Principle of biochemistry and fundamental of enzymology

Biochemistry
and
Metabolism
Dr. Kirpa Ram (Assistant Professor, Botany)
Ph. +91-9468393474, Mail- dr.kirparamjangra@gmail.com
Principle of Biochemistry And Fundamental of Enzymology

Principle of Biochemistry
And
Fundamental of Enzymology
Biochemistry by Dr. Kirpa Ram

Part - I
Principle of Biochemistry

What is Biochemistry
Biochemistry is the application of chemistry to the study of biological
processes at the cellular and molecular level. It emerged as a distinct
discipline around the beginning of the 20th century when scientists
combined chemistry, physiology, and biology to investigate the
chemistry of living systems.
"Biochemistry has become the foundation for understanding all
biological processes.
It has provided explanations for the causes of many diseases in
humans, animals and plants."

The study of life in its chemical processes
Biochemistry is both life science and a
chemical science
• It explores the chemistry of living
organisms and the molecular basis for
the changes occurring in living cells.
• It uses the methods of chemistry,
physics, molecular biology, and
immunology to study the found in
bistructure and behavior of the
complex moleculesological material
and the ways these molecules interact
to form cells, tissues, and whole
organisms.

Major Disciplines Related to Biochemistry?
Biochemistry is closely related to other biological sciences that deal
with molecules. There is considerable overlap between these disciplines:
1. Molecular Genetics
2. Pharmacology
3. Molecular Biology
4. Chemical Biology

Application and Importance of
Biochemistry in life science
1. Importance of biochemistry in MEDICINE
2. Importance of biochemistry in NURSING
3. Importance of biochemistry in AGRICULTURE
4. Importance of biochemistry in NUTRITION
5. Importance of biochemistry in PHARMACY
6. Importance of biochemistry in PLANTS

Importance of biochemistry in MEDICINE
• Biochemistry is a valuable subject in medicine without which
there would have been no such advancement in the field.
• Physiology: Biochemistry helps one understand the
biochemical changes and related physiological alteration in
the body.
• Pathology: Based on the symptoms described by the patient,
the physician can get a clue on the biochemical change and
the associated disorder. For example, if a patient complains
about stiffness in small joints, then the physician may predict
it to be gout and get confirmed by evaluating uric acid levels
in the blood as uric acid accumulation in blood results in
gout.
• Nutrition deficiency: In the present scenario, many people
rely on taking multivitamin & minerals for better health.
• Hormonal deficiency: There are many disorders due to
hormonal imbalance in especially women and children.

Importance of biochemistry in NURSING
• In nursing, the importance of clinical
biochemistry is invaluable. When a patient is in
the hospital nurses, need to keep a watch on how
his condition is progressing through clinical
biochemistry. That is the treatment for helping
him recover from said condition etc. Almost all
diseases or disorders have some biochemical
involvement. So the diagnosis of any clinical
condition is easily possible by biochemical
estimations.
• Kidney function test
• Blood test
• Liver function tests
• Serum cholesterol test

Importance of biochemistry in AGRICULTURE
In agriculture, biochemistry plays a valuable role
in farming, fishery, poultry, sericulture, beekeeping, etc.
• Prevent diseases: It helps for prevention, treatment of
diseases and also increases the production or yield.
• Enhance growth: Biochemistry gives an idea of how
the use of fertilizers can increase plant growth, their
yield, quality of food, etc.
• Enhance Yield: Some hormones promote growth,
while others encourage flowering, fruit formation, etc.
In fisheries, the use of substances to promote fish
growth, their reproduction, etc. can be understood.
• Adulteration: Even the composition of food material
produced, their alteration or adulteration for example
in honey can be found by biochemical tests.
Biochemistry tests help prevent contamination.

• Biochemical tests for the pesticide residues or
other toxic waste in plant, food grain and soil can
be evaluated. Hence during import and export of
food grains, a biochemical check of the toxic
residues is done to fix the quality.
• In animal husbandry, the quality of milk can be
checked by biochemical tests. It also helps
diagnose any disease condition in animals and
birds.
• In fisheries, the water quality is regularly
monitored by biochemical tests. Any drastic change
in water chemistry & composition of fishery ponds
can lead to the vast death of fishes and prawns.
Hence the tests are done regularly to see salt
content (calcium content), pH, accumulation of
waste due to not changing water for long, etc.

Importance of biochemistry in NUTRITION
• In nutrition, biochemistry describes food chemistry. For
maintenance of health, optimum intake of many
biochemicals like macro, micronutrients, vitamins,
minerals, essential fatty acids & water is necessary.
• Food chemistry components like carbohydrates,
proteins, fats, etc. and also the possible physiological
alteration due to their deficiency.
• The role of nutrients: Due to biochemistry the
importance of vitamins, minerals, essential fatty acids,
their contribution to health were known.
• The nutrients value of food material can also be
determined by biochemical tests.
• The physician can prescribe to limit usage of certain
food like excess sugar for diabetics, excess oil for heart
& lung problem prone patients, etc. As these
carbohydrate and fat diets can inhibit the recovery rate
from said disorder.

Importance of biochemistry in PHARMACY
• In a pharmacy, many drugs are stored for regular dispensing.
• Drug Constitution: Biochemistry gives an idea of
the constitution of the drug, its chances of degradation with
varying temperature, etc. How modification in medicinal
chemistry helps improve efficiency, minimize side effects, etc.
• The half-life: This is a test done on biochemical drugs to
know how long a drug is stable when kept at so and so
temperature.
• Drug storage: The storage condition required can be
estimated by the biochemical test. For example, many
enzymes, hormones are stored for dispensing. These get
deteriorated over time due to temperature or oxidation,
contamination and also due to improper storage.
• Drug metabolism: It also gives an idea of how drug
molecules are metabolized by many biochemical reactions in
the presence of enzymes. This helps to avoid drugs which
have a poor metabolism or those with excessive side effects
from being prescribed or dispensed to the patient.
• Biochemical tests: These tests help fix the specific half-life or
date of expiry of drugs. Biochemistry by Dr. Kirpa Ram

