Course Title: Biochemistry

Course Code: Biol.

Credit hour: 3(2+1)
Instructor: Hiwot worku

September, 2024
Haramaya University
 Assignment Question (15%)
1.Briefly explain about mechanism Biosynthesis of Carbohydrate in plants
and bacteria
A. Photosynthetic carbohydrate syntheses
B. Photorespiration and C4 and CAM pathways
C. Biosynthesis of starch and sucrose
D. Synthesis of plant and bacterial cell wall polysaccharides
Explain about Lipid biosynthesis
A. Biosynthesis of fatty acids and triglycerides
B. Biosynthesis of membrane phospholipids
C. Biosynthesis of cholesterol, steroids and isoprenoids
Discuss about the Biosynthesis of amino acids, nucleotides,
and other nitrogen containing compounds
A. Biosynthesis of amino acids
B. Biosynthesis and degradation of nucleotides
C. Regulation of nitrogen metabolism
 2.An overview of the cellular foundation of life

 cells is the microscopic units of all living things.

 They are structural, functional and biological units of all living
matter.
 Cells generally vary in size, shape, composition and function.
Cell size
 Plant cells (10 to 100 μm in diameter)
 Animal cells (5 to 30μm in diameter)
 Bacteria (1 - 2 μm in diam. But mycoplasmas =300 nm )

What are Organelles?

 Unit 3: Objectives

Describe the molecules of life

(Biomolecules)
Differentiate Amino acids, Peptides and
Proteins the structure and function.
Explain how Methods to study proteins
 3.The molecules of life (Biomolecules)

 A biomolecule, often known as a biological molecule.

 Large macromolecules like
proteins
carbohydrates
 lipids and
nucleic acids
tiny primary metabolites
 secondary metabolites are all biomolecule.
 Biomolecules are vital components of all living
things.
 Composition and Structure

 Carbon is the most abundant element in

biomolecules.
 H, O, N, P and S are also essential elements.
 Biomolecules have the same types of functional
groups as organic molecules.
 They have two categories based on molecular
weight and solubility.
 Micro molecule and Macromolecule.
 Amino acids, Peptides and Proteins

 Proteins are the most abundant biological macromolecules.

 Occurring in all cells and all parts of cells.
 Proteins also occur in great variety.
 Proteins are the molecular instruments through which genetic
information is expressed.
 All proteins are made up of different arrangements of the
same 20 amino acids.
 From these building blocks different organisms can make
such widely diverse products as
 enzymes,
 hormones
 Antibodies Etc…
 Cont…
 Amino acids are the monomers that make up proteins.
 Each amino acid has the same fundamental structure.
 consists of a central carbon atom bonded to an amino
group (NH2), a carboxyl group (COOH), a hydrogen
atom, and a variable “R” group.
 Cont…
 The same 20 common amino acids are present in proteins
from all species of life.
 Ten of these are considered essential amino acids in
humans.
 Because the human body cannot produce them and they
must be obtained from the diet.
 The chemical nature of the R group determines the nature
of the amino acid.
 For example, amino acids such as valine and methionine,
are nonpolar or hydrophobic in nature.
Structure of 20 amino acids
 Conti…
 Amino acids are linked together into linear chains
called polypeptides.
 Amino acids are attached to other amino acids by
covalent bonds, known as peptide bonds.
 formed by dehydration synthesis reactions.
 polypeptide chain has a peptide backbone (or carbon-
nitrogen backbone).
 Protein sequence and evolution
 the primary sequence of a protein dictates its
fold and function.
 cumulative changes in this sequence lead to
the evolution of protein structures and
functions.
 Protein Structure

1.Primary Structure
 The unique sequence of amino acids in a polypeptide chain is its
primary structure.
 Cont…
2.Secondary Structure
 The local folding of the polypeptide in some regions gives rise to the

secondary structure of the protein.

 The most common are the α-helix and β-pleated

sheet structures.
 Both structures are formed by hydrogen bonds
forming between parts of the peptide.
 Specifically, the oxygen atom in the carbonyl group
in one amino acid interacts with another amino acid
Secondary Structure
 Cont…
3.Tertiary Structure
 The unique three-dimensional structure of a polypeptide is
its tertiary structure.
 This structure is primarily due to interactions among R
groups.
 Tertiary structures are held together by several different types of side-chain
interactions.
 ionic interactions
 hydrophobic interaction (stronger in the centre
of the protein),
 hydrogen bonds stabilizing folding, and
 disulfide bridges.
Tertiary structure of proteins
 Cont…
4.Quaternary Structure
 In nature, some proteins are formed from several separate
polypeptides, known as subunits.

