UNIT THREE

ENZYMES
 Nature of enzymes
What are enzyme molecules like?
Enzymes can be defined more precisely as:
 An enzyme is a globular protein with a uniquely shaped active site.
 Enzymes are biological catalyst for a specific reaction, but
remain unaltered by the reaction.
Not all biological catalysts are proteins.

 Ribozymes: RNA molecules that exhibit catalytic

activity in the absence of any protein component.
A catalyst is a substance that speeds up a reaction; the reaction itself
is unaltered. There is no overall change to:
 the nature of the products
 the energy change that takes place during the reaction
 the catalyst itself
Cont…
Enzymes
   allow biochemical reactions inside cells to take
place quickly.
All enzymes are globular proteins.
 All globular proteins have unique tertiary structures which
give them a unique shape (Fig. 3.1).
It contains regions where there is:
 an -helix
 a β-pleated
 no folding into a secondary structure
 the active site(the part that binds with a substrate)
Cont..

Fig. 3.1 The human lipase enzyme

Heme is the deep red, non-protein portion of hemoglobin
that contains iron.
 Cont…
Enzyme molecules contain a special pocket or cleft called the active site.
The active site of an enzyme is shaped to allow binding with a particular
or only one substrate.
Such binding requires less energy than un catalyzed reactions.
The active site and the substrate(s) have complementary shapes.
The binding of substrate to enzyme is accomplished by the same types
of noncovalent interactions (ionic bonds, hydrogen bonds, hydrophobic
interactions) that determine the structure of the protein itself.
The active site contains amino acid side chain that participate in
substrate binding and catalysis.
The substrate binds the enzyme, forming an enzyme–substrate (ES)
complex.
Binding is thought to cause a conformational change in the active site of
enzyme (induced fit) that allows catalysis.
 ES is converted to an enzyme–product (EP) complex that subsequently
dissociates to enzyme and product.
Cont…
Example -The hydrolysis of sucrose by sucrase
E + SES E + P  
Sucrose + H2O sucrase Glucose +Fructose (Fig.
3.2)
Steps:
 1) Enzyme available with empty active site
 2) Substrate binds to form enzyme- substrate complex
 A molecule of water then reacts with the sucrose to hydrolyse the
molecule into glucose and fructose.
 3) Substrate is converted to product
 4) Products are released (glucose and fructose) and
make the active site free for another binding.
Fig.3.2 The hydrolysis of sucrose by sucrose
Cont…
The distinguishing feature of an enzyme-catalyzed
reaction is that it takes place within the confines of a
pocket on the enzyme called the active site.
The molecule that is bound in the active site and
acted upon by the enzyme is called the substrate.
 The properties of enzymes
With the exception of some RNA molecules, all enzymes
are globular proteins .
They are biological catalysts:
 they speed up a reaction without being used up, so they can be used over and over again.
They are specific in their action: catalyzing only one type of
chemical reaction.
 They are very specific for both the type of reaction they catalyze, and substrate
.
Why are enzymes specific?
 Because of the conformation of the active site.
 The specificity is détermine by the active site.
 Only substrates with the correct shapes can be bound by the enzyme,
minimizing the frequency of side reactions.
 Most enzymes are stereospecific and act only on one stereoisomeric form of
the substrate.
Cont…

