Enzymes

Our Objective
To provide comprehensive fundamental and applied
knowledge in all areas of Enzyme Technology including.

Important Text books
1. Fundaments of Enzymology : - Nicholas C Price and Stevens Oxford
Press. (1999).
2. Understanding Enzymes: Trevor Palmer.
3. Enzymes in Industry: Production and Applications: -W. Gerhartz
4. Enzymes : - Dixon and Webb. IRL Press.
5. Lehninger principles of Biochemistry: -Nelson & cox
6. Enzyme Technology by M.F. Chaplin and C. Bucke, Cambridge
University Press, Cambridge,1990.
7. Principles of Enzymology for technological Applications (1993): B
Heinemann Ltd. Oxford.

Introduction to Enzyme
 Enzyme, in Greek means in living (en= in, zyme =living).
 Coined by Kuhne in 1878.
 First enzyme extract from Yeast cells by Buchner (1897).
 First purified enzyme is Urease, by James B. Summer (1926).
(Hydrolysis of urea, forming ammonia and carbon dioxide).

 Biological catalysts.
 Protein in nature.
 Catalyze chemical reactions without being changed at the end of the
reaction.
 Enzymes can speed up the rate of biochemical reactions in the cells.
 Chemical reactions that occur within a living organism are called
metabolism.
 Metabolic reaction starts with the substrate and ends with product.
 The molecules that are affected by enzymes are called substrates.
(E. coli has 4288 proteins, 2656 of which are characterized, and 64% (1701) of the
characterized as enzymes).
What are Enzymes ?

 Enzymes lower the activation energy of a reaction so that it occurs more
readily.
 Enzymes (specialized proteins in our bodies) LOWER the activation energy of
specific reactions.
Enzymes as catalysts

Activation Energy
Imagine a chemical reaction
as the process of rolling a huge
stone (reactant) up a hill so
that it rolls down and breaks
into tiny pieces (products).
1

Activation energy is the
energy needed to roll the stone
up the hill.
Activation Energy
1
2

Once over the hill, the rest of
the reaction occurs.
Activation Energy
1
up the hill.
2
3

Activation Energy
1
up the hill.
2
3
The stone rolls down and breaks into
tiny pieces (products are formed).
4

The stone rolls down and breaks into
tiny pieces (products are formed).
The energy needed to start a chemical
reaction is called activation energy.
Activation Energy
1
up the hill.
2
3
4
5
Q: How much activation energy was needed to melt the ice cube? How did we change the
amount of activation energy needed?

 Speed up the rates of chemical reaction but remains unchanged at the end
of the reaction.
 Not destroyed by the reactions they catalyse.
 Enzymes are globular shaped (balled up) proteins.
 Enzymes work by temporarily bonding with substrate molecules.
 A single enzyme can be used multiple times.
 Needed in small quantities because they are not used up but released at
the end of reaction
 Enzyme-catalyses reaction are reversible.
 A single enzyme can induce millions of reactions per second.
 Can be slowed down or completely stopped by inhibitors.
-e.g. : heavy metals such as lead and mercury
Basic characteristic of enzymes

Properties of enzymes
I. Catalytic power
a. It increases the rate as much as 1017 fold.
b. It operates in moderate temperature and neutral pH (Enzymes from
archeabacteria are exceptions)
Extreme example is Nitrogen fixation (N2 to Ammonia), temp.700 ~
900K, high pressures (100 ~ 900atm) with iron catalysts vs. 300K, neutral
pH, pressure 1atm with iron and molybdenum in nitrogenase

I. Catalytic power
Rate constant K : is the number of molecules of substrate
converted to product by one enzyme site per second.

Properties of enzymes …………….ii. Specificity…
Extraordinary ability of enzyme to recognize a specific substrate
(1) Absolute specificity
Specificity of an enzyme towards a single substrate…
Urease
Urea Carbon dioxide + ammonia
(2) Dual specificity
Recognize two different substrates
Sucrase
Sucrose Glucose + fructose
Sucrase
Raffinose Fructose + Mellibiose

(3) Group Specificity
Specificity of an enzyme towards a group of closely related compounds…
Example: Alcohol dehydrogenase (ADH) act on ethanol and other higher alcohols
ADH
Ethanol + NAD+ Acetaldehyde + NADH + H+
ADH
Propanol + NAD+ Acetaldehyde + NADH + H+
Group specificity - the enzyme will act only on molecules that have specific
functional groups, such as amino, phosphate and methyl groups.
Ethanol
Acetaldehyde
or
Propanol
ADH