Importance of biochemistry in PLANTS
• Biochemistry of plants gave way to the
breakthrough of how food is synthesized in
them and the reason why they are autotrophs,
i.e., not dependent on other living beings for
food. Biochemistry in plants describes
• Photosynthesis: This describes how
carbohydrates are synthesized by the use of
sunlight, CO2, and water in the green leaves of
plants. It goes on to explain about different
complex enzymes involved in the process to
combine the energy of sun within the molecules
H2O+ CO2 in the form of carbohydrates.
• Respiration: By use of the above
photosynthesis pathway, plants leave out
Oxygen while taking up Carbon dioxide from
the air. This air is used to generate energy in a
cell like that of animal cells. Biochemistry by Dr. Kirpa Ram

• Different sugars: Biochemistry defines different types of
carbohydrates formed in plants like trioses (3 carbon
sugars, i.e., glyceraldehyde), tetroses (4), pentoses (5),
hexoses (6= glucose), heptoses (7), etc. Tetroses are the
carbohydrates which go on to form the nucleic acids, i.e.,
deoxyribonucleic acid (DNA), ribonucleic acid (RNA).
• Plants secondary metabolites: Biochemistry also
describes how the plant products like gums, tannins,
alkaloids, resins, enzymes, phytohormones are formed
inside the plant. Further, how the different plant cell parts
are involved in physiology. The conversion of different
biochemical over some time like lignin, chitin to harden
the dead vascular vessels, etc.
• Other functions: It also describes how plants fruits get
ripened, how to plant seed germinates, the respiration
process inside the plant cell, how proteins and amino
acids are formed on rough endoplasmic reticulum and fats
are formed on smooth ER.

Overview of Atomic Structure
What is Atomic Structure?
• The atomic structure of an element
refers to the constitution of its nucleus
and the arrangement of the electrons
around it.
• Primarily, the atomic structure of matter
is made up of protons, electrons, and
neutrons.
• Atoms are made up of particles called
protons, neutrons, and electrons, which
are responsible for the mass and charge
of atoms.

Key Points
• An atom is composed of two regions: the
nucleus, which is in the center of the atom
and contains protons and neutrons, and
the outer region of the atom, which holds
its electrons in orbit around the nucleus.
• Protons and neutrons have approximately
the same mass, about 1.67 × 10-24 grams,
which scientists define as one atomic
mass unit (amu) or one Dalton.
• Each electron has a negative charge (-1)
equal to the positive charge of a proton
(+1).
• Neutrons are uncharged particles found
within the nucleus.
Key Terms
• Atom: The smallest possible amount of
matter which still retains its identity as a
chemical element, consisting of a nucleus
surrounded by electrons.
• Proton: Positively charged subatomic
particle forming part of the nucleus of an
atom and determining the atomic number
of an element. It weighs 1 amu.
• Neutron: A subatomic particle forming
part of the nucleus of an atom. It has no
charge. It is equal in mass to a proton or it
weighs 1 amu.
• Mass number: The sum of the number of
protons and the number of neutrons in an
atom.
• Atomic number: The number of protons
in an atom.
• Atomic mass: The average mass of an
atom, taking into account all its naturally
occurring isotopes.

Atomic Structures of Some Elements
Hydrogen is the chemical element
with the symbol H and atomic number
1.
• With a standard atomic weight of
1.008, hydrogen is the lightest
element in the periodic table.
• Hydrogen is the most abundant
chemical substance in the universe,
constituting roughly 75% of all
baryonic mass.
• Electron configuration: 1s1
• Structure of Hydrogen atom: This
implies that it contains one proton,
one electron, and no neutrons (total
number of neutrons = mass number
– atomic number)

Atomic Structures of Carbon (C)
Carbon (from Latin: carbo "coal") is a
chemical element with the symbol C and
atomic number 6.
• It is nonmetallic and tetravalent
making four electrons available to
form covalent chemical bonds. It
belongs to group 14 of the periodic
table.
• Carbon makes up only about 0.025
percent of Earth's crust.
• Group: group 14 (carbon group)
• Carbon has two stable isotopes – 12C
and 13C. Of these isotopes, 12C has
an abundance of 98.9%. It contains 6
protons, 6 electrons, and 6 neutrons.

Oxygen (O), nonmetallic chemical element
of Group 16 (VIa, or the oxygen group) of
the periodic table.
• Oxygen is a colourless, odourless,
tasteless gas essential to living organisms,
being taken up by animals, which convert
it to carbon dioxide; plants, in turn, utilize
carbon dioxide as a source of carbon and
return the oxygen to the atmosphere.
• There exist three stable isotopes of
oxygen – 18O, 17O, and 16O. However,
oxygen-16 is the most abundant isotope.
• Structure of Oxygen atom: Since the
atomic number of this isotope is 8 and the
mass number is 16, it consists of 8 protons
and 8 neutrons. 6 out of the 8 electrons in
an oxygen atom lie in the valence shell.
Atomic Structures of Oxygen (O)

Molecules and Organic Compound

Molecules
Molecule, a group of two or more atoms that
form the smallest identifiable unit into which
a pure substance can be divided and still
retain the composition and chemical
properties of that substance.
Have you ever called water 'H2O'?
• That's a molecule. When two atoms of
hydrogen (H) and one atom of oxygen (O)
are bonded to each other, they make one
molecule of water.
• With just three atoms, it's one of the
smallest molecules you'll hear about in
biology. Others, like DNA, are made of
many, many, many atoms. But more about
those in a minute. Biochemistry by Dr. Kirpa Ram

Organic compounds
• Organic compound, any of a large
class of chemical compounds in which
one or more atoms of carbon are
covalently linked to atoms of other
elements, most commonly hydrogen,
oxygen, or nitrogen.
• The few carbon-containing compounds
not classified as organic include
carbides, carbonates, and cyanides.
• These compound are:-
• Molecular make-up: the main elements that make up the class of
compounds.
• Structural composition: how the monomers join up together to
form polymers.
• Biological role: importance of these molecules to animals and
plants.
• Chemical test: how to detect the presence of each class of
compounds

Carbohydrates
• Carbohydrates are the sugars, starches and fibers
found in fruits, grains, vegetables and milk products.
• Molecular make-up: Carbohydrates consist of carbon
(C), hydrogen (H) and oxygen (O).
• Structural composition
• Carbohydrates are made up of monomers known as
monosaccharides. The monosaccharide that makes up
most carbohydrates is glucose. Other monosaccharides
include fructose, galactose and deoxyribose (discussed
later). These monomers can be joined together by
glycosidic bonds. When two monosaccharides are
chemically bonded together, they form disaccharides.