 The structure of the active site accounts not only for the catalytic
activity of the enzyme, but also for its specificity.
 There are four distinct types of specificity:
 1. Absolute specificity:
 the enzyme will catalyze only one reaction.
 Few enzymes exhibit absolute specificity.
 2. Group specificity:
 the enzyme will act only on molecules that have specific functional groups,
such as amino, phosphate and methyl groups.
 3. Linkage specificity:
 the enzyme will act on a particular type of chemical bond regardless of the
rest of the molecular structure.
 4. Stereochemical specificity:
 the enzyme will act on a particular steric or optical isomer
 Cont…
They are effective in small amounts
 A small amount of enzyme can bring about a change in a large amount of
its substrate.
They do not change the positions of chemical equilibrium.
Enzymes do not start chemical reactions but they
accelerate them.
They do not alter the nature or the properties of the
product of the reaction.
Enzymes are affected by :
 pH and temperature.
 substrate concentration and
 inhibitor
Cont…
Enzymes are well suited to their roles in three major ways:
 they have enormous catalytic power,
 they are highly specific in the reactions they catalyze,
and
 their activity as catalysts can be regulated.
Class work
Why are several enzymes needed in
a typical metabolic pathway?
Describe the function of active site
of an enzyme?
 Naming and Classification of Enzymes
Differentenzymes are named in different ways.
Common or working enzyme nomenclature
(naming of enzymes)
Table 3.1Examples of enzymes and the reactions
they catalyze
Cont….
Among various criteria used for naming enzymes are:
Names given arbitrarily at the time of discovery
 Example: digestive enzymes(pepsin, rennin, trypsin and ptyalin).
 Enzymes name ending with ‘in’, indicating that they are basically proteins.
Most commonly enzymes are named by adding the suffix-
‘ase’ to part of the name of the substrate.
 E.g. Lipase (lipid hydrolyzing enzyme)
 Sucrase (sucrose hydrolyzing enzyme)
 Urease- controls the decomposition of urea to CO2 and NH3
 Restriction endonuclease-cut double-stranded DNA.

• Sometimes enzymes are named on the basis of the reaction

that they catalyze. Examples:

• Polymerase (in polymerization:joining similar units together)

 dehydrogenase(removal of hydrogen atoms ).
Cont…
 Some have been named based on the source from which
they were first identified.
E.g. Papayin- from papaya.

 Others are named according to where they act.

 E.g. Intestinal protease- acts on proteins in the intestine.
 The alternative name for pepsin is gastric protease. This
tells you that it acts on proteins and it does so in the
stomach.
Names given because of the property of the product formed.
Example: invertase(sucrase) forms inverted sugar.
EC System of Enzymes Naming
Because of the varied ways in which enzymes had
been named, a systematic nomenclature called the
Enzyme Commission (EC) system is used to name
them.
The International Union of Biochemistry (I.U.B.)
initiated standards of enzyme nomenclature which
recommend that enzyme names indicate both the
substrate acted upon and the type of reaction
catalyzed.
This is used to avoid random naming of enzymes.
All EC names end in –ase.
Modern Classification of enzymes
It is introduced by the International Union of Biochemistry
(I.U.B.) in 1961.
Enzymes has systematic name that specifies :
 the substrate of the enzyme (the substance acted on),
 the functional group acted on, and the type of reaction
catalyzed.
In the systematic naming system, enzymes are divided into
six major classes on the basis of the reaction which they
catalyze.
The systematic names are unambiguous and informative.
Table 3.2- Classification of enzymes
Cont…
Each class of enzymes contains several different,
but related, subclasses.
Each subclass is further divided into sub-subclasses.
Within the sub-subclasses, each enzyme has a
number.
In the systematic naming of enzymes, an enzyme will have
a ‘name’ such as EC 3.4.11.1
 EC stands for Enzyme Commission
 3- stands for main class the enzyme belongs(Hydrolases)
 4- indicates a subclass(tells enzyme action & clue about
substrate)
 11- indicates a sub-subclass(tell us nature of bond)
 1- is the serial number of the type enzyme in its sub subclass.
Cont…
Enzyme EC 3.4.11.1 is:
A hydrolase – all the enzymes in class three
hydrolyze some kind of bond.
A peptidase – all the enzymes in subclass 4 of class
3 are peptidase and hydrolyze peptide bonds
An amino-peptidase – all the enzymes in sub–
subclass 11 of sub-class 4 are amino- peptidases;
they hydrolyze peptide bonds at the amino end of a
polypeptide chain
Leucyl-amino-peptidase – this particular amino-
peptidase is number 1 of this sub-subclass
 Home work
visit site:
◦ www.chem.qmul.ac.uk/iubmb/enzyme/ and
find out more about the naming of enzymes.
What is enzyme 1.1.1.1?
 What do enzymes do for you?
Peoples have been using enzymes for thousands of years.
Peoples unknowingly, used enzymes (in yeast) to make bread
and beer.
These are almost certainly the first uses of ‘enzyme
technology’.
Use of enzyme has been seen in ancient Egyptians where they
were used for the preservation of food and beverages.
The oldest proven records of brewing are about 6000 years old
in the ancient country of Sumeria, in the Middle East.
A document 4000 years old is a Sumerian ‘Hymn (religious
song) to Ninkasi’, who was the goddess of brewing! The
‘hymn’ is also a recipe (instructions for making food) for
making beer.
 
