UNIT 3 ENZYMES

What are enzyme like?

 Enzymes are protein molecules that act as biological
catalysts (biocatalysts) and accelerate rate of chemical
reactions by lowering activation energy.
 Activation energy is the minimum amount of energy
required for the reactant to be converted to products.
 requires Energy (ATP)/Endorgonic
 generates Energy (ATP)/Exorgonic All cells contain
different enzymes depending on the type of the living
cell, which engage in biochemical activity called
metabolism.
 The metabolic processes in the cells require enzymes to
catalyze many biochemical reaction types at a rates fast
enough to sustain life.
 Enzymes act upon molecules (substrates), convert them
into products of different molecules, and remain
1 unchanged
Properties and Functions of Enzymes
Properties and Functions of Enzymes
• The general properties of enzymes are the
nature of both their physical and chemical
properties.
• They neither affect the nature of products
formed nor undergo any changes by the
reaction catalyzed.
A. The physical properties of enzymes
• The physical properties of enzymes include
1) Denaturation 5) Precipitation
2) Solubility 4) Biocatalysts
3) Colloids 6) Molecular weight
2
7) Enzyme activity
1) Denaturation is the process of breaking the intra and
inter-molecular noncovalent bonds that distort the
shape and active site of the enzymes.
• Enzymes are denatured by high heat (above 40ºC),
alternation in the pH (too low or too high), heavy metals
and high salt concentrations, solvents and other reagents.
2) Solubility is the property of enzymes that allow them to
be dissolved in water, diluted glycerol and alcohol causing
denaturation.
3) The colloidal nature of enzyme is the tendency of
having little or no dialysis cross the semipermeable
membrane due to the large size or high molecular
weight.
4) The biocatalyst property is the activity of enzymes in
which very small quantities or a small amount of
enzyme is enough to convert a large quantity of
3 substrate and remain unchanged after the reaction.
5) Enzyme precipitation is the separation of
enzymes for analysis using different aqueous or
ethanol solvents.
6) Molecular weights of enzymes are large
protein biomolecules that hold polypeptide
chains of various amino acid sequences in
enzymes having a high molecular weight.
7) Enzymatic activity is the general catalytic
properties of an enzyme. It depends on factors
such as temperature, pH, and enzyme
concentration and substrate concentration.
• Enzymes show the highest activity at optimum
temperature and pH that a low concentration of
enzymes and substrates slows down the
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enzymatic reaction.
 B. Chemical properties of enzymes
• Enzyme chemical properties are
1) Sensitivity 3) Specificity
2) Regulations 4) Catalysis 5) Reversibility
reactions
2) Heat and pH Sensitivity is an enzymatic
reaction to heat (temperatures) and pH
(acidity and basicity) activated at optimum
levels.
2. Regulation is the process of controlling the
activity of enzymes by activator and inhibitor
molecules.
• Enzyme regulation refers to the multiple
mechanisms available to control the activity of
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 . 3) Catalysis is the process of the acceleration of
a chemical reaction by a catalyst.
• They can transform about 100-10,000
substrates per second.
• The reactions catalyzed by the enzymes show a
103-108 times faster reaction rate in
comparison to the non-catalyzed reactions.
4) Reversibility is the ability of enzymatic
biomolecules to catalyze various metabolic
(anabolic and catabolic) reactions.
• It is the reaction to synthesize (build up new
molecules or products) and decompose (breaks
down different products) in which enzymatic
reactions catalyze biochemical reactions in both
6 forward and reverse directions.
. The function of enzymes
• Enzymes help speed up chemical reactions in
the human body.
• They are essential for respiration, digesting
food, the liver, muscle, and nerve function.
• Each cell in the human body contains thousands
of enzymes that provide help in facilitating
chemical reactions within each cell.
• The turn over number of molecules is the
number of substrates converted by one enzyme
molecule per second at saturated (fully
occupied) active sites.
• Enzymes are markers of the states (blood tests, the rise
or fall of enzyme levels can aid in the diagnosis of a
variety of conditions) of various diseases like
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• myocardial infraction:happens when a part of
the heart muscle doesn't get enough blood
• Jaundice: sign of a problem with the liver,
gallbladder, or
pancreas.pancreatitis:inflammation of the
pancreas may happen when digestive juices
or enzymes attack the pancreas cancer
• neurodegenerative disorders: Many of these
diseases are genetic. Sometimes the cause is
a medical condition such as alcoholism, a
tumor, or a stroke.
Each enzyme has an active site with a unique
shape that speeds up metabolism or chemical
reactions in our bodies and builds substances
in all living things.
 Protein structures
Amino Acids and Peptides
• Proteins are very large molecules – macro-
biopolymers – made from monomers called amino
acids.
• Amino acids can be joined via a peptide bond
An amino acid consists of a α-carbon atom bound to
four groups.
1. Amino group, —NH2 , —exist as NH3+ under
physiologic conditions).
2. Carboxylic acid group, —COOH (exist as —COO−
under physiologic conditions).
3. Simple hydrogen atom
4. Commonly denoted "—R" and is different for each
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amino acid.
 • In the above sections, we have discussed that enzymes
