent more energy than
carbohydrates which are heavily hydrated due to polar nature.
The presence of lipids in diet contributes considerably to palatability.

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Lipids contribute palatability in two ways. They induce olfactory responses, namely,
taste in the mouth and aroma through nose.
Secondly, they contribute to the texture of food and is responsible for the mouthfeel.
Lipids also supply the essential fatty acids which are not synthesised in human beings
but are essential for growth.
Lipids are essential for the effective absorption of fat soluble vitamins A, D, E and
K from intestine.
Many enzymes require lipid molecules for maximal activity. Examples are
microsomal enzyme, glucose 6-phosphatase and mitochondrial enzyme,
hydroxybutyrate dehydrogenase.
Adrenal corticosteroids, sex hormones and vitamin D3 (Cholecalciferol) are
synthesized from lipid derivative- cholesterol.
Much of the lipid of mammals is located subcutaneously and acts as insulation against
excessive heat loss to the environment.
The subcutaneous lipid deposits also insulate the important organs against mechanical
trauma.
List of Common Biological Fatty Acids:
4.1.1 Classification of Lipids
A) Simple lipids
i. Fat & oil (triacylglycerols)

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ii. Waxes – esters of fatty acids with alcohol (except glycerol). Alcohols may be aliphatic
(open chain) or alicyclic (both aliphatic and cyclic structure). Eg. Cetyl alcohol
B) Complex/compound lipids = fatty acid+ alcohol+ phosphate/carbohydrate/nitrogenous
base/protein/etc.
i. Phospholipids = fatty acid + alcohol + phosphoric acid + nitrogenous base
 Glycerophospholipids = glycerol as alcohol (eg. lecithin, cephalin)
 Sphingophospholipids = sphingosine as alcohol (sphingomyelin)
ii. Glycolipids/glycosphingolipids = fatty acid + alcohol (sphingosine) + carbohydrate
+ nitrogenous base (Cerebrosides, Gangliosides)
iii. Lipoproteins = macromolecular complexes of lipid and protein
iv. Other complex lipids
 Sulpholipids
 Aminolipid
 Lipopolysaccharide
C) Derived lipids = derived from hydrolysis of simple and complex lipids (Fat soluble
vitamins, Steroid hormones)
D) Miscelleneous lipids = carotenoids, squalene, terpenes, pentacosanes (in bee wax)
Neutral Lipids = Cholesterol; Mono, di, tri-acylglycerols, cholesteryl esters
Based on Polarity, lipids are classified into:
A. Polar lipids - soluble in polar solvents (acetone, alcohol) eg. phospholipids,
glyceroglycolipids, fatty acids
B. Non-polar lipids – soluble in non-polar solvents (ether, benzene, hexane). Eg.
Glycerides, sterols, sterol esters, Carotenoids, waxes, vitamins

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4.2 Structures and properties of fatty acids
Triacylglycerols
The fats and oils that occur in plants and animals consist largely of mixtures of triacylglycerols
(also referred to as triglycerides or neutral fats). These nonpolar, water-insoluble substances
are fatty acid triesters of glycerol:

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Triacylglycerols function as energy reservoirs in animals and are therefore their most
abundant class of lipids even though they are not components of biological membranes.
Common Structures of Glycerophospholipids
Structure of Sphingophospholipids

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Structure of Parent Compound of Steroid
4.3 Storage lipids and membrane lipids
4.3.1 Storage fats or oils
Triacylglycerols are widely distributed in the plant kingdom. They are found both in
vegetative as well as reproductive tissues.
Triacylglycerols are normally stored in the endosperm of the seed although some
plants store appreciable quantities of fat in the fleshy fruit mesocarp, for example,
avocado.
Some plants like the oil palm, store oils in both the mesocarp (Palm oil) and the
endosperm (Palm kernel oil).
The oil present as droplets in the cytoplasm of the seed cells.
These droplets are called as oil bodies and are surrounded by a membrane composed
of phospholipids and protein.
Most of the common edible oils (groundnut, sunflower, gingelly, soybean, safflower,
rice bran) contain limited number of the common fatty acids such as palmitic, stearic,
oleic, linoleic and linolenic acids.
Palm kernel and coconut oils contain higher amount of medium chain saturated fatty
acids.
Seed oils contain small amount of phospholipids, carotenoids, tocopherols,
tocotrienols and plant sterols depending on the species of plant and degree of
processing.
4.3.2 Membrane Lipids
Biological membranes are composed of proteins associated with a lipid bilayer matrix. Their
lipid fractions consist of complex mixtures that vary according to the membrane source and, to
some extent, with the diet and environment of the organism that produced the membrane.
Membrane proteins carry out the dynamic processes associated with membranes, and therefore
specific proteins occur only in particular membranes. Protein-to-lipid ratios in membranes

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vary considerably with membrane function, although most membranes are at least one-half
protein. The myelin membrane, which functions passively as an insulator around certain nerve
fibers, is a prominent exception to this generalization in that it contains only 18% protein.
4.4 Properties of Fats
4.4.1 Physical Properties
Fats are greasy to touch and leave an oily impression on paper.
They are insoluble in water and soluble in organic solvents.
Pure triacylglycerols are tasteless, odourless, colourless and neutral in reaction.
They have lesser specific gravity (density) than water and therefore float in water.
Though fats are insoluble in water, they can be broken down into minute dropletsand
dispersed in water. This is called emulsification.
A satisfactory emulsion is one highly stable and contains very minute droplets with
diameter less than 0.5 µm.
Examples of naturally occurring emulsions are milk and yolk of egg. But they are
not mere fat droplets in water.
They contain hydrophilic colloidal particles such as proteins, carbohydrates and
phospholipids which act as stabilizing agents.
Emulsification greatly increases the surface area of the fat and this is an essential
requisite for digestion of fat in the intestine.
4.4.2 Chemical Properties
The most important chemical reaction of neutral fat is their hydrolysis to yield three
molecules Alkali hydrolysis (saponification)The process of alkali hydrolysis is called
'saponification'.