Fig. 3.4 An ancient Egyptian tomb model showing a woman
brewing beer
Cont….
We still use yeast to brew alcoholic drinks, such as ‘tella’,
and bake breads, such as injera.
 When dough is baked to produce the bread,
 the tiny amount of alcohol formed is lost and
 the carbon dioxide expands to make the dough ‘rise’
to form the a loaf of bread.
When beer is brewed, it is the carbon dioxide that is lost
and the alcohol remains.
Cont…
The reasons for using enzymes in industry are:
◦ They allow the reactions to be carried out at much lower
temperatures.
◦ This means less energy (and therefore less money) is
spent on heating the reactants & lessCO2 released.
◦ This can benefit the environment as carbon dioxide is a
greenhouse gas and its accumulation in the atmosphere
can lead to global warming.
Table 3.3Some industrial uses of enzyme technology
Cont…
Using enzymes in industry can help the
environment. Because enzymes allow some
manufacturing processes to be carried out at lower
temperature, less carbon dioxide is produced in
raising the temperature of the reactants.
Fig. 3.6 shows the mass of carbon dioxide
emissions saved in some processes that now use
isolated enzymes rather than the traditional
method.
Fig. 3.6 Carbon dioxide emission reductions in some industrial processes that now use
enzymes
 
 Function of enzymes
How enzymes work? Mechanisms of Enzyme
Catalysis.
The activation energy (or Ea):
 the energy required to start off a chemical reaction.
 The difference between the energy levels of the ground state and the transition
state((high-energy intermediate).
 a higher activation energy corresponds to a slower reaction.
 the lower activation energy, the more molecules have
sufficient energy to pass through the transition state, and,
thus, the faster the rate of the reaction.
 Catalysts enhance reaction rates by lowering activation
energies.
 The role of enzymes is to accelerate the inter-conversion of
S and P by lowering activation energy.
Cont….
 For molecules to react, they must contain sufficient energy to
overcome the energy barrier of the transition state.

Fig. 3.7 Activation energy for unanalyzed reaction and the

same reaction with a catalyst
Cont’d…
Mechanisms of Enzyme Catalysis:
Three mechanisms by which enzymes accelerate
reactions:
◦ 1. maintaining precise substrate orientation
◦ 2. changing substrate reactivity by altering its electrostatic
structure.
 enzymes cannot change the pH of their medium, they do contain
numerous amino acids with acidic or basic side chains. These groups are
capable of donating or accepting protons to and from the substrate,
thereby altering the electrostatic character of the substrate, making it
more reactive.
◦ 3. exerting physical stress on bonds in the substrate to be
broken- Inducing Strain in the Substrate.
How do enzymes lower activation energy
There are two models of enzyme action. These are:
Lock-and-key model, first proposed in 1894 by a
German biochemist named Fischer.
Induced-fit model, proposed in 1958 by Koshland.
Both models suggest that the enzyme catalyzes the
reaction by lowering the activation energy.
However, they differ in the way:
 that they explain how this happens.
 explaining how the substrate binds to the active site of
the enzyme.
 
 Lock and key model
Itproposes that the shapes of the substrate molecules are complementary to that of the
active site.
The useful way of thinking the model is to think of an egg sitting in an egg cup
because the shapes are complementary (Fig.3.8).

A key is complementary to that of the lock it fits.