are proteins.
 Proteins have different structures.
• Protein structure is a polymer of amino acids joined by
peptide bonds with three-dimensional arrangements of
atoms in amino acid chain molecules.
• The protein complex macromolecules have four
structural levels: Primary, Secondary Tertiary and
Quaternary structure
1) The primary structure of proteins
• It is the sequence of amino acids linked together to
form a polypeptide chain through peptide bonds created
during the protein biosynthesis process
• Proteins with fewer than 50 sequences are peptides,
and proteins with longer than 50 sequences of amino
acids are polypeptides.
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 2) The secondary structure of protein
 is a folded structure formed within a polypeptide due to
interactions between atoms of the backbone based on
hydrogen bonding and containing a-helix and ß-sheet types of
strands
2.1 The a – Helix
The a-helix is a right-handed coiled strand
• The a–Helix structure is one of the most common ways in
which a polypeptide chain forms all possible hydrogen bonds by
twisting into a right-handed screw with the NH group of each
amino acid residue hydrogen-bonded to the CO of the adjacent
turn of the helix.
2.2 ß–pleated sheet
• The hydrogen bonding in the ß-sheet is between the inter-strands and intrastrands in
which the sheet conformation of the ß-sheet consists of pairs of strands lying side by-
side.
• All peptide schains stretch out to nearly maximum extension, laid side by side and
held together by intermolecular hydrogen bonds forming pleated folds of drapery
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 3. The tertiary structure of proteins
• The tertiary protein structure is the three
dimensional shape of protein molecules that bend and
twist to achieve the maximum stability or the lowest
energy state.
• It is fashioned by many stabilizing forces due to the
bonding interactions between the side-chain groups
of amino acids
4. . The quaternary structure of proteins
• A protein quaternary structure is the arrangement
of multiple folded protein subunits in a multi-subunit
complex.
• It is the association of several protein chains or
subunits into closely packed arrangements with their
own primary, secondary, or tertiary structures and
held together by the hydrogen bonds
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 Enzyme substrate models
Enzyme substrate models are models for enzyme
substrate interaction describing that the shapes of the
active site and the substrate complement to fit into the
binding active site perfectly.
• There are two different enzyme-substrate binding
models
1. lock and key model 2. induced fit model
1.lock and key model
• The lock and key model is when enzyme active sites
fill-in with a substrate to interact through non-covalent
interactions.
• The model explains on how the enzymes must bind to
substrates before they catalyze a chemical reaction.
• Once the reaction progresses to the transition state
and forms products, the active site will not be able to
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accommodate changes
 2. Induced fit model
• when the active site of an enzyme is not perfect to
perform the required function.
• The amino acid side chains that make up the active site
mold into the precise positions enable the enzyme to
perform its catalytic functions.
• The concept of induced fit states that when a substrate
binds to an enzyme, it brings about a change in the shape
of the enzyme, which either enhances or suppresses the
activity of the enzyme.
Enzyme regulation
 Enzyme regulation is a control system for enzymatic
activities in which enzymes are turned “on” or “off”
depending on the organisms need.
It adjust enzymatic activities by other molecules to either
increase or decrease the activities.
• It requires an extra activation process to pass through
some modifications and functions.
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 • Regulatory enzymes are of two types,
1. Allosteric enzymes 2.Covalently modulated enzymes
1. Allosteric enzymes
enzymes that have additional binding sites for effector
molecules other than the active site that cause
conformational changes, leading to changes of catalytic
properties.
• Allosteric enzymes contain two binding sites called
• active site/catalytic site for binding substrates
• allosteric site/regulatory site for binding effectors
Effectors are small molecules (inhibitor or activator)
change the enzyme activity and function through reversible
non-covalent binding of a regulatory metabolite in the
allosteric site or non-active site.
• Effectors lead to conformational changes in a actual part of
the enzyme that affect the overall conformation of the active
site, causing modifications in the activity of the reaction
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 2. Genetic and covalent modification
• The genetic and covalent modification modifies the
protein surface and facilitates intracellular delivery.
• Genetic modification of enzymes is to improve the
properties of enzymes and gain active and inactive forms.
• Covalent modulated enzymes are active and inactive
forms of the enzymes altered due to covalent modification
of structures catalyzed by other enzymes.
2. Genetic and covalent modification
• Covalent modifications are enzyme-catalyzed alterations
of synthesized proteins by the addition or removal of
chemical groups. • Modifications can target a single type of
amino acid or multiple amino acids and will change the
chemical properties of the site.
• Enzyme regulation occurs by the addition or elimination
of some molecules attaching to the enzyme protein.