Examples of carbohydrates based on the different classifications.
Sr. No CLASS EXAMPLES
1 Monosaccharides Glucose, fructose, galactose
2 Disaccharides Sucrose, lactose, maltose
3 Oligosaccharides
Fructo-oligosaccharides, malto-
oligosaccharides
4 Polyols Isomalt, maltitol, sorbitol, xylitol, erythritol
5 Starch polysaccharides Amylose, amylopectin, maltodextrins
6
Non-starch
polysaccharides
(dietary fibre)
Cellulose, pectins, hemicelluloses, gums,
inulin

Role of carbohydrates in animals and plants
The main function of carbohydrates is as energy storage molecules and as
substrates (starting material) for energy production. Carbohydrates are broken
down by living organisms to release energy.
Each gram of carbohydrate supplies about 17 kilojoules (kJ) of energy.
• Starch and glycogen are both storage polysaccharides (polymers made up
of glucose monomers) and thus act as a store for energy in living organisms.
• Starch is a storage polysaccharide in plants and glycogen is the storage
polysaccharide for animals.
• Cellulose is found in plant cell walls and helps gives plants strength.
• All polysaccharides are made up of glucose monomers, but the difference in
the properties of these substances can be attributed to the way in which the
glucose molecules join together to form different structures.

Lipids
• Lipids are molecules that contain
hydrocarbons and make up the building
blocks of the structure and function of
living cells.
• Molecular make-up: Lipids contain carbon
(C), hydrogen (H) and oxygen (O) but have
less oxygen than carbohydrates.
• A lipid is any of various organic compounds
that are insoluble in water. They include
fats, waxes, oils, hormones, and certain
components of membranes and function as
energy-storage molecules and chemical
messengers.
• Together with proteins and carbohydrates,
lipids are one of the principal structural
components of living cells.

Why are lipids important?
• Lipids are a diverse group of compounds and serve 37.8 kilojoules
(kJ) of energy per gram.
• At a cellular level, phospholipids and cholesterol are some of the
primary components of the membranes that separate a cell from its
environment.
• Lipid-derived hormones, known as steroid hormones, are important
chemical messengers and include testosterone and estrogens.
• At an organismal level triglycerides stored in adipose cells serve as
energy-storage depots and also provide thermal insulation.

Protein
s
• Proteins are large biomolecules or macromolecules
that are comprised of one or more long chains of
amino acid residues.
• Swedish chemist Jöns Jacob Berzelius, who in 1838
coined the term protein, a word derived from the
Greek prōteios, meaning “holding first place.”
• Molecular make-up: Proteins contain carbon (C),
hydrogen (H), oxygen (O), nitrogen (N) and may
have other elements such as iron (Fe), phosphorous
(P) and sulfur (S).
• Structural composition: Proteins are made of
amino acids. Amino acids are bonded together by
peptide bonds to form peptides. A long peptide
chain forms a protein, which folds into a very specific
dimensional shape.

Four different levels of protein structure
• Primary structure: This refers to the sequence
of amino acids joined together by peptide bonds
to form a polypeptide chain.
• Secondary structure: This is the first level of
three dimensional folding. It is driven completely
by hydrogen bonding.
• Tertiary structure: The secondary structures and
unstructured regions of the chain further fold into
a globular shape, driven by hydrophobic
interactions (non-polar regions trying to escape
the water in the cell environment) and
electrostatic interactions (polar and charged
regions wanting to interact with the water
environment and each other).
• Quaternary structure: Some proteins are
complex: two or more peptide chains fold into
their tertiary structures, then these complete
structures associate together by hydrophobic and
electrostatic interactions to form the final protein.

Role of protein
• Each gram of protein can be broken down to
release 17 kJ of energy. Certain proteins called
enzymes are important in catalysing cellular
reactions that form part of metabolism.
• Growth and Maintenance. Share on Pinterest
• Causes Biochemical Reactions
• Acts as a Messenger
• Provides Structure
• Maintains Proper pH
• Balances Fluids
• Bolsters Immune Health
• Transports and Stores Nutrients

Enzymes
• The term enzyme has a specific meaning: an
enzyme is a biological catalyst that speeds up
the rate of a chemical reaction without being
used up in the chemical reaction itself. Let us
analyze this definition in greater detail.
• Biological: Enzymes are protein molecules which
are made of long chains of amino acids. These fold
into unique three-dimensional structures with a
region known as an active site where reactions
take place.
• Catalyst: Enzymes speed up chemical reactions
without being used up in the reaction themselves.
All chemical reactions require a certain minimum
amount of energy to take place. This energy is
known as the free energy of activation. Enzymes
lower the energy of activation thus speeding up
chemical reactions

Enzymes in everyday life
• The properties of enzymes to control reactions have been widely used for
commercial purposes. Examples of some of these uses are listed below:
• Biological washing powders contain enzymes such as lipases (breaks down
lipids) and proteases (breaks down protein), which assist in the breakdown
of stains caused by foods, blood, fat or grease. These biological washing
powders save energy as they are effective at low temperatures.
• Meat tenderisers contain enzymes which are obtained from fruits such as
papaya or pineapple. When used in meat tenderisers these enzymes soften
the meat.
• Lactose-free milk is manufactured primarily for people who are lactose
intolerant. Lactose intolerant individuals lack the enzyme lactase that
digests lactose (milk sugar). Lactose is pre-digested by adding lactase to the
milk.