It explains the high specificity of enzyme activity.
The complementary substrate molecule binds with the active site of the enzyme to
form the enzyme–substrate complex.
The complex causes the reactants to enter a transition state in which the activation
energy of the reaction is lowered. The reaction takes place and the products formed
are released.
This lock-and key–model of enzyme action suggests that the enzyme lowers the
activation energy by providing the alternative pathways for the reaction.
Cont…
For example:
Non-catalyzed pathway:
Reactant A + Reactant B ➞ Product AB
Enzyme-catalyzed pathway:
Reactant A + Reactant B + Enzyme ➞ Intermediate ➞
Product AB + Enzyme
This model sees the enzyme–substrate complex as the
intermediate, which is part of a pathway that requires less
energy than the normal pathway.
However, a weakness of this model is that it does not
explain how the intermediate reduces activation energy
(Fig. 3.9 A and B).
 
Fig. 3.9 - The Lock-and-Key Model
 Induced fit model
 This model suggests that the active site and the substrate aren’t
naturally complementary in shape, but the binding of substrate
molecules produces a conformational change in the active site.
 enzymes have flexible conformations.
 This allows the substrate and active site to bind fully.
 The conformational change also puts the substrate molecules under
tension, so they enter ‘a transition state’
 So, bonds in the reactants are put under strain and break more easily
and rejoin with other bonds to form the products
 This is because of the lowered activation energy.
 Most biologists now prefer the induced-fit model over the lock and-
key model as it explains other properties of enzymes, such as enzyme
inhibition, in a more complete manner than the lock-and-key model.
 
 Efficiency of enzymes
The rate of a chemical reaction is the rate at which reactants
are converted into products.
In the case of enzyme- controlled reaction, this is
determined by how many molecules of substrate bind with
enzyme molecules to form enzyme-substrate complexes.
The number of molecules of reactants that form enzyme–
substrate complexes with each molecule of an enzyme, per
second is the turnover rate.
Table 3.4 The rate enhancement of some enzymes
Structural organization of enzymes
enzymes

Conjugated
(Holoenzyme) Simple enzyme

Apoenzyme(protein Cofactor(non-protein
part) part)

Co-enzymes Prosthetic group Mineral ions

Cont…
Sometimes an active enzyme has two parts these
are:
Apoenzyme-a protein portion that combines with a
cofactor, to form an active enzyme. The protein is
inactive on its own.
 Cofactor- a small non-protein particle essential for
the activity of some enzymes.
The cofactor combines with the apoenzyme to
produce an active enzyme, i.e.
 apoenzyme + cofactor  holoenzyme(conjugated)
(Fig.3.11).
Cont’d..
 Cofactors :
 Cofactors, unlike enzymes, are able at relatively high
temperature.
 Co-factors either remain unchanged at the end of reaction or be
generated by a later process.
 Neither apoenzyme nor the cofactors alone have catalytic
activity.
 cofactors include:
 Coenzymes-are non-protein organic molecules .
 Most are derived from vitamins and give catalytic
activity.
 Not tightly bound or not remaining with apoenzymes
between reaction
 thermostable
 Some coenzymes serve as transient carriers of specific atoms or
functional groups
Table 3.5 Common coenzymes and their functions

Coenzyme Vitamin Enzyme Function

     

Nicotinamide Niacin(Vitamin B3) Oxidoreductase Oxidation or

adenine   in respiration hydrogen transfer
dinucleotide in respiration
(NAD)  

Flavin adenine Riboflavin (vitamin B2) Oxidoreductase Oxidation or

dinucleotide   in respiration hydrogen transfer
(FAD)   in respiration
   
Cont…
Mineral ions- bind loosely with the enzyme to give
it its catalytic activity.
Includes K+, Fe++, Fe+++, Cu++, Co++, Zn++,
Mn++, Mg++, Ca++, and Mo+++.
Enzymes Mineral ions Function

Carbonic Zinc ions (Zn++) Causes CO2 to react with water to form
anhydrase   hydrogen carbonate
   