(5) Optical specificity
Specificity of enzymes towards chiral substances (D&L isomers).
Example:
Ala racemase
D-Alanine L- Alanine
(4) Geometrical Specificity
Recognition of Cis-trans isomer by enzymes is called geometrical specificity.
Example:
Fumarase
Fumarate (cis-form) Malate (trans-form)

(6) Bond specificity
Selectivity of enzymes to specific type of bonds in a compound for subsequent hydrolysis
Example
(1) Glycosidase cleaves glycosidic bonds.
(2) Proteases are a whole class of enzymes that all catalyze hydrolysis of peptide bonds.
Proteases
Glycosidase

Substrate Specificity -- proteases as an example
(A) Trypsin catalyzes hydrolysis of peptide bonds on carboxyl side of Lys and Arg
residues (digestive function in small intestine, cleaves just about any protein it
encounters after (eventually) every Lys and Arg).
(B) Thrombin (involved in blood clotting cascade) catalyzes hydrolysis of peptide
bonds between Arg and Gly residues in specific sequences in specific protein
substrates (activated only where blood needs to clot, works only on very
specific target protein)
Trypsin (Proteases)
Thrombin(Proteases)

Enzyme Specificity, continued
• Substrate specificity of proteases –Another example, chymotrypsin
– Cleaves on carboxyl side of aromatic and hydrophobic amino acid residues
– Genes for trypsin and chymotrypsin are homologous.
Specificity of reaction catalyzed:
Many proteases also catalyze hydrolysis of carboxylic ester bonds……..

III. Regulation ---Metabolic need of cells in human body.
a) Enzyme activities are regulated by small ions or small molecules (effectors),
such as phosphate or Ca2+.
b) The regulations are mediated by changing covalent structure and post –
translational modification, proteolytic cleavage .
c) Feedback inhibition is common in many biosynthetic pathway enzymes.
IV: Milder reaction conditions
Its works under milder condition of …
1. Temperature
2. Atmospheric Pressure
3. Neutral pH
Except for extremozymes (Psychrophiles or thermophiles)

Why do we need to digest our food?
• Starch, proteins and fats are very large.
• They cannot diffuse across cell membranes for absorption.
• Therefore, they must be digested into….
– Simpler, smaller and soluble substances.
– Diffusible across cell membranes.
Digestion: An Enzyme-Catalysed Process

• Anabolic processes
– Eg. Synthesis of proteins from amino acids.
• Catabolic processes
– Eg. Oxidation of glucose (tissue respiration).
• Catalase production
– Catalase catalyses the breakdown of toxic hydrogen peroxide
into harmless water and oxygen.
– Catalase is abundant in liver and blood.
Other applications of Enzymes

Denaturation of enzymes
 Enzyme are globular proteins.
 Their structure can be altered by change in pH
or temperature –if the shape of the active site is
changed considerably, they will not function.
 Denaturation is changing the structure of a
protein (enzyme) so that it cannot carry out its
function.
 High temp. cause
denaturation as the extra
energy leads to increased
vibration, breaking intra-
molecular bonds.
 Changes in pH cause
denaturation as hydrogen
bonds are broken.
 Both methods result in an
altered 3D structure of the
active site, and this
changes is irreversible.
Denaturation of protein

 Many enzymes, like chemotrypsin and triosephosphate isomerase, do not
require additional factor.
 Many others require non-protein component for enzyme activity
 Metal ions and organic cofactors (usually derived from B vitamins) are major
groups of cofactors.
 Tightly bound cofactors are called prosthetic groups (a. holoenzyme = enzyme +
cofactor); (b. apoenzyme = enzyme without cofator)
 Any other molecules that bind to an enzyme are called ligands (including
substrates and effectors)
Cofactors

Factors affecting enzyme activity
1. Temperature
2. pH
3. Substrate concentration

An enzyme is
less active at
very low
temperatures.
As the temperature rises, enzyme activity increases
as indicated by the increase in the rate of reaction it
catalyses. Usually the enzyme is twice as active for
every 10°C rise in temperature until the optimum
temperature is reached.
The optimum temperature
is reached. Enzyme is
most active.
A thermophile, such as bacteria at deep-sea
vents, is an organism that is able to
withstand much higher temperatures
before its enzymes denature
Effect of Temperature on the Rate of Reaction
Rate of reaction
(enzyme activity)
Enzyme has lost its
ability to catalyse
the reaction.