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Rancidity: Development of disagreeable odour and taste in fat or oil upon storage is
called rancidity.
Rancidity reactions may be due to hydrolysis of ester bonds (hydrolytic rancidity) or
due to oxidation of unsaturated fatty acids (oxidative rancidity).
Hydrolytic rancidity: This involves partial hydrolysis of the triacylglycerol to
mono and diacylglycerol.
The hydrolysis is hastened by the presence of moisture, warmth and lipases present
in fats or air.
In fats like butter which contains a high percentage of volatile fatty acids, hydrolytic
rancidity produces disagreeable odour and taste due to the liberation of the volatile
butyric acid. Butter becomes rancid more easily in summer.
Oxidative rancidity: The unsaturated fatty acids are oxidised at the double bonds to
form peroxides, which then decompose to form aldehydes and acids of objectionable
odour and taste.
Hydrogenation
 The degree of unsaturation of the fatty acids present in triacylglycerol determines
whether a fat is liquid or solid at room temperature.
 The presence of more unsaturated fatty acids lower the melting point.
 The presence of highly unsaturated fatty acids makes the oil more susceptible to
oxidative deterioration.
 The objective of hydrogenation is to reduce the degree of unsaturation and to
increase the melting point of the oil.
 The oil can be selectively hydrogenated by careful choice of catalyst and
temperature.
 Hydrogenation of unsaturated fats in the presence of a catalyst is known as
hardening.
 Normally the process of hydrogenation is partial so as to get desired characteristics and
to avoid products with high melting points.
 Hydrogenation is carried out in a closed container in the presence of finely powdered
catalyst (0.05 - 0.2% of nickel) at temperature as high as 180oC.
 The catalyst is usually removed by filtration.
 During hydrogenation process a proportion of the cis double bonds are isomerized to
trans double bonds and there is also migration of double bonds.

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 The hydrogenation process has made it possible to extend the food uses of a number of
vegetable oils and marine oils whose melting points are too low.
LESSION 5: PROTEINS
5.1 Amino Acids are Structural and Functional Units of Proteins
Twenty different amino acids are commonly found in proteins. The first to be discovered was
asparagine, in 1806. All the amino acids have trivial or common names, in some cases derived
from the source from which they were first isolated. Asparagine was first found in asparagus,
and glutamate in wheat gluten; tyrosine was first isolated from cheese (its name is derived from
the Greek tyros, “cheese”); and glycine (Greek glykos, “sweet”) was so named because of its
sweet taste. Some 300 additional amino acids have been found in cells. They have a variety of
functions but are not constituents of proteins. General structure of amino acid can be written
as:
Amino acids exists in zwitterionic form (hybrid form). A zwitterion can act as an acid or a
base. Substances having this dual nature are amphoteric and are often called ampholytes
(from “amphoteric electrolytes”).
Formation of Peptide Bond
The amino group of one amino acid (with R2 group) acts as a nucleophile to displace the
hydroxyl group of another amino acid (with R1 group), forming a peptide bond (shaded in
yellow). Amino groups are good nucleophiles, but the hydroxyl group is a poor leaving group

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and is not readily displaced. At physiological pH, the reaction shown does not occur to any
appreciable extent.
5.2 Classification of amino acids based on R group
Non-polar, aliphatic R group: Glycine, Alanine, Proline, Valine, Leucine, Isoleucine,
Methionine
Aromatic R group: Phenylalanine, Trytophan, Tyrosine
Polar, uncharged R group: Seine, Threonine, Cysteine, Glutamine, Asparagine
Positively charged R group: Histidine, Lysine, Arginine
Negatively charged R group: Aspartate, Glutamate
5.3 Chemical Properties of Amino acids
A) Reactions due to Amino Group
i. Reaction with Ninhydrin Reagent
Ninhydrin is a strong oxidizing agent. When a solution of amino acid is boiled with
ninhydrin, the amino acid is oxidatively deaminated to produce ammonia and a
ketoacid. The keto acid is decarboxylated to produce an aldehyde with one carbon atom
less than the parent amino acid. The net reaction is that ninhydrin oxidatively
deaminates and decarboxylates _amino acids to CO2, NH3 and an aldehyde. The
reduced ninhydrin then reacts with the liberated ammonia and another molecule of
intact ninhydrin to produce a purple coloured compound known as Ruhemann's
purple. This ninhydrin reaction is employed in the quantitative determination of amino
acids.
ii. Reaction with nitrous acid
Nitrous acid reacts with the amino group of amino acids to form the corresponding
hydroxyacids and liberate nitrogen gas.

29
B) Reactions due to Carboxyl Group
i. Decarboxylation: Amino acids undergo decarboxylation reaction to form
corresponding amines.
ii. Amino acids can form salts with bases and esters with alcohol.
5.4 Titration Curve of Amino Acids
Amino acids vary in their acid-base properties and have characteristic titration curves.
Monoamino monocarboxylic amino acids (with nonionizable R groups) are diprotic acids
(+
H3NCH(R)COOH) at low pH and exist in several different ionic forms as the pH is increased.
Amino acids with ionizable R groups have additional ionic species, depending on the pH of the
medium and the pKa of the R group.
Titration Curve of Glycine
At pH 5.97, the point of inflection between the two stages in its titration curve, glycine is
present predominantly as its dipolar form, fully ionized but with no net electric charge (Fig. 3–
10). The characteristic Ph at which the net electric charge is zero is called the isoelectric point
or isoelectric pH, designated pI. For glycine, which has no ionizable group in its side chain,
the isoelectric point is simply the arithmetic mean of the two pKa values:
Similarly, titration curve of histidine is shown

30
5.5 Classification of Proteins
1) Based on Chemical Nature and Solubility
I. Simple Proteins, consisting of only amino acids.
Globular Proteins: albumin, globulins, protamines, histones, globins, prolamines,
glutelins
Scleroproteins: collagen, elastin, keratin
II. Conjugated Proteins, containing amino acids and a non-protein moiety (often called
prosthetic group).
III. Derived Proteins, these are denatured product of simple and conjugated proteins.
Primary Derived Proteins: conjugated proteins, proteans, metaproteins