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 Enzyme inhibition
• Enzyme inhibition is a decrease in enzyme activity by
enzyme inhibitors.
• Enzyme inhibitors are molecule that binds to an
enzyme and blocks its activity.
• There are two types; these are
• Reversible inhibitors
• Irreversible inhibitors
Reversible inhibitor: inactivates an enzyme through
noncovalent easily reversed interactions.
It is characterized by a rapid dissociation of the
enzyme–inhibitor complex.
• Irreversible inhibitor: is a substance that permanently
blocks the action of an enzyme.
Usually covalently modify an enzyme, and inhibition
can therefore not be reversed
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 Reversible inhibitors can be
1. Competitive inhibitor is a molecule that
blocks the binding of the substrate to the
active site.
2.Noncompetitive inhibitor binds to the
enzyme already bound the substrate and
decreases the effectiveness of the enzyme.
3. Uncompetitive inhibitor binds only to the
enzyme – substrate complex, but not to the
free enzyme.
The uncompetitive inhibitor’s binding site is
created only when the enzyme binds the
substrate.
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 Enzyme structural classification
• The structural classification of enzymes deals with the
separation of an enzyme into
1. simple proteins (active), contain only a polypeptide chain of
linked amino acids
2. conjugated proteins (holoenzymes), contain non amino acid
components
• Then, the conjugated protein (holoenzyme) is divided into two
parts
• The protein part (apoenzyme: inactive) responsible for the
specificity of enzymes to their substrates.
• The non-protein part (cofactor) necessary for the catalytic
function of the enzymes.
E.g. - Inorganic molecules metal ions (Mg2+, Fe3+, Zn2+), -
organic molecules or coenzymes (NAD+, NADP+, FAD2+)
Finally, the non-protein part (cofactor) separates into
• Firmly attached metal ion (prosthetic group)
• Loosely attached mostly vitamines (coenzyme) groups
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 Basic classification of enzymes
• Enzymes are composed of six classes based on
• what and how they react,
• The types of reactions they catalyzed,
• Most enzymes are named for their substrates and for the
reactions that they catalyze, with the suffix “ase” added.
• Peptide hydrolase is an enzyme that hydrolyzes peptide
bonds
• ATP synthase is an enzyme that synthesizes ATP.
The followings are basic classes of enzymes.
1. Oxidoreductases are a class of enzyme that catalyzes
oxidation-reduction reactions.
It catalyzes the transfer of electrons from one molecule
(oxidant) to other molecule (reductant) reactions in the
following pattern:
• where A is the oxidant and B is the reductant.
2. Transferase is an enzyme that transfers functional groups
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(like methyl) from one donor molecule to acceptor molecule.
 3. Hydrolases are enzymes that catalyze the hydrolysis
of various bonds by reaction with/addition of water
4. Lyases are enzymes that cleave bonds by other
means rather than hydrolysis or oxidation in which two
or more substrates are involved in one reaction (an
addition or elimination reaction).
• What is the difference between lyase and hydrolase?
Hydrolases are able to break chemical bonds, while
lyases create new bonds by removing or adding
functional groups and usually forms a new double
bonds
5. Isomerases are a general class of enzymes that
catalyze reactions involving a structural rearrangement
of a molecule in which bonds are broken and formed
6. Ligases (synthetases) are enzymes that catalyze the
joining of two molecules with concomitant hydrolysis of
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the di-phosphate bond in ATP.
 Factors affecting enzyme action
1Temperature: while all enzymes work best within
the specific ranges of optimum temperatures,
low or high temperature causes an enzyme to
lose its activity and ability to bind a substrate
and denatured.
(Denaturation means a protein loses its shape)
• Once enzymes denatured, they cannot be
renatured.
Optimal temperature.
As you increase temperature, the kinetic energy of
reactants increases. This increases the rate in two
ways:
• More frequent collisions – the reactant particles
move faster, collide more often with enzymes
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 • More successful collisions -leading to a reaction
• As you increase or decrease temperature further,
• bonds in the active site begin to break
• the tertiary structure is disrupted & this changes the
specific shape of the active site
• so it may no longer be complementary to the substrate.
2. pH:
enzymes function at optimum pH (the potential of
hydrogen ions) that ranges from too low (strong acid) to
too high (too alkaline) pH.
• Such extreme pH cause an enzyme to lose its ability to
bind into a substrate.
Optimal pH. Most proteins and enzymes optimally function