Enzymes only operate within a
narrow range of pH values. This is
called an optimum pH.
 If there is a deviation from the
optimum pH, the hydrogen bonds
between amino acids in the structure
of the enzyme are broken.
 This results in the loss of the shape of
the active site of the enzyme, so it
dies not function.
 This is usually a permanent change.
Effect of pH on enzyme activity

Interpreting pH vs. Rate of Enzyme Activity graphs:
1.) Enzyme activity
increases with
increasing pH until
it reaches its
optimum pH.
2.) Optimum pH (‘peak
efficiency’) helps to maintain 3º
structure, i.e. H-bonds where
the enzyme is most active and
therefore maximum ES
complexes, product’s and rate
of enzyme activity.
3.) Further increase of pH
disrupts the H-bonds,
changes the 3º structure,
and denaturation occurs.
Therefore fewer active sites
are available for the reaction
and fewer complexes are
formed.

Most enzymes of the body have an optimum pH of about 7.4.
 In certain organs, enzymes operate at lower and higher optimum pH values.
Optimum pH Values

The effect of substrate concentration on enzyme activity
Increasing substrate concentration
increase the rate of reaction At the optimum concentration of
substrate molecules, all active
sites are full and working at
maximum efficiency.
Any increase in concentration
beyond the optimum will have
no added effect as there are no
extra active site to be used

Effect of Oxidation
 Oxidation of sulphydryl group (-SH) in the active site by the oxidizing agent
leads to disulphide bridging (S-S), resulting in loss of enzyme activity.
Effect of radiation
 Exposure to high (short wavelength) radiation like X-rays, β-rays and γ- rays
leads to conformational change and loss of enzyme activity.
 UV rays also inactivate enzyme

Enzyme units
 Actual molar amount of the enzyme can not be determine , it is unknown. The
amount can be expressed in term of its activity.
Four ways of expressing enzyme activity.
1. International units:
One IU of an enzyme….. is the amount of enzyme that catalyzes the formation
of one micromole of product in one minute under optimal condition of pH,
temperature, and ionic strength.
2. Katal:
One katal……. Is the amount of enzyme catalyzing the conversion of one mole
substrate to product in one second. 1 Katal= 6 x 107IU
3. Specific activity(U/mg)
Specific activity is the number of units of enzyme activity per milligram of
total protein present.

Turn over number (kcat)
 Maximum number of moles of substrate that an enzymes can convert to
product per catalytic site per unit time.
 It is also called molecular activity.
Example:
Carbonic anhydrase (Enzymes) can produce up 400,000 S-1, which
means that each carbonic anhydrase molecule can produced up to 400,000
molecule of product (CO2) per second
Bicarbonate (CH2CO3)n (CO2)n + (H2O)n
Carbonic anhydrase
Molecule 400,000 S-1,

Nomenclature and classification of Enzyme

Nomenclature of Enzyme
 In most cases, enzyme names end in –ase
 The common name for a hydrolase is derived from the substrate.
a. Urea: remove -a, replace with -ase = urease
b. Lactose: remove -ose, replace with -ase = lactase
 Other enzymes are named for the substrate and the reaction catalyzed
a. Lactate dehydrogenase
b. Pyruvate decarboxylase
 Some names are historical - no direct relationship to substrate or reaction type
a. Catalase
b. Pepsin
c. Chymotrypsin
d. Trypsin

Major classification
 According to the International Enzyme Commission constituted by the
International Union of Biochemistry (1956).
 Enzymes are divided into six major classes depending on the type of reaction
catalyzed.
 Each major class is further divided into subclass and each subclass is redivided
into subsubclass.
Class I: Oxidreductase
Subclasses:
1.Dehydrogenase
2. Oxidase
3. Peroxidase
4. Hydroxylase
5. Oxygenase
6. Oxidative deaminase

1.Dehydrogenase
Removal of two hydrogen atoms with double bond formation
Example:
a) Lactate dehydrogenase:
b) Malate dehydrogenase

2. Oxidase : Reduction of Oxygen
Example:
Cytochrome oxidase
3. Peroxidase
Example: Catalase…… Reduction of H2O2
4. Hydroxylase…… Introduction of Oh groups
Example: Phenylalanine-4-hydroxylase
Enzyme Cytochrome oxidase
Catalase
Phenylalanine-4-hydroxylase
2H2O2 H2O + O2

5. Oxygenase…………Incorporation of Molecular Oxygen
Example:FADH2 Monooxygenase
FADH2
Monooxygenase
6. Oxidative deaminase :Oxidation of amino acid with libration of NH3
Example: Amino acid oxidase Amino acid oxidase

Class II: Oxidreductase
Subclasses:
1. One carbon transferase
2. Aldehyde and ketone transferase
3. Acyl transferase
4. Glycosyl transferse
5. Alkyl tranasferase
6. N-transferase
7. Phospho tranasferase
8. Sulpho transferase
1. One carbon transferase
a) Methyl transferse
b) Hydroxyl methyl transferse
c) Formyl transferase
Hydroxyl methyl
transferse
Formyl transferase
Transfer of one-carbon group
Phenylethanolamine N-methyltransferase