31
Secondary Derived Proteins: peptide, polypeptide, peptones
2) Based on Function
i. Structural Protein: keratin, collagen
ii. Catalytic Proteins: hexokinase, pepsin
iii. Transport Proteins: haemoglobin, serum albumin
iv. Storage Proteins: ovalbumin, glutelin
v. Hormonal Proteins: insulin, growth hormones
vi. Contractile Proteins: actin, myosin
vii. Toxic Proteins: ricin in castor bean is toxic to higher animals even in small amount,
snake venom, enzyme inhibitors, bacterial toxin, lectin in legumes agglutinates RBC.
viii. Exotic Proteins: anti-freeze glycoproteins present in Antarctic fishes
ix. Secretory Proteins: fibroin
5.6 Protein Conformation
Levels of Structure in Proteins
The primary structure consists of a sequence of amino acids linked together by peptide bonds
and includes any disulfide bonds. The resulting polypeptide can be coiled into units of
secondary structure, such as an α- helix. The helix is a part of the tertiary structure of the
folded polypeptide, which is itself one of the subunits that make up the quaternary structure
of the multisubunit protein, in this case hemoglobin.
I. Primary structure
Primary structure of protein refers to the number of amino acids and the order in
which they are covalently linked together.

32
It also refers to the location of disulfide bridges, if there are any, in a polypeptide chain.
The peptide bond is covalent in nature, quiet stable and referred as backbone of the
protein.
They can be disrupted by chemical or enzymatic hydrolysis but are not directly
influenced by salt concentration, change in pH or solvent.
Frederick Sanger in 1953 determined the complete amino acid sequence of insulin for
the first time.
II. Secondary structure
Secondary structure refers to the steric relationship of amino acids that are close to
one another in the linear sequence.
The folding of a linear polypeptide chain occurs to form a specific coiled structure.
Such coiling or folding is maintained by hydrogen bonds and hydrogen bond is the
only bond responsible for secondary structure.
X-ray studies of several polypeptides by Linus Pauling and Robert Corey revealed
that the peptide group has a rigid, planar structure which is a consequence of
resonance interactions that give the peptide bond a 40% double bond character.
Peptide groups mostly assume the trans-conformation in which successive C2 atoms
are on opposite sides of peptide bond joining them.
The cis configuration creates steric interference.
If a polypeptide chain is twisted by the same amount each of its C atoms, it assumes a
helical conformation.
a) α-Helix Structure
The α-helix is the most stable arrangement of polypeptides.
The helix structure of proteins is stabilized by intramolecular hydrogen bonding.
In this structure, hydrogen bonds are formed between the C=O group of one peptide
bond and the N-H group of another after 3 amino acid units.
The polypeptide chain constituted by L-amino acids form a right-handed helix, whereas
the polypeptide chains made up of D-amino acids form a left-handed helix.
In the α -helical conformation, all the side chains lie outside the helix whereas C, N, O
and H of the peptide bond lie in the same plane.
Certain amino acids tend to disrupt the α -helix. Among these are proline (the N atoms
is part of the rigid ring and no rotation of the N-C bond can occur) and amino acid with
charged or bulk R groups that either electrostatically or physically interferes with helix
formation.

33
b) β-pleated Sheet Structure
β-conformation organizes polypeptide chains to sheets.
This is a more extended conformation of polypeptide chains, and its structure has been
confirmed by x-ray analysis.
In conformation, the backbone of the polypeptide chain is extended into a zigzag rather
than helical structure.
The zigzag polypeptide chains can be arranged side by side to form a structure
resembling a series of pleats.
In this arrangement, hydrogen bonds are formed between adjacent segments of
polypeptide chain.
Adjacent polypeptide chains in a sheet can be either parallel or antiparallel (having the
same or opposite amino-to-carboxyl orientations, respectively).
The structures are somewhat similar, although the repeat period is shorter for the
parallel conformation (6.5 Å, versus 7 Å for antiparallel) and the hydrogen bonding
patterns are different.
Right Handed Helix Structure

34
Beta Pleated Sheet Structure: Parallel and Anti-parallel
III. Tertiary structure
Tertiary structure refers to the steric relationship of amino acid residues that are far
apart in the linear sequence.
This leads to the twisting of polypeptide chains into specific loops and bends which
are maintained chiefly by five kinds of bonds – H-bond, ionic bond, disulphide bond,
hydrophobic bond, dipole-dipole interaction.
IV. Quaternary structure
Proteins that have more than one subunit or polypeptide chains will exhibit
quaternary structure.
Quaternary structure refers to a functional protein aggregate (organization) formed
by interpolypeptide linkage of subunits or polypeptide chains.
These subunits are held together by noncovalent surface interaction between the polar
side chains.
Proteins formed like above are termed oligomers and the individual polypeptide chains
are variously termed protomers, monomers or subunits.

35
The most common oligomeric proteins contain two or four protomers and are termed
dimers or tetramers, respectively.
Myoglobin has no quaternary structure since, it is composed of a single polypeptide
chain.
Important Points to Remember about Tertiary and Quaternary Structure
 Tertiary structure is the complete three dimensional structure of a polypeptide chain.
There are two general classes of proteins based on tertiary structure: fibrous and
globular.
 Fibrous proteins serve mainly structural roles, have simple repeating elements of
secondary structure.
 Globular proteins have more complicated tertiary structures, often containing several
types of secondary structure in the same polypeptide chain. The first globular protein
structure to be determined, using x-ray diffraction methods, was that of myoglobin.
 Complex structures of globular proteins can be analyzed by examining stable
substructures called supersecondary structures, motifs, or folds. The thousands of
known protein structures are generally assembled from a repertoire of only a few
hundred motifs.
 Regions of a polypeptide chain that can fold stably and independently are called
domains.
 Quaternary structure results from interactions between the subunits of multisubunit
(multimeric) proteins or large protein assemblies.
 Some multimeric proteins have a repeated unit consisting of a single subunit or a group
of subunits referred to as a protomer. Protomers are usually related by rotational or
helical symmetry.
 For proteins that consist of a single polypeptide chain, monomeric proteins, tertiary
structure is the highest level of organization.
 Multimeric proteins contain two or more polypeptide chains, or subunits, held together
by noncovalent bonds. Quaternary structure describes the number (stoichiometry) and
relative positions of the subunits in a multimeric protein. Hemagglutinin is a trimer of
three identical subunits; other multimeric proteins can be composed of any number of
identical or different subunits.
 Many proteins contain one or more motifs built from particular combinations of
secondary structures. A motif is defined by a specific combination of secondary