at a natural pH of around 7.
However, certain enzymes can tolerate higher or lower pH
levels.
23• For example, the human protein, pepsin, which is found in
the stomach, works best at a pH of 2, which is highly acidic.
 Denaturation can occur at low or high pH.
• The enzyme is affected due to disruption of the ionic and
hydrogen bonds in the tertiary structure, which leads to an
alteration in the specific shape of the active site.
3.Enzyme Concentration
• Increasing [enzyme] initially increases rate.
• increasing the number of enzymes increases the rate by
increasing the amount of collisions between enzymes and
substrates.
• After a certain point, if the amount of substrate is kept
constant, the rate of the reaction will not increase with
increasing enzyme concentration.
• If the supply of substrate is unlimited, addition of enzymes
will continue to result in increased reaction rates.
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(The dotted line represents a reaction with unlimited
4. Substrate concentrations: enzymes require a
maximum
limit of substrate concentration to bind.
• Increasing concentration of substrate initially
increases rate of reaction Since it increase the rate
of collisions, so there will be more successful
collisions per second. (assuming a constant enzyme
concentration)
• After a while the enzyme active sites are
saturated.
• After a certain point (the saturation point),
increasing the concentration of a substrate, while
keeping the enzyme concentration constant, no
longer increases the rate of reaction.
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 1. Radiation damages enzyme activities by reducing
in enzymatic efficiency and
creating disorders in the macromolecules.
2. Water: affects the performance of enzymes’
activity beyond its optimum level.
3. The salt concentration: Each enzyme has an
optimal salt concentration.
Changes in the salt concentration may also denature
enzymes
4. End product (Feedback) inhibition is a cellular
control mechanism in that the end product inhibit
enzyme's activity.
• In feedback inhibition, the endproduct binds to the
allosteric site of the enzyme and change the
structure of the active site and this prevents the
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enzyme to sperform its activity.
 3.8 Enzyme kinetics
 Enzyme kinetics describes the rates of
chemical reactions that are catalyzed by
enzymes and the binding affinities of
substrates, inhibitors and the maximal
catalytic rates achieved.
 Enzyme kinetics explains that enzymes speed
up reactions by lowering the activation energy
of the reactants and turning them into
products.
 Hence, the concentration of enzyme and
substrates determines the rate of the
reactions or production volumes per unit time.
 one of the most known models of enzyme
27 kinetics is the Michaelis-Menten formula that
takes a form of equation describing the rate of
 3.9 Application of enzymes in industries and their
benefits
3.9.1 Uses of enzyme application
The application of enzymes are widely used in food, feed, textile, papermaking, leather
and detergents, pharmaceutical and other industrial productions. Examples:
1. Enzymes break down larger complex molecules into simpler molecules in our body
where they can be used to fuel our digestive systems and cellular respirations.
2. Most enzymes used for food industry were extracted from the internal organs of
animals and plants, but now most enzymes are obtained by microbial fermentation.
3. Enzymes cause billions of chemical reactions to happen at lightning speed inside the
cells of our body.
4. Enzymes improve the utilization of feed rate of starch, protein, and minerals and
degrade the anti-nutritional factors in animal feed, prevent animal indigestion and
improve feed digestibility.
5. In the pharmaceutical industry, enzymes are used in drugs, antibiotics, household
products to speed up chemical reactions and synthesis.
6. Enzymes are powerful tools in sustaining a clean environment in several ways.
7. Washing powders are enzymes used to break down protein, starch and fat stains on
clothes
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 3.10 Malting in Ethiopian tradition
 Malting (sprouting) is a widely applied traditional
technology.
 It is the process of steeping, germinating and drying
grain to convert it into malt.
 Malting is the limited controlled germination of grains
in moist air, which results in the mobilization of
amylases, proteases and other enzymes that hydrolyze
and modify the grain components and its structure
3.10.1 Steps of modern malting
There are three steps to modern malting, steeping,
germinating and kilning, and these will be discussed in
this section.
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1. Steeping – is the process of cleaning the grain kernels and
bringing it to life with water and oxygen by immersing it in the
water and air for a specified time period.
 The water activates naturally the existing enzymes in grains and