Alcohol Zinc ions (Zn++) Oxidizes alcohol

dehydrogenase    
 

Cytochrome Copper ions Transfers electrons to oxygen during

oxidase (Cu++ or Cu+) respiration
     

Table 3.6 Enzymes that require mineral ions as cofactors

Cont…
Prosthetic group: thermostable
A coenzyme or metal ion that is very tightly or even
covalently bound to the enzyme protein is called a
prosthetic group.
Examples include, flavin mononucleotide (FMN),
flavin adenine dinucleotide (FAD), thiamin
pyrophosphate, and biotin.
Some enzymes require both a coenzyme and one or
more metal ions for activity.
Factors affecting the functions of enzymes
 Enzyme concentration
 Substrate concentration
 Temperature
 pH
 Salinity
 the presence of Inhibitors or activators
 Temperature
Optimum Temperature :
Temperature at which an enzyme works most efficiently.
 Greatest number of molecular collisions.
 ‘Free’ particles move around more quickly.
 ↑temperature, ↑kinetic energy leads more molecular collision between enzyme and
substrate.
 Human enzymes = 35°- 40°C
Enzymes do not all have the same optimum temperature.
They are adapted to work most efficiently within the organism in
which they are found.
For example, the optimum temperature for enzymes:
Organism optimum temperature
◦ In human beings - 37 °C
◦ in plants growing in the Arctic - < 5 °C
◦ thermophilic bacteria - > 90 °C.
Cont’d
Different enzymes function in different organisms in
different environments.
 Cont’d
Increase T° beyond optimum T°:
◦ Particles within a molecule vibrate more energetically.
◦ Increased energy level of molecules disrupts or strains on
bonds in enzyme & between enzyme & substrate.
◦ Bonds begin to break.
In the case of an enzyme, the shape of the molecule
and the active site in particular, begin to change
The enzyme begin to lose its tertiary structure and
denature
Denaturation = lose 3D shape (3° structure)
The addition of heat can cause a change in the tertiary
structure of a protein (mainly by breaking H-bonds)
Cont’d
Extreme temperature causes the native folded
structure of proteins to uncoil into random
configuration.
The new shape results in a change in the chemical
properties of the protein.
The protein is denatured if the shape change causes
it to lose its normal biological activity.
Heat-induced denaturation is not usually reversible.
PH-induced denaturation is usually reversible .
Denaturation in living cells this is reversible.
 Cont’d
cold: decrease T° below optimum:
 Molecules move slower
 decrease collisions between enzyme & substrate
 Enzymes become inactive
 The PH scale
PH is a measure of the acidity /alkality or hydrogen ion concentration
of a solution.
 It is measured on a scale of 0-14 with pH values .
 below 7 being acidic,
 values above 7 being alkaline and
 a value of 7 being neutral.

The highest H+ concentration and is the most acid.
The lowest H+ concentration and is the most alkaline
As the pH changes away from the optimum toward the acidic
range of the pH scale, an enzyme tends to gain hydrogen ions
from the solution.
As the pH changes away from the optimum toward the
alkaline range of the pH scale, the enzyme tends to lose
hydrogen ions to the solution.
Cont’d
In both cases, changes are produced in the weak
interactions that maintain the shape of the enzyme
molecule and the charge of amino acids that form the
active site.
Significant changes in PH can affect an enzyme
molecule by:
 Breaking ionic bond that hold the tertiary structure
in place and denature enzymes.
 Altering the charge of amino acids that form the
active site.
Therefore substrate can no longer bind to the active
site and so enzyme action decreases
Cont’d
Optimum PH:
 Enzyme action is greatest within a narrow range of pH,
because all enzymes are active .
 Each enzyme has an optimum pH that works more
efficiently.
 Above or below an enzyme’s optimum pH, its activity is
lower.
 The optimum pH of a particular enzyme corresponds to the
pH of its natural environment or depends on localized
conditions.
 For many enzymes, this corresponds to pH values of around
7.
 Majority of enzymes in most mammals function most
efficiently within the PH range of 6-8.
Cont’d
The PH of human gut enzymes :
Human gut region Enzymes optimum PH
Mouth Salivary amylase b/n 5 & 7.5
Stomach Pepsin PH 1.5 and 3.
Small intestine Trypsin PH 8

PH in the mouth varies from being slightly

alkaline(PH 7.5) to acidic(PH5)
 Depends on whether or not we have eaten and also what
we have eaten.
The PH in the stomach can be as low as PH 1.5.
The PH of small intestine is slightly alkaline at PH 7.5
Cont’d

Figure 3.16 The optimum pHs of some human digestive enzymes

Human intracellular enzymes work best at 37°C and pH 7.