Nucleic acids
• Nucleic acids are the main information-carrying molecules of the cell, and,
by directing the process of protein synthesis, they determine the inherited
characteristics of every living thing.
• The building blocks of nucleic acids are called nucleotides. Each nucleotide
is made up of a sugar, a phosphate and a nitrogenous base.
• Nucleotides are joined together by phosphodiester bonds, which join the
phosphate of one nucleotide to the sugar of the next.
• The phosphate-sugar-phosphate-sugar strands form a "backbone" upon
which the nitrogen-containing bases are exhibited.
• Nucleic acids are therefore polymers made up of many nucleotides. DNA is
a double-stranded polymer, due to hydrogen bonding between the
nitrogenous bases of two complementary strands.
• RNA is a single-stranded polymer. Nucleic acids do not need to be obtained
from the diet because they are synthesised using intermediate products of
carbohydrate and amino acid metabolism.

Nucleic acids include
• Deoxyribonucleic acid (DNA):
which contains the 'instructions' for
the synthesis of proteins in the form
of genes. DNA is found in the
nucleus of every cell, and is also
present in smaller amounts inside
mitochondria and chloroplasts.
• Ribonucleic acid (RNA): is
important in transferring genetic
information from DNA to form
proteins. It is found on ribosomes, in
the cytoplasm and in the nucleus.

Chemical Bonding
and
Buffers

Chemical bonds
• Chemical bonds hold molecules
together and create temporary
connections that are essential to
life. Types of chemical bonds
including covalent, ionic, and
hydrogen bonds and London
dispersion forces.
• A chemical bond is a lasting
attraction between atoms, ions or
molecules that enables the
formation of chemical
compounds. The bond may result
from the electrostatic force of
attraction between oppositely
charged ions as in ionic bonds or
through the sharing of electrons
as in covalent bonds.

Types of Chemical Bonds
The type of chemical bonds formed vary in strength and properties.
There are 4 primary types of chemical bonds which are formed by
atoms or molecules to yield compounds. These types of chemical bonds
include:
1. Ionic Bonds
2. Covalent Bonds
3. Hydrogen Bonds
4. Polar Bonds
These types of bonds in chemical bonding are formed from the loss,
gain, or sharing of electrons between two atoms/molecules.

Ionic Bond
• As the name suggests, ionic bonds are a result of
the attraction between ions. Ions are formed when
an atom loses or gains an electron.
• Ionic bonds are bonds formed between ions with
opposite charges.
• These types of bonds are commonly formed
between a metal and a nonmetal.
• Electropositive chemical elements have a
tendency to lose one or more electron particles.
But the electronegative elements have a tendency
to gain these electron particles.
• As a result of mutual electrostatic attraction
between positive and negative ion establishes the
formation of ionic bonding in chemical
compounds.
Examples
1. Sodium (Na) and chlorine (Cl)
combine to form stable crystals of
sodium chloride (NaCl), also known
as common salt.
2. Magnesium (Mg) and oxygen (O)
combine to form magnesium oxide
(MgO).
3. Potassium (K) and chlorine (Cl)
combine to form potassium chloride
(KCl)
4. Calcium (Ca) and fluorine (F)
combine to form calcium fluoride
(CaF2)

Covalent bonds
In the case of a covalent bond, an atom shares one or
more pairs of electrons with another atom and forms a
bond.
• This sharing of electrons happens because the atoms
must satisfy the octet (noble gas configuration) rule
while bonding.
• Such type of bonding is common between
two nonmetals.
• The covalent bond is the strongest and most common
form of chemical bond in living organisms.
• Together with the ionic bond, they form the two most
important chemical bonds.
• A covalent bond can be divided into a nonpolar
covalent bond and a polar covalent bond.
• In the case of a nonpolar covalent bond, the electrons
are equally shared between the two atoms. On the
contrary, in polar covalent bonds, the electrons are
unequally distributed between the atoms.
Examples
1. Two atoms of iodine (I) combine to form
iodine (I2) gas.
2. One atom of carbon (C) combines with
two atoms of oxygen (O) to form
a double covalent bond in carbon dioxide
(CO2).
3. Two atoms of hydrogen (H) combine with
one atom of oxygen (O) to form a polar
molecule of water (H2O).
4. Boron (B) and three hydrogens (H)
combine to form the polar borane (BH3).

Hydrogen Bond
• A hydrogen bond is a chemical bond between a
hydrogen atom and an electronegative atom.
• However, it is not an ionic or covalent bond but
is a particular type of dipole-dipole attraction
between molecules.
• First, the hydrogen atom is covalently bonded to
a very electronegative atom resulting in a
positive charge, which is then attracted towards
an electronegative atom resulting in a hydrogen
bond
• Tendency for the hydrogen to be attracted
towards the negative charges of any neighbouring
atom. This type of chemical bonding is called a
hydrogen bond and is responsible for many of the
properties exhibited by water.
Examples
1. Hydrogen atom from one molecule of
water bonds with the oxygen atom from
another molecule.
2. In chloroform (CH3Cl) and ammonia
(NH3), hydrogen bonding occurs between
the hydrogen of one molecule and
carbon/nitrogen of another.
3. Nitrogen bases present in DNA are held
together by a hydrogen bond.

Metallic Bonds
‘Metallic bond’ is a term used to describe the collective
sharing of a sea of valence electrons between several
positively charged metal ions.
• A metallic bond is a force that holds atoms together in
a metallic substance.
• Such solid consists of tightly packed atoms, where the
outermost electron shell of each metal atoms overlaps
with a large number of neighboring atoms.
• The valence electrons move freely from one atom to
another. They are not associated with any specific pair
of atoms. This behavior is called non-localization.
• The factors that affect the strength of a metallic bond
include:
1. Total number of delocalized electrons.
2. Magnitude of positive charge held by the metal
cation.
3. Ionic radius of the cation
Examples
1. Sodium metal
2. Aluminum foil
3. Copper wire

Other Types of Chemical Bonds
Van der Waals Bond
• Neutral molecules are held together by weak electric forces known as Van
der Waals forces. Van der Waals force is a general term used to define the
attraction of intermolecular forces between molecules. This type of
chemical bond is the weakest of all bonds.
• Examples include hydrogen bonding, dispersion forces, and dipole-dipole
forces.
Peptide Bond
• Within a protein, multiple amino acids bonds are formed by a biochemical
reaction that extracts a water molecule as it joins the amino group of one
amino acid to the carboxyl group of neighboring amino acids. Examples
include polypeptides like insulin and growth hormone.
Glycosidic bond
• Glycosidic linkage is a type of covalent bond that joins a carbohydrate
(sugar) molecule to another group, which may or may not be another
carbohydrate.