2. Aldehyde and ketone transferase …… transfer of aldehyde/keto group
Example: Acetaldehyde transferase
3. Acyl transferase….. Transfer of acyl/acetyl group to a suitable acceptor.
Example: Carnitine acyltransferase
Keto group

4. Glycosyl transferse. (key enzyme in glycogen synthesis)
…….Transfer of glycosyl group
Example: Hexosyl transferse
Hexosyl transferse
5. Alkyl transferase………. Transfer of alkyl other then methyl group.
Example: Ethyl transferse:

For example:
Aspartate aminotransferase (Glutamic-oxaloacetic transaminase or simply GOT).
6. N-transferase …….Transfer of nitrogenous group
Glutamic-
oxaloacetic
transaminase
7. Phospho tranasferase…… Transfer of phosphoryl group
Example: Glycerol kinase
Glycerol kinase

Example: Choline sulphokinase
8. Sulpho transferase : Transfer of sulfur containing group
3'-Phosphoadenosine
5'-phosphosulfate
Choline
Choline sulfate
Adenosine 3',5'-
bisphosphate
Choline sulphokinase
Class III: Hydrolase
Subclasses:
1.Estrase
2. Peptidase
3. Glycosidase
4. Phosphatase
5. Deaminase
6. Deamidase
1.Estrase ……. Hydrolysis of ester
Example: Acetylcholin esterase
Estrase

2. Peptidase
..Hydrolysis of peptide bond
Example: Trypsin catalyzes hydrolysis of peptide bonds on carboxyl side of Lys
and Arg residues
3. Glycosidase…. Hydrolysis of glycosidic bond
Example: Lysozyme
4. Phosphatase
Hydrolysis of phosphate bond.
Example:
Alkaline phosphatase
Glycosidase
Trypsin

5. Deaminase…. Hydrolysis of amines
Example: Glutaminase, Glucosamine-6-P-deaminase.
6. Deamidase ..Hydrolysis of amide
Example: Nicotinamide deamidase
Nicotinamide deamidase

Class IV: Lyase
Subclasses:
1. C=C Lyase
2. C=O Lyase
3. C=N Lyase
1. C=C Lyase ...Cleavage of carbon-carbon bond
Example.. Aldolase
2. C=O Lyase...Cleavage of carbon-Oxygen bond
Example: DNA lyase or AP lyase
3. C=N Lyase .Cleavage of carbon- nitrogen bond
Example: Histidine Ammonia lyase
Aldolase
AP site:apurinic/apyrimidinic site)
Histidine Ammonia lyase

Class V: Isomerase
Subclasses:
1. Recamase
2. Epimerase
3. Cis –trans isomerase
1. Recamase ..Interconversion of optical isomers
Example: Succinylamino acid racemase
2. Epimerase… Interconversion of epimers
Example: D-Xylulose-5-phosphatease epimerase
Two compounds is the relative position of
the H (hydrogen) group and OH (hydroxyl) group
3. Cis –trans isomerase.. Interconversion
of geometrical Isomer
Example:Fumarase
Succinylamino acid racemase
L D

Class VI: Ligase
Subclasses:
1. C=O Ligase
2. C=S Ligase
3. C=N Ligase
4. C=C Ligase
1. C=O Ligase… Formation of C-O bond
Example: T-4-RNA ligase
2. C=S Ligase… Formation of C-S bond
Example: Acetate CoA ligase
acetazolamide

3. C=N Ligase… Formation of C-N bond
Example: NAD+ Synthase, Tryptophan synthase
NAD+ Synthase
Tryptophan synthase

4. C=C Ligase … Formation of Carbon- carbon bond
Example: Pyruvate carboxylase

Enzyme Commission
Each enzyme is assigned a code by the Enzyme commission.
The code number is called the enzyme code. It has four digits.
1. First integer represent the major class.
2. Second integer represents the subclass.
3. Third integer represent the sub-subclass.
4. Forth integer represent the individual serial number of that enzyme in the
sub-subclass.
Thus, a series of four number serves to the specify a particular enzyme , to
illustrate, considered the enzyme that enzyme catalyzes this reaction.