stimulates the production of enzymes in which water temperature

and aeration are vital for producing high quality malt.
 Although the process can vary depending on the grain type and

size, malting occurs over a period of 24–48 hours.

 The steeping will be complete when the barley has reached a

sufficient moisture level to allow a uniform breakdown of starches

30 and proteins.
 2. Germinating - is to continue the process with the growth and
modification of the grain.
 Modification is the breakdown of protein and carbohydrates,

resulting in the opening up of the seeds’ starch reserves within

four to six days as Green Malt.
 The control of temperature and moisture levels with regulated

airflow and the uniformity of water spray enables achieve a high

quality and consistent germination process.
 Malting is partly an art and partly a science that can be gauged in

the degree of modification with the eyes, sense of smell and

hands
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 3. Kilning (Heating) –
is the heating treatment of germinated grain to dry the green malt and prevent
from further germination.
 If germination continued, the kernel would keep growing and

the growing plant would use all of the starch reserves needed
by the brewer.
 Removing moisture from the germinated grain is initially for

withering. Additional drying further reduces the moisture

content and prepares the malt for flavor and color
development.
 The kilning process achieves enzymatic activity and friability,

a wide ranges of malt colors and flavors and distinctive ales

32 and lagers.
 3.10.2 Why is malting for?
Malting aims to convert or modify the physical

structure of the barley grain and allow synthesis or

activation of a series of enzymes to produce malt
for uses in the subsequent purposes (brewing,
distilling or food production).
Barley is the most common cereal used for the

production of malt because of its high starch-to-

protein ratio and adhering husk that contribute to
the economic yield, ease of processing and

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production.
  Barley malting in Ethiopia is practiced for the production of

traditional beer (Tella) and uses as in ingredient in porridge

making or drinks.
 The most common enzymes used in the malting process are

beta-glucanase, alpha amylase, protease (breaks down

proteins) and beta-amylase.
 The main purpose of malting is to produce enzymes such as

the α-amylase and βamylase useful for modifying and

converting grains’ starches into simple sugar , complex sugar
and malt sugar (maltose) and higher sugars called
maltodextrines.
 Then after, the yeast uses these monosaccharides to to

produce alcohol by alcoholic fermentation.

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 3.10.3 Traditional malting for local alcohol production
Traditional malting is the process of sprouting barley grains
for the production of enzymes (α-amylase and β-amylase)
to process fermentation drinks such as Tella. The steps of
traditional malting process include:
1. Soaking barley grains (steeping)
2.Germinating (sprouting)
3. Kilning (Heating) the malt
The most commonly used grains for malting are barley,
maize, millet, sorghum and the like.
However, barley is the most preferable grain to produce
local drinks in local
 Malted barley The main purpose of malt production is to
produce alcoholic beverages drinks for consumptions and
income generation to support the livelihood of the people.
 Malting requires raw materials like barley for
fermentation of alcoholic drinks like Tella.
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 People in Ethiopia produces local drinks like Booka, Cheka,
Keribo, Korefe, Shameta, Borde and Teji in different occasions
(holidays, wedding ceremonies, and celebrations).
3.11 Renowned Biochemists in Ethiopia
 A biochemist is a scientist who studies the chemical processes
transformations in living organisms including DNA, proteins and
cell parts.
 A biochemist also conducts research on how certain chemical
reactions happen in cells, tissues and organisms and record the
effects of products in food additive and medicines.
 he study of biochemistry deals with all aspects of the immune
systems, expressions of genes, isolation, analysis and synthesis
of products.
 It also concerned with studying mutations that leads to cancers,
scientific procedures used to manage and monitor laboratory
works.
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