36
structures that has a particular topology and is organized into a characteristic three-
dimensional structure.
 The tertiary structure of large proteins is often subdivided into distinct globular or
fibrous regions called domains. Structurally, a domain is a compactly folded region
of polypeptide. For large proteins, domains can be recognized in structures determined
by x-ray crystallography or in images captured by electron microscopy. These discrete
regions are well distinguished or physically separated from other parts of the protein
but connected by the polypeptide chain. Hemagglutinin, for example, contains a
globular domain and a fibrous domain
5.7 Protein Denaturation
The low conformational stabilities of native proteins make them easily susceptible to
denaturation by altering the balance of the weak nonbonding forces that maintain the native
conformation. When a protein in solution is heated, its conformationally sensitive properties,
such as optical rotation, viscosity, and UV absorption, change abruptly over a narrow
temperature range.
pH variations alter the ionization states of amino acid side chains, which changes
protein charge distributions and H bonding requirements.
Detergents, some of which significantly perturb protein structures at concentrations as
low as 10-6
M, hydrophobically associate with the nonpolar residues of a protein,
thereby interfering with the hydrophobic interactions responsible for the protein’s
native structure.
High concentrations of water-soluble organic substances, such as aliphatic alcohols,
interfere with the hydrophobic forces stabilizing protein structures through their own
hydrophobic interactions with water. Organic substances with several hydroxyl groups,
such as ethylene glycol or sucrose, however, are relatively poor denaturants because
their H bonding ability renders them less disruptive of water structure.

37
LESSION 6: ENZYMES
6.1 Nature of Enzymes
Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical
reactions in living organisms, and which can be extracted from cells and then used to
catalyse a wide range of commercially important processes. For example, they have
important roles in the production of sweetening agents and the modification of
antibiotics, they are used in washing powders and various cleaning products, and they
play a key role in analytical devices and assays that have clinical, forensic and
environmental applications.
The word ‘enzyme’ was first used by the German physiologist Wilhelm Kühne in
1878, when he was describing the ability of yeast to produce alcohol from sugars, and
it is derived from the Greek words en (meaning ‘within’) and zume (meaning ‘yeast’).
In the late nineteenth century and early twentieth century, significant advances were
made in the extraction, characterization and commercial exploitation of many enzymes,
but it was not until the 1920s that enzymes were crystallized, revealing that catalytic
activity is associated with protein molecules. For the next 60 years or so it was believed
that all enzymes were proteins, but in the 1980s it was found that some ribonucleic acid
(RNA) molecules are also able to exert catalytic effects. These RNAs, which are called
ribozymes, play an important role in gene expression.
In the same decade, biochemists also developed the technology to generate antibodies
that possess catalytic properties. These so-called ‘abzymes’ have significant potential
both as novel industrial catalysts and in therapeutics.
As catalysts, enzymes are only required in very low concentrations, and they speed up
reactions without themselves being consumed during the reaction. We usually describe
enzymes as being capable of catalysing the conversion of substrate molecules into
product molecules as follows:

38
6.2 Classification of Enzymes
International Union of Biochemistry set up the Enzyme Commission (EC) to classify enzymes.
The main classes of enzymes are as follows:
Secondary classes of oxidoreductase enzymes in EC system.
Tertiary classes of oxidoreductase enzymes in EC system
For example, lactate dehydrogenase with the EC number 1.1.1.27 is an oxidoreductase
(indicated by the first digit) with the alcohol group of the lactate molecule as the hydrogen
donor (second digit) and NAD+
as the hydrogen acceptor (third digit), and is the 27th enzyme
to be categorized within this group (fourth digit).

39
6.3 Mechanism of Enzymatic Action
Emil Fischer’s “Lock and Key Hypothesis”, 1894: Only a key of the correct size and
shape (the substrate) fits into the keyhole (the active site) of the lock (the enzyme).
Later by X-ray crystallography, it became clear that enzymes are not rigid structures,
but are quite flexible in shape.
In the light of this finding, in 1958 Daniel Koshland extended Fischer’s ideas and
presented the Induced-fit Model of substrate and enzyme binding, in which enzyme
molecule changes its shape slightly to accommodate the binding of substrate.
Substrate approaches active site of enzyme
Shape of active site changes to fit precisely around substrate – substrate induces active
site to change shape
The reaction is catalysed and product is formed.
Product diffuse away from active site, and active site reverts to original shape.
Enzyme reduce the activation energy required to start a reaction.
6.4 Enzyme Kinetics
In 1913, Leonor Michaelis and Maud Menten, derived the equation:
Where, Km is known as Michaelis constant. It should be noted that enzymes which catalyse the
same reaction, but which are derived from different organisms, can have widely differing Km
values. Furthermore, an enzyme with multiple substrates can have quite different Km values for
each substrate.
A low Km value indicates that the enzyme requires only a small amount of substrate in order
to become saturated. Therefore the maximum velocity is reached at relatively low substrate
concentrations. A high Km value indicates the need for high substrate concentrations in order
to achieve maximum reaction velocity. Thus we generally refer to Km as a measure of the
affinity of the enzyme for its substrate—in fact it is an inverse measure, where a high Km

40
indicates a low affinity, and vice versa. A low Km value indicates that the enzyme requires
only a small amount of substrate in order to become saturated. Therefore the maximum velocity
is reached at relatively low substrate concentrations. A high Km value indicates the need for
high substrate concentrations in order to achieve maximum reaction velocity. Thus we
generally refer to Km as a measure of the affinity of the enzyme for its substrate—in fact it is
an inverse measure, where a high Km indicates a low affinity, and vice versa.
Double Reciprocal Plot
Because the plot of V0 vs [S] for an enzyme-catalyzed reaction asymptotically approaches the
value of Vmax at high [S], it is difficult to accurately determine Vmax (and thereby, Km) from
such graphs. The problem is readily solved by converting the Michaelis-Menten kinetic
equation to the so-called double-reciprocal equation (Lineweaver-Burk equation) which
describes a linear plot from which Vmax and Km can be easily obtained. The Lineweaver-Burk
equation is derived by first taking the reciprocal of both sides of the Michaelis-Menten
equation.
1/V0 = (Km + [S])/Vmax[S]
Separating the components of the numerator on the right side of the equation gives
1/V0 = Km/Vmax[S] + [S]/Vmax[S]
Which simplifies to
1/V0 = Km/Vmax[S] + 1/Vmax.
The plot of 1/V0 vs 1/[S] gives a straight line, the y-intercept of which is 1/Vmax and the x-
intercept of which is -1/Km.