Example:
Class: A phosphate group is transferred from ATP to the –OH group of the sixth
carbon of glucose, so the enzyme is transferase (Class 2).
Subclass: Enzyme transferring phosphorus-containing groups are called
phosphotransferase which comes under seventh category of transferase class, So
subclass is 7.
Sub-subclass: If an alcohol group- to phosphate (1), aldehyde may be (2 )… is
the acceptor of the phosphate group.
It refers to the first category of the phosphotransferse. Therefore, the sub-sub
class is 1 . Entry 2 in this-subclass is ATP (donor1).
EC: 2.7.1.2
D-Glucose
-6-phosphotransferase

Catalyze cleavege of bonds by addition of water

Catalyxe racemization of optical or geometric
Isomers and intramolecular oxidation-reduction
reactions

Ligases are usually referred to as synthetases

5.3 Properities and catalytic mechanisms of
enzymes
5.3.1 properities of enzyme catalytic reactions
5.3.2 catalytic mechanisms of enzymes

73
Properities of enzyme catalytic reactions
1).High catalytic activity of enzymes:
(a) Enzymes can decrease activation energy i.e.
molecules are activated using activation energy.
By decreasing activation energy,enzymes promotes
chemical reactions
(fig 5-5) .

 Intracellular : Synthesised and retained in the cell for the use of cell itself
: found in the cytoplasm, nucleus, mitochondria and chloroplast
Example : 1) oxydoreductase catalyse biological oxidation
2) reduction in the mitochondria
 Extracellular : Synthesised in the cell but secreted from the cell to work
externally
Example : 1) digestive enzyme produced by the pancreas are not used
by cells in the pancreas but are transported to the duodenum
Intracellular and extracellular enzyme

 Protein/Enzymes are synthesized in the ribosomes are transported through
the spaces between the rough endoplasmic reticulum.
 Protein depart from the RER wrapped in vesicles that bud off from the
sides of the RER.
 These transport vesicles fuse with the membrane of the Golgi apparatus.
 Secretory vesicles containing these modified protein bud off from the
Golgi membrane and travel to the plasma membrane.
 These vesicles will then fuse with the plasma membrane before releasing
the proteins outside the cells as enzymes.
Production of extracellular enzymes

Mechanisms of Enzyme action
There are two proposed methods by which enzymes bind to their
substrate molecules:
a) Lock and Key model
b) Induced Fit model

77
The Active Site of an Enzyme
1. The active site is the region that binds the substrates (&
cofactors if any)
2. It contains the residues that directly participate in the making &
breaking of bonds (these residues are called catalytic groups)
3. The interaction of the enzyme and substrate at the active site
promotes the formation of the transition state
4. The active site is the region that most directly lowers G‡ of the
reaction - resulting in rate enhancement of the reaction

78
Common Features of Active Sites
Enzymes differ widely in, structure, specificity, &
mode of catalysis, yet, active site have common features
1. The active site is a 3-dimensional cleft formed by groups that
come from different parts of the amino acid sequence
2. The active site takes up a relatively small part of the total
volume of an enzyme. Why are enzymes so big? Scaffolding,
regulatory sites, interaction sites for other proteins, & channels
3. Active sites are clefts or crevices - exclude H2O
4. Substrates are bound to enzymes by multiple weak attractions
ES complexes have K’eq of 10-2 to 10-8 M, & G of interaction
from about -3 to -12 kcal mol-1 (electrostatic interactions, hydrogen
bonds, Van der Waals forces, & hydrophobic interactions can be Involved)
5. The specificity of binding depends on the precisely defined
arrangement of atoms at the active site

79
Active sites - distant residues

80
Enzyme - substrate: hydrogen bonds
Ribonuclease:
cleaves RNA

Active site enclose and sequester substrate
• Functional groups (mostly aa residues) of active s. align (match) those of substrate.
• Charles-Adolphe Wurtz (1880) enzyme-substrate complex.

1. Lock-and-Key Model
 Enzymes have a 3º structure (recall a 3-D shape held together by covalent,
ionic, hydrogen and peptide bonds).
 The portion of the enzyme involved in a reaction is the active site.
 It is the old view of enzyme specificity, that there was an exact match
between the active site and the substrate.
 The active site has a rigid shape.
 Only substrates with the matching shape can fit.

2. Induced-fit Model
 The induced- fit model better explains enzyme activity.
 If the lock-and key model were true, one enzyme would only catalyase one
reaction.
 In actually, some enzymes can catalyse multiple reaction.
 As the substrate approaches the enzyme, it induces a conformational changes
in the active site-it changes shape to fit the substrate.
 This stress the substrate, reducing the activation energy of the reaction.
 The active site is flexible, not rigid. There is a greater range of substrate
specificity. The enzyme returns to original shape so that it can be used again.
Conformational Change in
active site of enzyme

Enzyme Cooperativity
 Some enzymes have multiple active site.
 It has been observed that when one substrate molecule binds to a
single active site in the inactive form or tense state of the enzyme.
 A configurational change occurs in the other active sites making them
more receptive to other substrate molecules.

Isoenzymes
 Isoenzymes catalyze the same reaction in different tissues in the body.
 Lactate dehydrogenase, which converts lactate to pyruvate, (LDH)
consists of five isoenzymes.