 The interaction of these subunits forms the quaternary

structure of a protein.

 Weak interactions between the subunits help to stabilize the

overall structure.
 Denaturation and Protein Folding

 Any physical or chemical agent that destroys these

stabilizing structures changes the conformation of the
protein.
 If a protein is subject to changes in temperature, pH, or exposure to
chemicals, it may lose its shape, a process called denaturation.
 Different proteins denature under different conditions.
Modes of Protein Denaturation (Destruction
of Secondary and Higher Structures)
 Methods to study proteins
 Protein purification isolates a specific protein of interest from a complex
mixture of biological material.
 To obtain a highly pure form of the desired protein while removing
contaminants

 What are the Objectives of Protein Purification?

 The primary objectives of protein purification are as follows:

1.Obtain high purity in samples

2. Separate from contaminants
3. Maintain protein integrity
4. Quantify and concentrate
5. Reliable and reproducible results
 Cont…
 Here's an overview of common techniques used in each category

A. Cell Lysis and Extraction

 Mechanical disruption (e.g., sonication, homogenization)

 Chemical lysis (e.g., detergents, enzymes)

 Each extraction method offers distinct advantages and

limitations.
 The choice of method depends on

 the type of cells being used,

 the properties of the target protein and

 the subsequent purification steps planned.

 Conti…
Homogenization:
 This technique employs mechanical shear force to break open cells.
 It's highly effective for tough plant and animal tissues and it is
scalable.
 The process can generate heat, which may denature sensitive
proteins.
Sonication
 Utilizes ultrasonic waves to disrupt cell membranes.
 This is a quick approach commonly used for bacterial cells.
 It is very effective for small volumes.
 It requires cooling to prevent protein denaturation due to the
generated heat.
 Cont…
Detergents
 Solubilize cell membranes by disrupting lipid bilayers.
 Some common detergents include Triton X-100 and SDS.
 This process is easy to use and very effective for
membrane proteins.
 It may require removal before further purification.
Enzymatic treatment
 lysozymes, are used to break down bacterial cell walls.
 This approach is very specific and maintains protein
integrity
 requires additional steps for a complete cell lysis.
 Protein purification techniques
 Protein purification involves several techniques
1. Centrifugation
 separates components based on their density by spinning
samples at high speeds.

 During the process, heavier particles, such as cellular debris,

sediment at the bottom,

 allowing the lighter supernatant, which contains the proteins,

to be collected.

 This technique is often the first step in purification

 Cont…
2. Precipitation
 involves altering the solubility of proteins to cause them to
aggregate and precipitate out of solution.
 This can be achieved using salts (salting out).
 Ammonium sulfate, organic solvents or changes in pH.
3. Chromatography

 Comprises of a varied set of versatile and widely used purification

techniques.

 can be used to separate proteins based on various properties.

 Cont…
Affinity chromatography:- exploits specific interactions
between a protein and a ligand attached to a resin.
 This technique is suitable for proteins with known binding
partners or tags.
Ion exchange chromatography:- separates proteins based on
their charge.
Size exclusion chromatography (SEC) :- ) separates
proteins based on size by passing them through a column
filled with porous beads.
 This technique is usually used for separating monomers
from aggregates
 Cont…

4. Ultrafiltration and dialysis

 uses semipermeable membranes to concentrate and desalt
protein solutions by applying pressure.
 dialysis is a process that involves placing the protein
solution inside a membrane with selective permeability.
 Allowing small molecules to diffuse out to a surrounding
solution.
 These methodologies are used to remove small
contaminants.
 Exchange buffers and concentrate proteins.

.
 Cont…

5.Electrophoresis
 separates proteins through a gel matrix based on size and
charge.
 by applying an electric field

 One of the most common variants is SDS-PAGE.

 separate them by size through a polyacrylamide.

 Electrophoresis both a technique for protein separation and

the analytical assessment of protein purity and molecular

 Methods and Techniques for Protein Sequencing

 What is Protein Sequencing?

A. Edman Degradation

 One of the earliest methods for protein sequencing.

 This method selectively removes and identifies the N-terminal
amino acid of a protein.
Use Phyenylisothiocynate reagent
 Need large quantities of pure protein and challenges with repetitive
sequences.
B.Mass Spectrometry
 Emerged as a powerful tool for protein sequencing.
 Conti…
 It involves ionizing protein fragments and measuring their mass-to-
charge ratios.
 Enable the identification of peptides and their sequences.
 Offers high sensitivity and can handle complex mixtures of
proteins.