41
Double Reciprocal Plot
6.5 Allosteric Enzymes
An allosteric protein is one in which the binding of a ligand to one site affects the binding
properties of another site on the same protein. The term “allosteric” derives from the Greek
allos, “other,” and stereos, “solid” or “shape.” Allosteric proteins are those having “other
shapes,” or conformations, induced by the binding of ligands referred to as modulators. The
conformational changes induced by the modulator(s) interconvert more-active and less-active
forms of the protein. The modulators for allosteric proteins may be either inhibitors or
activators.
Allosteric enzymes function through reversible, noncovalent binding of regulatory compounds
called allosteric modulators or allosteric effectors, which are generally small metabolites or
cofactors. Other enzymes are regulated by reversible covalent modification.
LESSION 7: NUCLEIC ACID
7.1 DNA, RNA are the two Nucleic Acids
There are two classes of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA). DNA is the hereditary molecule in all cellular life-forms, as well as in many viruses.
It has but two functions:
1. To direct its own replication during cell division.
2. To direct the transcription of complementary molecules of RNA.
RNA, in contrast, has more varied biological functions:

42
1. The RNA transcripts of DNA sequences that specify polypeptides, messenger RNAs
(mRNAs), direct the ribosomal synthesis of these polypeptides in a process known as
translation.
2. The RNAs of ribosomes, which are about two-thirds RNA and one-third protein, have
functional as well as structural roles.
3. During protein synthesis, amino acids are delivered to the ribosome by molecules of transfer
RNA (tRNA).
4. Certain RNAs are associated with specific proteins to form ribonucleoproteins that
participate in the posttranscriptional processing of other RNAs.
5. A variety of short RNAs participate in the control of eukaryotic gene expression and in
protection against viruses, a phenomenon known as RNA interference (RNAi).
6. In many viruses, RNA, not DNA, is the carrier of hereditary information.
7.1 Chemical Structures of Ribonucleotide and Deoxy-ribonucleotide are given below:
7.1.1 Chemical Structures of Nitrogenous Bases: Purines & Pyrimidies
7.1.2 Structures of Nucleosides and Nucleotides
Nucleosides = Pentose + Nitrogenous Base
Nucleotides = Nucleosides + Phosphate

43
Names and Abbreviations of Nucleic Acid Bases, Nucleosides, and Nucleotides
7.2 DNA’s Base Composition Is Governed by Chargaff’s Rules
The base composition of DNA generally varies from one species to another.
DNA specimens isolated from different tissues of the same species have the same base
composition.
The base composition of DNA in a given species does not change with an organism’s
age, nutritional state, or changing environment.
In all cellular DNAs, regardless of the species, the number of adenosine residues is
equal to the number of thymidine residues (that is, A = T), and the number of guanosine
residues is equal to the number of cytidine residues (G = C). From these relationships
it follows that the sum of the purine residues equals the sum of the pyrimidine residues;
that is, A +G=T+C.

44
7.3 DNA Double Helical Structure
Double helical DNA has three major helical forms, B-DNA, A-DNA, and Z-DNA.
Watson and Crick, 1953 reported the structure of B-DNA.
Differences between the three forms of DNA:
A-DNA B-DNA Z-DNA
Helical sense Right Handed Right Handed Left Handed
Diameter 26 Å 20 Å 18 Å
Base pairs per
helical turn
11.6 10 12 (6 dimers)
Helical twist per
base pair
31° 36° 9° for pyrimidine–
purine steps; for
pyrimidine–purine
steps; 51° for
purine–pyrimidine
steps
Helix pitch (rise per
turn)
34 Å 34 Å 44 Å
Helix rise per base
pair
2.9 Å 3.4 Å 7.4 Å per dimer
Base tilt normal to
helix axis
20° 6° 7°
Major groove Narrow and deep Wide and deep Flat
Minor groove Wide and shallow Narrow and deep Narrow and deep
Sugar pucker C3’-endo C2’-endo C2’-endo for
pyrimidines; C3’-
endo for purines
Glycosidic bond Anti Anti Anti for
pyrimidines; syn for
purines

45
7.4 Separation of DNA Strands
Denaturation: At 900
C or above, ds DNA is denatured and strand separation takes
place. Absorbance measured at 260 nm.
Absorbance of DNA at 260 nm increases as DNA becomes denatured – Hyperchromic
shift.
Temperature at which 50% of DNA is melted is called Melting Temperature (Tm)
and depends on nature of DNA.
Tm is highest for those DNA molecule consisting highest proportion of C, G residues
and Tm actually be used to estimate percentage of C+G in DNA sample.
Renaturation: If melted DNA is cooled, it is possible for the separated strands to re-
associate, a process reverse to denaturation.
Annealing: Small ss fragments of DNA (oligonucleotides, upto 40 bases in length) can
hybridize to a denatured sample of DNA, pending on base sequence of oligontd. and
salt concentration of sample.
7.5 Types of RNA: mRNA, rRNA, tRNA, microRNA, snRNA, RNAi
Messenger RNA (mRNA):
It contains only 4 major bases
It is synthesized in nucleus during transcription
Some mRNA also formed in mitochondria
Occurs in many distinctive form which vary greatly molecular weight and base
sequence
Each of thousands of different protein synthesized by the cell is coded by a specific
mRNA or segment of mRNA molecule