90
Coenzymes
What are coenzymes?
• Some enzymes require a coenzyme to be bound
to them before they can catalyse reactions.
• Usually, coenzymes are non-protein organic
compounds.
– Eg. Vitamins, especially the B complex vitamins.

91
Coenzymes
• Coenzymes are altered in some way by
participating in enzyme reaction.

Effects on Enzyme Activity
Introduction
• Enzyme action occurs when the enzyme and
substrate collide. During the collision the
substrate slots into the active site of the
enzyme.
• Collisions happen because of the rapid
random movement of molecules in liquids.

Factors affecting the rate
• The following factors affect the rate of enzyme
activity and therefore the amount of products
produced:
– pH
– temperature
– [substrate]
– [enzyme]
– competitive inhibitors
– non-competitive inhibitors, e.g. heavy metals

pH
• Each enzyme has an optimum pH that
maintains the 3º shape (and its active site!).
• E.g. Stomach pH ~ 2 - 3
Pepsinogen  Pepsin
(Inactive) (Active)

More Examples of pH
• Small intestines pH ~ 8.5 – 9
Peptidase, lipase, maltase, trypsin, pancreatic
amylase etc.
• Blood pH ~ 7.4
Carbonic anhydrase
• Mouth pH ~ 7
Salivary amylase

pH
• Deviations from optimum pH will denature
the enzyme (destroy the H-bonds, 3º structure
and active site). Loss of 3º structure and
ability of active site to bond with the
substrate; enzyme is inactive.
– (Note: Denaturation is not usually reversible.
Some denatured proteins do renature when their
normal pH conditions are restored.)

Interpreting pH vs. Rate of Enzyme Activity
graphs:

Interpreting graph
• 1.) Enzyme activity increases with increasing
pH until it reaches its optimum pH.
• 2.) Optimum pH (‘peak efficiency’) helps to
maintain 3º structure, i.e. H-bonds where the
enzyme is most active and therefore
maximum ES complexes, P’s and rate of
enzyme activity.

Interpreting pH vs. Rate of Enzyme Activity
graphs:
• 3.) Further increase of pH disrupts the H-
bonds, changes the 3º structure, and
denaturation occurs. Therefore fewer active
sites are available for the reaction and fewer
complexes are formed

Temperature
• Each enzyme has an optimum temperature
where maximum activity of ES complexes is
achieved. E.g. the body’s optimum
temperature is 37ºC.
– (Recall from way back: changes in Tº will cause the
speed of molecules/ molecular movement to
increase/decrease & therefore molecular
collisions)

Temperature
• Deviations from optimum Tº will affect enzyme
activity rate and alter its shape.
• Too high of a temperature will cause denaturation
where H-bonds break, lose it’s 3º structure &
changes the shape. The enzyme no longer has an
active site to bond with the substrate & is inactive.
– (Note: Denaturation is not usually reversible. Some
denatured proteins do renature when their normal
temperature conditions are restored.)

Interpreting Temperature vs. Rate of
Enzyme Activity graphs:
37

Temperature Vs. Time for O2 Production

About the graph
1.) Enzyme activity increases with increasing
temperature; movement/collisions of enzyme
and substrate molecules increases and more
active sites are filled until it reaches the
optimum temperature.

About the graph
2.) Maximum rate of reaction, maximum ES
complexes formed at optimum temperature.
3.) Enzyme activity decreases with increasing
temperature as H-bonds break, alters the 3º
structure and denaturation occurs, loss of
active sites, fewer ES complexes; enzyme is
inactive.

 Substrate concentration
 Increase in substrate concentration, more substrate molecule are available to bind
the active sites of the enzyme
 Hence, more products will be produced
 Because more chances of collision between the substrate molecule and the
enzyme molecules for a catalytic reaction to take place
 In increase in substrate concentration will only speed up the reaction if there are
enough enzyme molecules to catalyse the additional substrate molecules
 The rate of reaction is directly proportional to the substrate concentration until the
reaction reaches a maximum rate
new

 Enzyme concentration
 When the concentration of an enzyme increases,more enzyme molecules are
available
 The rate of of reaction will increase only if there is abundant supply of substrate
molecules and other factors are constant because more active sites are made
available for the catalytic reaction.
 The rate of reaction is directly proportional to the concentration of the enzyme
present until a maximum rate is achieved.
 After the maximum rate,the doncentration of substrate becomes a limiting factor.
 If the concentration of enzyme is doubled,the amount of substrate molecules also
doubled.
NEW

Interpreting Substrate Concentration vs.
Rate of Enzyme Activity graph:

About the Substrate graph
1.) Enzyme activity increases as [substrate] increases
and reaction rate increases to a point.
2.) Enzyme activity slows down and levels off reaching
the maximum rate. The substrate exceeds the
number of enzymes and active sites are all occupied.
E.g. All maltase activity sites are in use.
Note: Adding more enzymes (see ‘3’ in graph above) will further
increase the rate of enzyme activity as there are more
available enzymes and active sites for the substrate.