C. Next-Generation Sequencing
 Have expanded their application beyond genomics to proteomics.

 NGS-based methods, like RNA-seq and ribosome profiling.

 Relies on mRNA data and may not provide direct protein sequence information.
 Protein detection

 Protein detection :-is used for clinical diagnosis, treatment and

biological research.
 Detection evaluates the concentration and amount of different proteins in
a particular specimen.
 There are different methods and techniques to detect protein in different
organisms.
1. Western Blotting: Uses antibodies to detect specific proteins after
separation by gel electrophoresis.
2. ELISA (Enzyme-Linked Immunosorbent Assay) : Uses antibodies and
color change to detect the presence of a protein.
3. Fluorescence-Based Methods: Fluorescence Microscopy: Uses
fluorescently labeled antibodies or tags to detect proteins in cells or
 Unit: 4 OBJECTIVES
• Defining the Enzymes
• Differentiate types of Enzymes, How
Enzymes Work
• Explain Enzyme kinetics
• Explain Mechanism of Regulation of enzyme
catalyzed reactions
 4.Enzymes

Definition
A reusableprotein molecule that brings
about chemical change while remaining
unchanged itself.
 Enzymes, the catalysts of biological systems.
 Catalysis takes place at a particular site on the
enzyme called the active site.
 Properties of Enzymes
 Catalyst - speeds up attainment of reaction
equilibrium
 Enzymatic reactions - 103 to 1017 faster than the
corresponding uncatalyzed reactions
 Substrates - highly specific reactants for enzymes
 Stereospecificity - many enzymes act upon only
one stereoisomer of a substrate
 Reaction specificity - enzyme product yields are
essentially 100% (there is no formation of wasteful
byproducts)
 Active site - where enzyme reactions take place
 Conti…
 Nearly all known enzymes are proteins.
 However, some enzymes Ribozyme origin of
RNA.
A. Characteristics of Enzymes
– Highly Specific
– Sensitive to the environment
– May function in enzyme systems or pathways
– Enzyme names end in –”ase”
 C. Classification of Enzymes
Enzyme Classification
 Basedon where the enzyme activity occurs.
– Exoenzymes
– Endoenzymes
 Based on type of enzyme function

-The six major enzyme categories are

1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases
 How Enzymes Work
Mechanism of activity

– Active Site

– Substrate

–Products
_Enzymes work by lowering the activation
energy.
 Enzyme kinetics
 The study of the chemical reactions that are catalysed by
enzymes.
 In enzyme kinetics, the reaction rate is measured and how
get changes in response to changes in experimental
parameters.
 Such as substrate concentration, enzyme concentration etc.
 The initial rate (or initial velocity), designated V0, when
[S] is much greater than the concentration of enzyme [E]
 can be measured by Michaelis–Menten kinetics.
 It is one of the simplest and best-known models of enzyme
kinetics.
 A General Enzymatic reaction

V0 is determined by the breakdown of ES to form product, which is determined by [ES]:

Where as k1= rates of formation of ES, K-1 + k2 = breakdown of ES

Rate of formation of [ES] =k1[E] [S]
Rate of dissociation of [ES]= k-1[ES] + k2[ES]
So in the steady sate , k1[E] [S]= k-1[ES] + k2[ES] ------------- 3
[E] [S]/[ES] = (k-1 + k2)/k1 ------------- 4
k-1 + k2)/k1 =Km
Note# Here E, S, ES and P symbolize the enzyme, substrate, enzyme-substrate complex and products
respectively. Note# Km is called Michaelis constant, it is a substrate concentration at which V0= 1⁄2Vmax
and half of the active sites on the enzymes are filled . Different enzymes have different Km values. They
typically range from 10-1 to 10-7 M. The unit of Km is concentration. Km is affected by: pH, temperature,
ionic strengths and the nature of the substrate. If V0= 1⁄2Vmax, then Km=[S]. Km is a measure of a
substrate’s affinity for the enzyme.
 Cont…
•Since the enzyme is not consumed, the conservation equation on the
•Enzyme yields: [E] = [E0]-[ES]
•Km= [E] [S]/[ES] ------------- 5
•So putting the value of [E] in to the equation no -5
•Km= [E0]-[ES] [S]/[ES]
•[ES]= [E0]-[ES] [S]/ Km
•So finally, [ES]= [E0][S] / Km +[S] -----------6
•Then putting the value of [ES] from equation no-5 to equation no -2, the rate V0 can be expressed in terms of
[S].
•V0 = k2 [E0][S] / Km +[S]-------------7
•Vmax= k2 [E0]
•Finally, V0 = Vmax [S] / Km +[S]-------------8
•The equation -8 is called the Michaelis-Menten equation, for a one-substrate enzyme-catalyzed reaction. Note
# when V0 is exactly one-half Vmax:
•V0/2 = Vmax [S] / Km +[S]
•Graphical Representation of Michaelis-Menten equation
 Cont…
 Lineweaver-Burk plot, has the great advantage of allowing a
more accurate determination of Vmax, and Km.
 D. Influences on Enzyme Activity

enzymes work best at an optimum

 Temperature:

temperature.