46
In eukaryotes, mRNA contains long sequence of adenylate residues in 3’ end (polyA
tail) which plays a role in processing or transport of mRNA from nucleus to cytoplasm.
This polyA tail provides stability and prevents attacks by 3’ exonucleases
5’ end of mRNA is capped by 7-methylguanosine triphosphate, which prevent attacks
of 5’ exonucleases
5’cap also helps in recognition of mRNA for protein synthesis
Transfer RNA (tRNA):
i. The large dots on the backbone represent nucleotide residues; the blue lines represent
base pairs.
ii. Characteristic and/or invariant residues common to all tRNAs are shaded in pink.
iii. Transfer RNAs vary in length from 73 to 93 nucleotides.
iv. At the end of the anticodon arm is the anticodon loop, which always contains seven
unpaired nucleotides.
v. The D arm contains two or three D (5,6-dihydrouridine) residues, depending on the
tRNA.
vi. In some tRNAs, D arm has only three hydrogen-bonded base pairs.
Abbreviations used in tRNA structure: Pu, purine nucleotide; Py, pyrimidine nucleotide;
G*, guanylate or2-O-methylguanylate.

47
7.6 Wobble allows some tRNAs to Recognize More than One Codon
When several different codons specify one amino acid, the difference between them usually
lies at the third base position (at the 3’end). For example, alanine is coded by the triplets GCU,
GCC, GCA, and GCG. The codons for most amino acids can be symbolized by or
.The first two letters of each codon are the primary determinants of specificity, a feature that
has some interesting consequences.
Transfer RNAs base-pair with mRNA codons at a three-base sequence on the tRNA called the
anticodon. The first base of the codon in mRNA (read in the 5’ to 3’direction) pairs with the
third base of the anticodon. If the anticodon triplet of a tRNA recognized only one codon triplet
through Watson-Crick base pairing at all three positions, cells would have a different tRNA for
each amino acid codon. This is not then case, however, because the anticodons in some tRNAs
include the nucleotide inosinate (designated I), which contains the uncommon base
hypoxanthine.
Inosinate can form hydrogen bonds with three different nucleotides (U, C, and A), although
these pairings are much weaker than the hydrogen bonds of Watson-Crick base pairs (G-C and
A-U). In yeast, one tRNAArg
has the anticodon (5’) ICG, which recognizes three arginine
codons: (5’) CGA, (5’) CGU, and (5’) CGC. The first two bases are identical (CG) and form
strong Watson-Crick base pairs with the corresponding bases of the anticodon, but the third
base (A, U, or C) forms rather weak hydrogen bonds with the I residue at the first position of
the anticodon.

48
Examination of these and other codon-anticodon pairings led Crick to conclude that the third
base of most codons pairs rather loosely with the corresponding base of its anticodon; to use
his picturesque word, the third base of such codons (and the first base of their corresponding
anticodons) “wobbles.” Crick proposed a set of four relationships called the wobble
hypothesis.
LESSION 8: METABOLISM OF CARBOHYDRATES
8.1 Glycolysis
By 1940, the efforts of many investigators had come to fruition with the elucidation of the
complete pathway of glycolysis. The work of three of these individuals, Gustav Embden, Otto
Meyerhof, and Jacob Parnas, has been commemorated in that glycolysis is alternatively known
as the Embden–Meyerhof–Parnas pathway (E-M-P Pathway).
Glucose enters most cells by specific carriers that transport it from the exterior of the cell into
the cytosol. The enzymes of glycolysis are located in the cytosol, where they are only loosely
associated, if at all, with cell structures such as membranes. Glycolysis converts glucose to two
C3 units (pyruvate) of lower free energy in a process that harnesses the released free energy
to synthesize ATP from ADP and Pi. This process requires a pathway of chemically coupled
phosphoryl transfer reactions. Thus the chemical strategy of glycolysis is:
i. Add phosphoryl groups to the glucose.
ii. Chemically convert phosphorylated intermediates into compounds with high
phosphate group-transfer potentials.
iii. Chemically couple the subsequent hydrolysis of reactive substances to ATP synthesis.
The 10 enzyme-catalyzed reactions of glycolysis complete the pathway. Note that ATP is used
early in the pathway to synthesize phosphoryl compounds but is later resynthesized. Glycolysis
may therefore be considered to occur in two stages:
Stage I (Reactions 1–5): A preparatory stage in which the hexose glucose is phosphorylated
and cleaved to yield two molecules of the triose glyceraldehyde-3-phosphate. This process
utilizes two ATPs in a kind of energy investment.
Stage II (Reactions 6–10): The two molecules of glyceraldehyde- 3-phosphate are converted
to pyruvate, with concomitant generation of four ATPs.

49
Glycolysis therefore has a net profit of two ATPs per glucose: Stage I consumes two ATPs;
Stage II produces four ATPs but is later resynthesized (Reactions 7 and 10). The overall
reaction of glycolysis may be summarized as:
Fate of Pyruvate
Fate of Pyruvate in anaerobic condition: Fermentation to ethanol in yeast

50
Glycolytic Pathway
8.2 TCA cycle
The pyruvate formed in glycolytic pathway enters mitochondria and form acetyl CoA. The
conversion of pyruvate to acetyl groups, then the entry of those groups into the citric acid

51
cycle, also called the tricarboxylic acid (TCA) cycle or the Krebs cycle (after its discoverer,
Hans Krebs).
TCA Cycle
TCA cycle accounts for the major portion of carbohydrate, fatty acid, and amino acid oxidation
and generates numerous biosynthetic precursors. The citric acid cycle is therefore amphibolic,
that is, it operates both catabolically and anabolically.
Steps of TCA cycle
1. Citrate synthase catalyzes the condensation of acetyl-CoA and oxaloacetate to yield
citrate, giving the cycle its name.