About the Enzyme graph
• Reaction rate increases as [enzyme] increases
(to the same increasing [substrate]). The same
amount of products will be produced.

Competitive Inhibitor
• Chemicals that have the same shape as the substrate
and will compete for the active site.
• Enzyme cannot react with the “look-a-like”. This
effectively reduces the [of available enzyme] and
inhibits/decreases the reaction.
• The effect of competitive inhibitors can be overcome
by increasing the [substrate].

120
Characteristics
Functions Mode of Action
Limiting factors
affected by
Enzymes

122
Biological catalysts, which are mainly made of proteins. They speed up the rate of chemical
reactions without themselves being chemically changed at the end of the reactions.
Enzymes

124
Functions
• Building up or synthesising complex
substances
• Breaking down food substances in cells
to release energy (cellular respiration)
• Breaking down poisonous substances in
cells
Enzymes

125
Characteristics
Functions
Enzymes

126
Characteristics
Functions
• Speed up chemical reactions
• Required in small amounts
• Highly specific
• Work best at an optimum
temperature and pH
• May need coenzymes for activity
• Some catalayse reversible reactions
Enzymes

127
Characteristics
Enzymes

128
Characteristics
• Lower the activation
energy of a reaction
• Interact with the substrate
according to lock and key
hypothesis to form an
enzyme-substrate complex
Enzymes

129
Characteristics
affected by
Enzymes

130
Characteristics
Limiting factors
Factors that directly affect the rate at which a chemical reaction occurs if their quantity is
changed. The value of a limiting factor must be increased in order to increase the rate of reaction.
affected by
Enzymes

131
Characteristics
Limiting factors
Temperature / pH
e.g.
affected by
Enzymes

132
Characteristics
Limiting factors
Temperature / pH
e.g.
• Increase in temperature increases
the rate of enzyme reaction until
optimum temperature is reached
• Increase in pH increases the rate
of enzyme reaction until optimum
pH is reached
affected by
Enzymes

133
Characteristics
Limiting factors
Temperature / pH
e.g.
Classes
affected by
Enzymes

134
Characteristics
Limiting factors
Temperature / pH
e.g.
Classes
based on the
type of reaction
catalysed e.g.
Hydrolases
affected by
Enzymes

135
Characteristics
Limiting factors
Temperature / pH
e.g.
Classes
based on the
type of reaction
catalysed e.g.
Hydrolases
Oxidation-reduction
enzymes
affected by
Enzymes

144
5.2 Classification of Enzymes
Enzymes are classified
• according to the chemical reaction involved in:
– Enzymes that catalyse hydrolysis reactions are called
hydrolases.
Example of hydrolases:
Carbohydrases, proteases, lipases.
– Enzymes involved in oxidation of food as called
oxidation-reduction enzymes.

145
Learning Objectives
Candidates should be able to:
• Explain enzyme action in terms of the ‘lock
and key’ hypothesis.
• Investigate and explain the effects of
temperature and of pH on the rate of
enzyme catalyzed reactions .

146
5.3 Characteristics of Enzymes
• Enzymes alter or speed up the rates of chemical
reaction that occur in a cell.
• Enzymes are required in minute amounts.
– Since enzymes are not altered in a chemical
reaction, a small amount can catalyse a huge
reaction.

147
Enzymes are specific
• Specificity of enzyme is due to its shape
(or surface configuration).
• The substrate will fit into an enzyme,
forming an enzyme-substrate complex.
• The product will then be released.

148
Lock and key hypothesis
What is the ‘lock and key’ hypothesis?
• It is the old view of enzyme specificity,
that there was an exact match
between the active site and the
substrate.

150
active sites
A
B
enzyme molecule
(the ‘lock’)
substrate molecules
( A and B) can fit
into the active sites
Lock and Key
Hypothesis

151
active sites
A
B
enzyme molecule
(the ‘lock’)
enzyme-substrate
complex
substrate molecules
( A and B) can fit
Lock and Key Hypothesis

152
Lock and Key Hypothesis
active sites
A
B
AB
enzyme molecule
(the ‘lock’)
enzyme-substrate
complex
substrate molecules
( A and B) can fit
enzyme molecule is free
to take part in another
reaction
a new substance (product) AB
leaves the active sites

153
Induced fit hypothesis
What is induced fit hypothesis?
• shape of the active site adjusts to fit the substrate.

154
Induced fit hypothesis
How did induced fit hypothesis come about?
- recent imaging technology demonstrated
changes in the 3-D conformation of
enzymes when interacting with their
substrates.