 An increase in temperature provides more kinetic energy

to the molecules involved.

 The numbers of collisions between enzyme and substrate

will increase so the rate will too.

 Above the optimum temperature, and the enzymes are

denatured.

 Bonds holding the structure together will be broken and

the active site loses its shape and will no longer work.
Temperature
 The effect of change in pH.

 pH: as with temperature, enzymes have an

optimum pH.
 If the pH changes much from the optimum, the
chemical nature of the amino acids can change.

This may result in a change in the bonds and so

the tertiary structure may break down.
 The active site will be disrupted and the enzyme
will be denatured.
pH.
 The effect of change in
concentration
 Enzyme concentration

 At low enzyme concentration there is great competition for the

active sites and the rate of reaction is low.
 As the enzyme concentration increases, there are more active

sites and the reaction can proceed at a faster rate.

 increasing the enzyme concentration beyond a certain point has

no effect.
 Because the substrate concentration becomes the limiting factor.
 Cont…
 Substrate concentration

 At a low substrate concentration there are many active sites that are

not occupied.
 This means that the reaction rate is low.

 When more substrate molecules are added, more enzyme-substrate

complexes can be formed.

 As there are more active sites, and the rate of reaction increases.

 increasing the substrate concentration yet further will have no effect.

 The active sites will be saturated so no more enzyme-substrate

complexes can be formed.

Substrate concentration
 Inhibitors on Enzyme Activity (Competitive and Non-
Copetitive Inhibitors )
Inhibitor: Any molecule that acts directly on an enzyme to lower
its catalytic rate. These can be cellular metabolites, or foreign
substances such as drugs or toxins that have either a therapeutic
or toxic (can be lethal) effect.
There are two major types of inhibition:

(1) Irreversible inhibition

(2) Reversible inhibition
a) Competitive
b) Un-competitive
c) Mixed
 Cont…
(a)Competitive inhibition:

Inhibitor has close structural similarities to the normal substrate

and therefore competes with the substrate for the active site.
 Cont…
• An uncompetitive inhibitor binds at a site other than the active
site and, binds only to the ES complex
 Cont….
Mixed
• Inhibitor binds at a site other than the active site (E or ES) and
causes changes in the overall 3-D shape of the enzyme that
leads to a decrease in activity
 Regulation of enzyme catalyzed reactions

 The activity of proteins, including enzymes, often must be

regulated.
 So, that they function at the proper time and place.
 The rates of enzyme-catalyzed reactions are altered by

activators and inhibitors (a.k.a. effector molecules).

 The biological activity of proteins is regulated in four

principal ways:
 Cont…

(1) Allosteric enzymes:

 have more than one site, where effector binding at one site induces a

conformational change in the enzyme.

 Altering its affinity for a substrate.

 An allosteric activator increases enzyme rate of activity,

 an allosteric inhibitor decreases its activity.

Regulation mechanism:

Reversible, noncovalent binding of allosteric effectors.

Covalent modification (phosphorylation, adenylation, etc.).

Binding by separate regulatory proteins.

 Cont…

(2) Feedback inhibition: An enzyme,

early in the metabolic pathway, is
inhibited by an end-product. Often
takes place at the committed step
of the pathway, the step which commits
a metabolite to a pathway.
 Cont…
(3 )Reversible Covalent Modification
is the making and breaking of a covalent bond between a
non-protein group and an enzyme that affects its activity.
 Cont…
4.Proteolytic activation:
 Some enzymes are synthesized as larger inactive.

 precursor forms called proenzymes or zymogens.

 Activation involves the irreversible hydrolysis of one or more peptide bonds, resulting in an active form.

 The enzymes controlled by some of these mechanisms cycle between active and inactive states.

 A different regulatory motif is used to irreversibly convert an inactive enzyme into an active one.

.
 Cont…
In this example, A activates B, B activates C, D, and E, C activates F and G, D
activates H and I, and E activates K and L. Cascades also help to serve as
control points for certain process. In the protease cascade, the activation of B
is really important because it starts the cascade.