52
2. The strategy of the cycle’s next two steps is to rearrange citrate to a more easily oxidized
isomer and then oxidize it. Aconitase isomerizes citrate, a not readily oxidized tertiary
alcohol, to the easily oxidized secondary alcohol isocitrate.The reaction sequence
involves a dehydration, producing enzyme-bound cis-aconitate, followed by a
hydration, so that citrate’s hydroxyl group is, in effect, transferred to an adjacent carbon
atom.
3. Isocitrate dehydrogenase oxidizes isocitrate to the α-keto acid intermediate
oxalosuccinate with the coupled reduction of NAD+
to NADH; oxalosuccinate is then
decarboxylated, yielding α-ketoglutarate. This is the first step in which oxidation is
coupled to NADH production and also the first CO2-generating step.
4. The multienzyme complex α-ketoglutarate dehydrogenase oxidatively
decarboxylates α-ketoglutarate to succinyl-coenzyme A. The reaction involves the
reduction of a second NAD+
to NADH and the generation of a second molecule of CO2.
At this point in the cycle, two molecules of CO2 have been produced, so that the net
oxidation of the acetyl group is complete. Note, however, that it is not the carbon atoms
of the entering acetyl-CoA that have been oxidized.
5. Succinyl-CoA synthetase converts succinyl-coenzyme A to succinate.The free energy
of the thioester bond is conserved in this reaction by the formation of “high-energy”
GTP from GDP + Pi.
6. The remaining reactions of the cycle serve to oxidize succinate back to oxaloacetate in
preparation for another round of the cycle. Succinate dehydrogenase catalyzes the
oxidation of succinate’s central single bond to a trans double bond, yielding fumarate
with the concomitant reduction of the redox coenzyme FAD to FADH2 (the molecular
formulas of FAD and FADH2 and the reactions through which they are interconverted.
7. Fumarase then catalyzes the hydration of fumarate’s double bond to yield malate.
8. Finally, malate dehydrogenase reforms oxaloacetate by oxidizing malate’s secondary
alcohol group to the corresponding ketone with concomitant reduction of a third NAD+
to NADH. Acetyl groups are thereby completely oxidized to CO2 with the following
stoichiometry:

53
8.3 Glyoxylate cycle
After germinating, oil-containing seeds metabolize stored triacylglycerols by converting lipids
to sucrose. Plants are not able to transport fats from the endosperm to the root and shoot tissues
of the germinating seedling, so they must convert stored lipids to a more mobile form of carbon,
generally sucrose. This process involves several steps that are located in different cellular
compartments: oleosomes, glyoxysomes, mitochondria, and cytosol.
 Korenberg and Krebs (1957) framed a cycle which is known as Glyoxylic Acid Cycle
or Glyoxylate Cycle through which the fats could be converted into sucrose
(carbohydrates) during the germination of fatty seeds in plants.
 Sucrose translocated to growing regions of young germinating seedling till it develops
green leaves to manufacture its own food.
8.4 Electron transport chain
The mitochondrion is the aerobic organelle in which the final stage of the oxidation of food
occurs. It is the site of the citric acid cycle, fatty acid oxidation and oxidative phosphorylation,
processes that are responsible for the formation of ATP under aerobic condition. The two most
important energy transductions in the biological systems are the oxidative phosphorylation
(ATP synthesis driven by electron transfer to oxygen) and photophosphorylation (ATP
synthesis driven by light). Oxidative phosphorylation is the process in which ATP molecules
are formed as a result of the transfer of electrons from the reducing equivalents, NADH or
FADH2 (produced by glycolysis, the citric acid cycle and fatty acid oxidation) to oxygen by a

54
series of electron carriers in the form of a chain located in the inner membrane of mitochondria.
This is the final reaction sequence of respiration.
The chemiosmotic theory states that the coupling of oxidation to phosphorylation is indirect.
According to this, the hydrogen ions (protons) generated by the oxidation of components in the
respiratory chain are ejected to the outside (matrix) of the inner membrane. The
electrochemical potential difference resulting from the asymmetric distribution of the hydrogen
ions (protons or H+
) is used to drive a membrane-located ATP synthase which in the presence
of Pi + ADP forms ATP.
Inhibitors of ETC
Compounds such as barbiturates, amytal, rotenone prevent the transfer of electron from
FeS centre to ubiquinone. Carboxin specifically inhibits transfer of reducing
equivalents from succinate dehydrogenase to ubiquinone.
Antimycin A blocks electron transfer from cytochrome b to cytochrome c1.
Substances such as cyanide (CN-), azide (N3-) and carbon monoxide inhibit
cytochrome c oxidase by binding to heme group and are extremely poisonous.
Oligomycin inhibits ATP synthase.
Uncouplers of ETC
In the presence of the uncouplers such as dicoumarol and 2,4-dinitrophenol, oxidation proceeds
without phosphorylation (dissociation of oxidation in the respiratory chain from
phosphorylation) releasing energy in the form of heat rather than in the form of ATP.
LESSION 9: METABOLISM OF LIPIDS
9.1 Beta-oxidation of Fatty Acids
In 1904 Franz Knoop proposed that the breakdown of fatty acid occurs by a mechanism known
as β-oxidation in which the fatty acid’s Cβ atom is oxidized. There are three main steps to
describe the β-oxidation oxidation of fatty acids:
1. Fatty Acid Activation
Before fatty acids can be oxidized, they must be “primed” for reaction in an ATP-dependent
acylation reaction to form fatty acyl-CoA.This activation process is catalyzed by a family of
at least three acyl-CoA synthetases (also called thiokinases) that differ according to their
chain-length specificities. These enzymes, which are associated with either the endoplasmic
reticulum (ER) or the outer mitochondrial membrane, all catalyze the reaction:

55
2. Transport across Mitochondrial Membrane
a) The acyl group of a cytosolic acyl-CoA is transferred to carnitine, thereby releasing
the CoA to its cytosolic pool.
b) The resulting acyl-carnitine is transported into the mitochondrial matrix by the
transport system.
c) The acyl group is transferred to a CoA molecule from the mitochondrial pool.
d) The product carnitine is returned to the cytosol.
3. Beta-oxidation
Fatty acids are dismembered through the β-oxidation of fatty acyl-CoA, a process that occurs
in four reactions:
i. Formation of a trans- α, β double bond through dehydrogenation by the flavoenzyme
acyl-CoA dehydrogenase (AD).
ii. Hydration of the double bond by enoyl-CoA hydratase (EH) to form a 3-L-
hydroxyacyl CoA.
iii. NAD+
-dependent dehydrogenation of this _- hydroxyacyl-CoA by 3-L-hydroxyacyl-
CoA dehydrogenase (HAD) to form the corresponding _-ketoacyl-CoA.
iv. Cα¬Cβ cleavage in a thiolysis reaction with CoA as catalyzed by β-ketoacyl-CoA
thiolase (KT; also called just thiolase) to form acetyl-CoA and a new acyl-CoA
containing two less C atoms than the original one.
Stepwise reaction of β-oxidation of fatty acids is given below:

56
Energetics of β-oxidation of fatty acids
β-oxidation of palmitic acid will repeat 7 cycles producing 8 molecules of Acetyl
CoA
In each cycle, FADH2 and NADH + H+
will be produced and transported to ETC
FADH2 1.5 ATPs
NADH + H+
2.5 ATPs
So, 7 cycles (1.5+2.5) x7 =28 ATPs
Each Acetyl CoA which is oxidized in citric acid cycle gives 10 ATPs, i.e., 8 x10 =
80 ATPs [each acetyl CoA in citric acid cycle gives 3 NADH (=7.5 ATPs), 1
FADH2(=1.5 ATPs) and 1 ATP (in plant) or 1 GTP (in animals)]
Two ATPs are utilized in activation of fatty acid (occurs only once)
Net ATP gain = (28+80) – 2 = 106 ATPs

57
Comparison of Mitochondrial and Peroxisomal β- Oxidation
Plant Peroxisomes and Glyoxysomes Use Acetyl-CoA from β- Oxidation as a
Biosynthetic Precursor
In plants, fatty acid oxidation does not occur primarily in mitochondria but in the peroxisomes
of leaf tissue and in the glyoxysomes of germinating seeds. Plant peroxisomes and
glyoxysomes are similar in structure and function; glyoxysomes, which occur only in
germinating seeds, may be considered specialized peroxisomes. The biological role of β
oxidation in these organelles is to use stored lipids primarily to provide but biosynthetic
precursors, not energy. During seed germination, stored triacylglycerols are converted into
glucose, sucrose, and a wide variety of essential metabolites. Fatty acids released from the
triacylglycerols are first activated to their coenzyme A derivatives and oxidized in
glyoxysomes by the same four-step process that takes place in peroxisomes. The acetyl-CoA
produced is converted via the glyoxylate cycle to four-carbon precursors for gluconeogenesis.
Glyoxysomes, like peroxisomes, contain high concentrations of catalase, which converts the
H2O2 produced by β oxidation to H2O and O2.
The peroxisomal/glyoxysomal system differs from the
mitochondrial system in two respects:
(1) In the first oxidative step electrons pass directly to O2,
generating H2O2, and
(2) The NADH formed in the second oxidative step cannot be
re-oxidized in the peroxisome or glyoxysome, so reducing
equivalents are exported to the cytosol, eventually entering
mitochondria.
The acetyl-CoA produced by peroxisomes and glyoxysomes
is also exported; the acetate from glyoxysomes (organelles
found only in germinating seeds) serves as a biosynthetic
precursor. Acetyl-CoA produced in mitochondria is further
oxidized in the citric acid cycle.

58
9.2 Fatty Acid Biosynthesis
Fatty Acid Synthesis Occurs in the Cytosol of Many Organisms but in the Chloroplasts
of Plants
In the photosynthetic cells of plants, fatty acid synthesis occurs not in the cytosol but in the
chloroplast stroma. This makes sense, given that NADPH is produced in chloroplasts by the
light reactions of photosynthesis. Usually, NADPH is the electron carrier for anabolic
reactions, and NAD+
serves in catabolic reactions. The high [NADPH] / [NADP+
] ratio
provides the reducing environment that favors reductive anabolic processes such as fatty acid
synthesis.
Fatty acid biosynthesis occurs through condensation of C2 units, the reverse of the β oxidation
process. Through isotopic labeling techniques, David Rittenberg and Konrad Bloch
demonstrated, in 1945, that these condensation units are derived from acetic acid. Acetyl-CoA
was soon proven to be a precursor of the condensation reaction, but its mechanism remained
obscure until the late 1950s when Salih Wakil discovered a requirement for bicarbonate in
fatty acid biosynthesis and malonyl-CoA was shown to be an intermediate.
Comparison of fatty acid _ oxidation and fatty acid biosynthesis
Differences occur in:
1. cellular location
2. acyl group carrier
3. electron acceptor/ donor
4. stereochemistry of hydration/dehydration reaction
5. the form in which C2 units are produced/donated

59
Subcellular localization of lipid metabolism in Animals & Plants
Steps for de novo synthesis of fatty acids
1. Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonyl CoA by
acetyl CoA carboxylase, with the consumption of ATP.

60
2. In a subsequent reaction, CoA is exchanged for acyl carrier protein (ACP). ACP
contains a serine residue to which a pantetheine residue is linked via a phosphate group.
Since the pantetheine residue is also a functional constituent of CoA, ACP can be
regarded as a CoA, which is covalently bound to a protein.
3. The enzyme b-ketoacyl-ACP synthase III catalyzes the condensation of acetyl CoA
with malonyl-ACP.
4. The liberation of CO2 makes this reaction irreversible.
5. The acetoacetate thus formed remains bound as a thioester to ACP and is reduced by
NADPH to b-D-hydroxyacyl-ACP.
6. Following the release of water, the carbon-carbon double bond formed is reduced by
NADPH to acyl-ACP.
7. The product is a fatty acid that has been elongated by two carbon atoms.

61
References
1. Plant Biochemistry, 3rd
Edition, Hans-Walter Heldt
2. Lehninger Principles of Biochemistry, 5th
Edition, D. L. Nelson & M. M. Cox
3. Biochemistry, 4th
Edition, D. Voet & J. Voet
4. Biochemistry, U. Satyanarayana & U. Chakrapani

Complete E material on Fundamentals of Biochemistry [2+1]; (32 Lectures)

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Complete E material on Fundamentals of Biochemistry [2+1]; (32 Lectures)