155
Effect of temperature
• At low temp:
– Rate of reaction is slow.
– Enzymes are inactive at low temp.
– Every 10oc rise in temp, rate of reaction
increases by double
(till it reaches optimum temp).

156
Effect of temperature
• At optimum temp:
– Rate of reaction is the highest.
– Enzymes are most active.
• Beyond optimum temp:
– Rate of enzyme activity decreases
sharply.
– Enzymes are being denatured.
– Hydrogen bonds are easily disrupted
by increasing temperature.

158
Temperature
Rate of reaction
(enzyme activity)
0 K (optimum temperature) D
At point D, the enzyme
has lost its ability to
catalyse the reaction.
An enzyme
is less active
at very low
temperatures.
1
As the temperature rises,
enzyme activity increases as
indicated by the increase in
the rate of reaction it
catalyses. Usually the
enzyme is twice as active
for every 10°C rise in
temperature until the
optimum temperature is
reached.
2
The optimum temperature is reached.
Enzyme is most active.
3
Beyond the optimum
temperature, enzyme
activity decreases.
4
5
Effect of Temperature on the Rate of Reaction

159
Effect of pH
• Enzymes have an optimum pH.
• Deviation from the optimum pH will decrease enzyme
activity.

160
Effect of pH on Enzyme Activity

161
Effects of substrate and enzyme
concentration on rate of reaction
• Increasing substrate concentration will increase rate
of reaction until a certain limit.
• Cause:
– Enzyme molecules are saturated.
• Enzyme concentration is now the limiting factor.

162
What is a limiting factor?
• Any factor that directly affects the rate of a
process if its quantity is changed
• The value of the limiting factor has to be
increased in order to increase the rate of the
process.

163
Coenzymes
What are coenzymes?
• Some enzymes require a coenzyme to be bound
to them before they can catalyse reactions.
• Usually, coenzymes are non-protein organic
compounds.
– Eg. Vitamins, especially the B complex vitamins.

164
Coenzymes
• Coenzymes are altered in some way by
participating in enzyme reaction.

165
Enzymes
• catalyse reversible reactions
A D
B C
+ +
reactants products
reactants
reactants

166
Characteristics
Limiting factors
affected by
Enzymes

168
Biological catalysts, which are mainly made of proteins. They speed up the rate of chemical
reactions without themselves being chemically changed at the end of the reactions.
Enzymes

170
Functions
• Building up or synthesising complex
substances
• Breaking down food substances in cells
to release energy (cellular respiration)
• Breaking down poisonous substances in
cells
Enzymes

171
Characteristics
Functions
Enzymes

172
Characteristics
Functions
• Speed up chemical reactions
• Required in small amounts
• Highly specific
• Work best at an optimum
temperature and pH
• May need coenzymes for activity
• Some catalayse reversible reactions
Enzymes

173
Characteristics
Enzymes

174
Characteristics
• Lower the activation
energy of a reaction
• Interact with the substrate
according to lock and key
hypothesis to form an
enzyme-substrate complex
Enzymes

175
Characteristics
affected by
Enzymes

176
Characteristics
Limiting factors
Factors that directly affect the rate at which a chemical reaction occurs if their quantity is
changed. The value of a limiting factor must be increased in order to increase the rate of reaction.
affected by
Enzymes

177
Characteristics
Limiting factors
Temperature / pH
e.g.
affected by
Enzymes

178
Characteristics
Limiting factors
Temperature / pH
e.g.
• Increase in temperature increases
the rate of enzyme reaction until
optimum temperature is reached
• Increase in pH increases the rate
of enzyme reaction until optimum
pH is reached
affected by
Enzymes

179
Characteristics
Limiting factors
Temperature / pH
e.g.
Classes
affected by
Enzymes

180
Characteristics
Limiting factors
Temperature / pH
e.g.
Classes
based on the
type of reaction
catalysed e.g.
Hydrolases
affected by
Enzymes

181
Characteristics
Limiting factors
Temperature / pH
e.g.
Classes
based on the
type of reaction
catalysed e.g.
Hydrolases
Oxidation-reduction
enzymes
affected by
Enzymes

 Ribosomes are attached to the roughed endoplasmic reticulum.
 Information for the synthesis of enzyme is carried by DNA.
 The different of bases in DNA are codes to make different protein.
 RNA is formed to translate the codes into a sequence of amino acids.
 Amino acids are bonded together to form specific enzyme according to the
DNA’s codes.
The sites of enzyme synthesis

Enzymes

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