Buffers
A buffer is a solution containing either a weak
acid and its salt or a weak base and its salt,
which is resistant to changes in pH.
• In other words, a buffer is an aqueous solution
of either a weak acid and its conjugate base or
a weak base and its conjugate acid.
• A buffer may also be called a pH buffer,
hydrogen ion buffer, or buffer solution.
• Buffers are used to maintain a stable pH in a
solution, as they can neutralize small
quantities of additional acid of base.
• An example of a common buffer is a solution
of acetic acid (CH3COOH) and sodium
acetate. In water solution, sodium acetate is
completely dissociated into sodium (Na+) and
acetate (CH3COO-) ions.
The general form of a buffer chemical reaction
is:
HA ⇌ H+ + A−
Examples of Buffers
 Blood - contains a bicarbonate buffer system
 TRIS buffer
 Phosphate buffer Biochemistry by Dr. Kirpa Ram

Types of Buffer Solution
The two primary types into which buffer solutions are broadly classified into are acidic and alkaline buffers.
Acidic Buffers
• As the name suggests, these solutions
are used to maintain acidic
environments. Acid buffer has acidic
pH and is prepared by mixing a weak
acid and its salt with a strong base. An
aqueous solution of an equal
concentration of acetic acid and
sodium acetate has a pH of 4.74.
• The pH of these solutions is below
seven
• These solutions consist of a weak acid
and a salt of a weak acid.
• An example of an acidic buffer
solution is a mixture of sodium acetate
and acetic acid (pH = 4.75).
Alkaline Buffers
• These buffer solutions are used to
maintain basic conditions. Basic buffer
has a basic pH and is prepared by
mixing a weak base and its salt with
strong acid. The aqueous solution of
an equal concentration of ammonium
hydroxide and ammonium chloride has
a pH of 9.25.
• The pH of these solutions is above
seven
• They contain a weak base and a salt of
the weak base.
• An example of an alkaline buffer
solution is a mixture of ammonium
hydroxide and ammonium chloride
(pH = 9.25).

Preparation of buffer
• If the dissociation constant of the acid (pKa) and of the base (pKb) are
known, a buffer solution can be prepared by controlling the salt-acid or
the salt-base ratio.
• An example of this method of preparing buffer solutions can be given by
the preparation of a phosphate buffer by mixing HPO4
2- and H2PO4-.
• The pH maintained by this solution is 7.4.
• Determine the exact amount of acid and conjugate base needed to make
a buffer of a certain pH, using the Henderson-Hasselbach equation:
pH=pKa+log([A−][HA])

Handerson-Hasselbalch Equation
Preparation of Acid Buffer
• Consider an acid buffer solution,
containing a weak acid (HA) and its
salt (KA) with a strong base(KOH).
Weak acid HA ionizes, and the
equilibrium can be written as-
• HA + H2O ⇋ H+ + A−
• Acid dissociation constant = Ka = [H+]
[A–]/HA
• Taking, negative log of RHS and LHS:
Preparation of Base Buffer
• Consider base buffer solution,
containing a weak base (B) and its salt
(BA) with strong acid.
pOH, can be derived as above,
• pOH of a basic buffer = pKb + log ([salt]/[acid])
• pH of a basic buffer = pKa – log ([salt]/[acid])
Significance of Handerson Equation
Handerson Equation can be used to:
1. Calculate the pH of the buffer prepared
from a mixture of the salt and weak
acid/base.
2. Calculate the pKa value.
3. Prepare buffer solution of needed pH

pH= 𝒑𝑲𝒂 + 𝒍𝒐𝒈
[𝒄𝒐𝒏𝒋𝒖𝒈𝒂𝒕𝒆 𝒃𝒂𝒔𝒆]
[𝒘𝒆𝒆𝒌 𝒂𝒄𝒊𝒅]
pOH= 𝒑𝑲𝒃 + 𝒍𝒐𝒈
[𝒄𝒐𝒏𝒋𝒖𝒈𝒂𝒕𝒆 𝒂𝒄𝒊𝒅]
[𝒘𝒆𝒆𝒌 𝒃𝒂𝒔𝒆]
For week Acid
For week Base

Uses of Buffer Solutions
1. There exists a few alternate names that are used to refer buffer
solutions, such as pH buffers or hydrogen ion buffers.
2. An example of the use of buffers in pH regulation is the use of
bicarbonate and carbonic acid buffer system in order to regulate the
pH of animal blood.
3. Buffer solutions are also used to maintain an optimum pH for
enzyme activity in many organisms.
4. The absence of these buffers may lead to the slowing of the enzyme
action, loss in enzyme properties, or even denature of the enzymes.
This denaturation process can even permanently deactivate the
catalytic action of the enzymes.

Part – II
Fundamental of Enzymology

Enzymology
Enzymology is the study of enzymes, their kinetics, structure, and function, as well as
their relation to each other.
Enzymes - Biological catalysts
• By definition a Catalyst:- Accelerates the rate of chemical reactions
• Capable of performing multiple reactions (recycled)
• Final distribution of reactants and products governed by equilibrium properties
• Enzymes are biological catalysts:- Proteins, (a few RNA exceptions)- Orders of
magnitude faster than chemical catalysts- Act under mild conditions (temperature
and pressure)- Highly Specific- Tightly Regulated.
• A catalyst is defined as a substance that increases the velocity or rate of a chemical
reaction without itself undergoing any change in the overall process.
Enzymes may be defined as biocatalysts synthesized by living cells. They are protein
in nature, colloidal and thermolabile in character, and specific in their action. In
recent years, certain non-protein enzymes (chemically RNA) have also been
identified.

Nomenclature and Classification:
Enzymes are sometimes considered under
two broad categories:
Intracellular enzymes:
• They are functional within cells where
they are synthesized.
Extracellular enzymes:
• These enzymes are active outside the cell;
all the digestive enzymes belong to this
group.

The International Union of Biochemistry (IUB-1961)
The International Union of
Biochemistry (IUB)
appointed an Enzyme
Commission in 1961.
• This committee made a
thorough study of the
existing enzymes and
devised some basic
principles for the
classification and
nomenclature of enzymes.
• Since 1964, the IUB
system of enzyme
classification has been in
force. Enzymes are divided
into six major classes (in
that order).

1. Oxidoreductases: Enzymes
involved in oxidation-
reduction reactions.
2. Transferases: Enzymes that
catalyse the transfer of
functional groups.
3. Hydrolases: Enzymes that
bring about hydrolysis of
various compounds.
4. Lyases: Enzymes specialised
in the addition or removal of
water, ammonia, CO2 etc.
5. Isomerases: Enzymes
involved in all the
isomerization reactions.
6. Ligases: Enzymes catalysing
the synthetic reactions
(Greek: ligate—to bind)
where two molecules are
joined together and ATP is
used.

Chemical Nature of Enzymes
• All the enzymes are invariably proteins. In recent years,
however, a few RNA molecules have been shown to
function as enzymes. Each enzyme has its own tertiary
structure and specific conformation which is very
essential for its catalytic activity.
• The functional unit of the enzyme is known as
holoenzyme which is often made up of: -
• Apoenzyme (the protein part) and
• Coenzyme (non-protein organic part).
Holoenzyme (active enzyme) → Apoenzyme (protein
part) + Coenzyme (non-protein part).
The term prosthetic group is used when the non-protein
moiety tightly (covalently) binds with the apoenzyme. The
coenzyme can be separated by dialysis from the enzyme
while the prosthetic group cannot be.
According to Holum, the
cofactor may be:
1. A coenzyme - a non-
protein organic substance
which is dialyzable,
thermostable and loosely
attached to the protein part.
2. A prosthetic group - an
organic substance which is
dialyzable and thermostable
which is firmly attached to
the protein or apoenzyme
portion.
3. A metal-ion-activator -
these include K+, Fe++,
Fe+++, Cu++, Co++, Zn++,
Mn++, Mg++, Ca++, and
Mo+++.

Factor affecting of enzyme Activity
The contact between the enzyme and substrate is the most essential pre-
requisite for enzyme activity. The important factors that influence the
velocity of the enzyme reaction are discussed here under.
1. Concentration of enzyme: As the concentration of the enzyme is
increased, the velocity of the reaction proportionately increases.
2. Concentration of substrate: Increase in the substrate concentration
gradually increases the velocity of enzyme reaction within the limited
range of substrate levels.
3. Order of reaction: When the velocity of the reaction is almost
proportional to the substrate concentration (i.e. [S] is less than Km,), the
rate of the reaction is said to be first order with respect to substrate.
When the ISJ is much greater than Km, the rate of reaction is
independent of substrate concentration, and the reaction is said to be
zero order. (depending on Km)

Enzyme Kinetics
Enzyme kinetics and K, value: The enzyme (E) and
substrate (S) combine with each other to form an
unstable enzyme-substrate complex (ES) for the
formation of product (P).
Here kl, k2 and k3 represent the velocity constants
for the respective reactions, as indicated by arrows.
Km the Michaelis-Menten constant (or Brigrs and
Haldane's constant), is given by the formula.
The following equation is obtained after suitable
algebraic manipulation
Where v= Measured velocity
VMax = Maximum velocity,
S= Substrate concentration,
Km= Michaelis- Menten constant
Let us assume that the measure velocity (v) is equal to1/2 VMax. Then
the equation (1) may be substituted as follows
K stands for a constant and m
stands for Michaelis (in Km.)
Km or the Michaelis-Menten constant is defined as the
substrate concentration (expressed in moles/L) to produce half-
maximum velocity in an enzyme catalyzed reaction. Lt indicates
that half of the enzyme molecules (i.e. 50%) are bound with the
substrate molecules when the substrate concentration equals the
Km value.
1. Km value is a constant and a characteristic feature of a given enzyme.
2. It is a representative for the measuring the strength of ES complex.
3. A low Km value indicates a strong affinity between enzyme and substrate,
whereas a high Km value reflects a weak affinity between them.
4. For majority of enzymes, the Km. values are in the range of 10-5 to 10-2
moles. It may however, be noted that Km is not dependent on the
concentration of enzyme.

Lineweaver-Burk
Lineweaver-Burk double reciprocal plot: For the
determination of K, value, the substrate saturation
curve (Fig 6.2) is not very accurate since VMax is
approached asymptotically. By taking the
reciprocals of the equation (1), a straight line
graphic representation is obtained.
Therefore, a plot of the
reciprocal of the velocity
(1/v) vs. the reciprocal of
the substrate concentration
(1/[s]) gives a straight line.
Here the slope is Km/VMax
and whose y intercept is 1/
VMax.
The Lineweaver-Burk plot is
shown in Fig. 6.3. it is much easier
to calculate the Km from the
intercept on x-axis which is -(l/Km).
Further, the double reciprocal plot
is useful in understanding the effect
of various inhibitions

4. Effect of Temperature
• Each enzyme shows its highest activity
at a particular temperature called the
optimum temperature.
• Enzyme activity declines both below
and above the optimum value.
• Low temperature preserves the enzyme
in a temporarily inactive state whereas
high temperature destroys enzymatic
activity because proteins are denatured
by heat.
5. Effect of pH
• Each enzyme has a pH value that it works at
with maximum efficiency called the optimal pH.
• If the pH is lower or higher than the optimal pH,
the enzyme activity decreases until it stops
working.
• For example, Acid phosphatase works at a low
pH, i.e, it is highly acidic, while Alkline
phosphatase works at a high pH, i.e, it is basic.
Most enzymes work at neutral pH 7.4 (Salivary
Amylase).

6. Effect of Activators
• Some of the enzymes
require certain inorganic
metallic cations, like Mg2+,
Mn2+, Zn2+, Ca2+, Co2+,
Cu2+, Na+, K+ etc., for their
optimum activity. Rarely,
anions are also needed for
enzyme activity, e.g. a
chloride ion (CI–) for
amylase.

Nature of Enzyme Action
Enzymes are very specific and it was suggested by Fischer in 1890 that this was
because the enzyme had a particular shape into which the substrate or substrates
fit exactly.
E+ S→ ES→ EP→ E+ P
1. The substrate binds to the active site of the enzyme, fitting
into the active site (E+S).
2. The binding of the substrate induces the enzyme to alter its
shape, fitting more tightly around the substrate (ES).
3. The active site of the enzyme, now in close proximity of
the substrate breaks the chemical bonds of the substrate
and the new enzyme-product complex (EP) is formed.
4. The enzyme releases the products of the reaction (E+P)
and the free enzyme is ready to bind to another molecule
of the substrate and run through the catalytic cycle once
again. Biochemistry by Dr. Kirpa Ram

Model
According to this model, shape of active site of enzyme
is complementary to the shape of substrate molecules. Ie.
the substrate is like a key whose shape is complementary
to the enzyme which is supposed to be lock and they fit
perfectly.
1. Enzymes catalyze only those substrates which fit
perfectly on the active site of that enzyme.
2. Most enzymes are far larger than the substrates
molecules that act on and the active site is usually a
very small portion of the enzyme, between 3 and 12
amino acids. The remaining amino acids which make
the bulk of the enzyme, function to maintain the
correct globular shape of the enzyme.
3. Once the product is formed, they no longer fit into
the active site and escape into surrounding medium.
4. According to lock and key model, enzymes behave
as rigid molecules. However, most enzymes are
globular and are flexible with varying shape.
Lock and Key Mechanism

Induced fit Mechanism
In 1959, Koshland suggested a modification to the ‘Lock and Key’ hypothesis which is
known as ‘Induced fit’ hypothesis.
1. Working from evidence that suggested that some enzymes and their active site are more
flexible. To this, he proposed that the active site can modify its shape as the substrate
interact with the enzyme.
2. The amino acids which make up the active site are mounded into precise shape which
enable the enzyme to perform its catalytic function most efficiently.
3. For instance, a suitable analogy to describe Induced fit model would be that of a hand
changing the shape of the glove as the individual put on the glove. Therefore in this case,
glove is the active site of enzyme and the hand is substrate
Model
4. However, in some cases, the
substrate molecules changes
slightly as it enters the active
site before binding.

Substrate strain theory
• In this model, the substrate is strained due to the induced conformation
change in the enzyme.
• It is also possible that when a substrate binds to the preformed active site, the
enzyme induces a strain to the substrate. The strained substrate leads to the
formation of product.
• In fact, a combination of the induced fit model with the substrates train is
considered to be operative in the enzymatic action.

Active Site
The active site is region of an enzyme where
substrate molecules bind and undergo a chemical
reaction. The active site consists of amino acid
residues that form temporary bonds with the
substrate (binding site) and residues that catalyse
a reaction of that substrate (catalytic site).
• Active site occupies only ~10–20% of the
volume of an enzyme.
• Enzymes bind substrates to their active site and
stabilize the transition state of the reaction.
• The active site of the enzyme is the place where
the substrate binds and at which catalysis
occurs.
• The active site binds the substrate, forming an
enzyme-substrate(ES) complex.Biochemistry by Dr. Kirpa Ram

Salient Features of Active Site
1. The existence of active site is due to the tertiary structure of protein resulting in
three dimensional native conformation.
2. The active site is made up of amino acids (known as catalytic residues) which
are far from each other in the linear sequence of amino acids (primary structure
of protein).
3. The active site is not rigid in structure and shape. It is rather flexible to promote
the specific substrate binding.
4. Generally, the active site possesses a substrate binding site and a catalytic site.
The latter is for the catalysis of the specific reaction.
5. Of the 20 amino acids that could be present in enzyme structure, only some of
them are repeatedly found at the active sites of various enzymes. These amino
acids are serine, aspartate, histidine, cysteine, lysine, arginine, glutamate,
tyrosine etc. Among these amino acids, serine is the most frequently found.
6. The substrate [S] binds the enzyme (E) at the active site to form enzyme-
substrate complex (ES). The product (P) is released after the catalysis and the
enzyme is available for reuse. Biochemistry by Dr. Kirpa Ram

Enzyme regulation
Enzymes can be regulated by other molecules that either
increase or reduce their activity. Molecules that increase the
activity of an enzyme are called activators, while molecules
that decrease the activity of an enzyme are called
inhibitors.
1. Regulatory molecules. Enzyme activity may be turned
"up" or "down" by activator and inhibitor molecules
that bind specifically to the enzyme.
2. Cofactors. Many enzymes are only active when bound
to non-protein helper molecules known as cofactors.
3. Compartmentalization. Storing enzymes in specific
compartments can keep them from doing damage or
provide the right conditions for activity.
4. Feedback inhibition. Key metabolic enzymes are often
inhibited by the end product of the pathway they
control (feedback inhibition).
“Process, by which cells can turn on, turn off, or modulate the activities of various
metabolic pathways by regulating the activity of enzyme”

Enzyme Inhibition
Enzyme inhibitor is defined as a substance which binds with the enzyme
and brings about a decrease in catalytic activity of that enzyme.
The inhibitor may be organic or inorganic in nature.
There are two broad categories of enzyme inhibition:
I. Reversible inhibition.
II. Irreversible inhibition.

1. Reversible Inhibition
The inhibitor binds non-covalently with enzyme and the enzyme inhibition can be reversed if the inhibitor is
removed. The reversible inhibition is further sub-divided into:
I. Competitive inhibition
• Inhibitor bind to active site of enzyme and block binding of the substrate has a similar shape to the
substrate molecule
• Competitive Inhibition is usually temporary and depends on the relative concentrations of substrate
II. Non-competitive inhibition
• Inhibitor doesn’t block the substrate from binding to the active sitebut block a site other than the active
site
• Binding of the inhibitor causes a conformational change to the enzyme’s active site
• The active site and substrate no longer share specificity, meaning the substrate cannot bind and
inhibitor is not in direct competition with the substrate, increasing substrate levels cannot mitigate the
inhibitor’s effect
III. Allosteric Inhibition
• Alosteric means other site- These enzymes have two receptor sites, One site fits the substrate like other
enzymes one other site fits an inhibitor and Accumulation of the final product of the reaction is capable of
inhibiting the step of reaction

Feedback Inhibition
• Feedback inhibition is a cellular control mechanism in which an enzyme’s activity is
inhibited by the enzyme’s end product.
• This mechanism allows cells to regulate how much of an enzyme’s end product is produced.
1. In end-product inhibition, the final product in a series
of reactions inhibits an enzyme from an earlier step
in the sequence
2. The product binds to an allosteric site and
temporarily inactivates the enzyme (via non-
competitive inhibition)
3. As the enzyme can no longer function, the reaction
sequence is halted and the rate of product formation
is decreased.
4. If product levels build up, the product inhibits the
reaction pathway and hence decreases the rate of
further product formation
5. If product levels drop, the reaction pathway will
proceed unhindered and the rate of product formation
will increase

Irreversible Inhibition
• The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible.
These inhibitors are usually toxic substances which may be present naturally or man-made.
• Iodoacetate is an irreversible inhibitor of the enzymes like papain and glyceraldehyde 3-
phosphate dehydrogenase, Iodoacetate combines with sulfhydryl (-SH) groups at the active
site of these enzymes and makes them inactive. Diisopropyl fluorophosphate (DFP) is a
nerve gas developed by the Germans during Second World War. DFP irreversibly binds
with enzymes containing serine at the active site, e.g. serine proteases, acetylcholine
esterase.
• Suicide inhibition: In this type of irreversible inhibition, the original inhibitor is converted
to a more potent form by the same enzyme that should to be inhibited e.g., allopurinol, an
inhibitor of xanthine oxidase, gets converted to alloxanthine, a more effective inhibitor of
the enzyme.

Cofactors
• Enzymes are composed of one or several
polypeptide chains (Proteins).
• The non protein part of the enzymes is called
cofactor, which make the enzyme
catalytically active.
• If the cofactor is removed from a complete
enzyme (holoenzyme), the protein component
(apoenzyme) no longer has catalytic activity.
• A cofactor that is firmly bound to the
apoenzyme and cannot be removed without
denaturing the latter is termed a prosthetic
group; most such groups contain an atom of
metal such as copper or iron.

CO-FACTORS
CO-ENZYME
PROSTHETIC
GROUP
METAL ION
ENZYME SIDE GROUPS

Coenzyme
• The non-protein, organic, low
molecular weight and dialysable
substance associated with enzyme
function is known as coenzyme.
• Their association with the apoenzyme
is only transient, usually occurring
during the course of catalysis.
• The essential chemical component of
many coenzymes are vitamins, e.g.,
coenzyme nicotinamide adenine
dinucleotide (NAD) and NADP
contain the vitamin niacin.

Coenzymes play a decisive role in enzyme function
• Coenzymes from B-complex vitamins: Most of the coenzymes are the
derivatives of water soluble B-complex vitamins. In fact, the
biochemical functions of B-complex vitamins are exerted through their
respective coenzymes.
• Non-vitamin coenzymes: Not all coenzymes are vitamin derivatives.
There are some other organic substances, which have no relation with
vitamins but function as coenzymes. They may be considered as non-
vitamin coenzymes e.g. ATP, CDP, and UDP etc.
• Nucleotide coenzymes: Some of the coenzymes possess nitrogenous
base, sugar and phosphate. Such coenzymes are, therefore, regarded as
nucleotides e.g. NAD+, NADP+, FMN, FAD, coenzyme A, UDPG etc.

Prosthetic group
• A prosthetic group is a tightly bound,
specific non-polypeptide unit required for
the biological function of some proteins.
• The prosthetic group may be organic
(such as a vitamin, sugar, or lipid) or
inorganic (such as a metal ion), but is not
composed of amino acids.
• In peroxidase and catalase, which
catalyze the breakdown of hydrogen
peroxide to water and oxygen, haem is
the prosthetic group.

Metal Ions
• A number of enzymes require metal
ions for their activity.
• Zinc is a cofactor for the proteolytic
enzyme carboxypeptidase.
• Some of the enzymes require
certain inorganic metallic cations,
like Mg2+, Mn2+, Zn2+, Ca2+, Co2+,
Cu2+, Na+, K+ etc., for their
optimum activity.

Isoenzymes
• Isozymes (also known as isoenzymes or more generally as
multiple forms of enzymes) are enzymes that differ in amino
acid sequence but catalyze the same chemical reaction.
• These enzymes usually display different kinetic parameters
(e.g. different KM values), or different regulatory properties.
• Isozymes were first described by R. L. Hunter and Clement
Markert (1957).
• Who defined them as different variants of the same enzyme
having identical functions and present in the same
individual.
• A single gene can make different variations of the enzyme called
allozymes.
• Different genes can make different variations of the enzyme called
isozymes.

They, differ in their physical and
chemical properties which include
• The Structure,
• Electrophoretic and Immunological
Properties,
• Km and Vmax Values,
• Ph Optimum,
• Relative Susceptibility to Inhibitors
and
• Degree Of Denaturation.
e.g. lactate dehydrogenase (5 isoenzymes),
creatine kinase (3 isoenzymes).

Principle of biochemistry and fundamental of enzymology

Principle of biochemistry and fundamental of enzymology

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