 Substrate concentration
At low substrate concentration the reaction proceeds slowly.
This is because:
 there are no enough substrate molecules to occupy all of
the active sites on the enzyme.
As substrate concentration increases, the reaction rate
increase and more enzyme substrate complexes formed.
If the concentration of enzymes remains constant,
increasing the substrate concentration increases the rate of
reaction until all the active sit are occupied.
 As ↑ substrate = ↑ reaction rate
 more substrate = more frequently collide with enzyme
Cont’d…
Each enzyme molecule could be working at maximum
turnover, so the activity of enzyme is likely to remain
constant.
Reaction rate levels off:
 When all of enzymes active site are engaged/occupied
or enzyme is saturated by substrate.
 maximum rate of reaction
 Enzymes concentration
At low enzyme concentrations there are more substrate molecules
than there are available active sites.
Increasing the number of active sites by increasing the concentration
of the enzyme, therefore, effectively increases the rate of the reaction.
At constant large supply of substrate molecules, each enzyme
molecule will work at maximum turnover. Therefore, the reaction rate
will increase. Because the reaction rate will be directly proportional
to the concentration of the enzyme.
as ↑ enzyme = ↑ reaction rate
 more enzymes = more frequently collide with substrate
However, increasing the concentration of the enzyme will not
increase the activity of the enzyme.
Each enzyme molecule will be working at maximum turnover, so the
activity of the enzyme is likely to remain constant.
Cont’d
If the substrate concentration is high and
constant, increasing the enzyme
concentration increases the reaction rate.
Reaction rate levels off:
When substrate molecule becomes limiting
factor therefore, increasing concentration of
the enzyme will not increase the activity of
the enzyme or rate of reaction.
 Inhibitors
Inhibitors: are substances that bind to
enzymes and prevent enzymes from forming
enzyme–substrate complexes and, as a result,
stop, or slow down, the reaction.
They are usually specific and they work at
low concentrations
They block the enzyme but they do not
usually destroy it
There are two main types of inhibitors :
 Reversible inhibitor
 Irreversible inhibitor
 Cont’d
Irreversible inhibitors:
Bind tightly and permanently to enzymes, usually by a
covalent bond
Permanently alter the structure of the enzyme and
inactivating it.
They work at very low concentration of inhibitors.
Examples:
1. the painkiller aspirin binds with the enzyme cyclooxidase-
2, which is an important enzyme in producing
prostaglandins which give the sensation of pain.
Prostaglandin is an unsaturated fatty acid found in all
mammals that performs a similar function to that of
hormones in controlling smooth muscle contraction, blood
pressure, inflammation, and body temperature.
 Cont’d
2. very small concentration of heavy metal ions such
as:
 Mercury(Hg2+)
 Silver(Ag+)
 Arsenic (As+) bind with (-SH) groups in the active

 Lead(Pb2+) site.
3. The nerve gas DFP(Diisopropylfluoro phosphate)-
 Used in warfare
 It combines with serine amino acid at the active site of
enzyme acetyl cholinesterase.
 This enzyme deactivates the
acetylcholine(neurotransmitter) .
 Cont’d
 If acetylcholinesterase is inhibited, acetylcholine
accumulates and nerve impulses can not be stopped,
causing prolonged muscle contraction.
 Paralysis occurs and death may results since the
respiratory muscles are among those affected.
4. a number of insecticides and drugs
Enzymes themselves can act as poisons if they get into the
wrong compartment of the body.
 Reversible inhibitors
Bind only weakly to enzymes and the bond that
holds them breaks easily releasing the inhibitor
Temporarily alter the structure of enzyme. This
allows the enzyme to become active again.
When inhibitor is removed, the enzyme activity is
restored.
There are two main kinds of reversible inhibitors:
 Competitive inhibitors, and
 Non-competitive inhibitors
 Competitive reversible inhibition
Their molecules have shapes which are
complementary to all, or part, of the active site of
an enzyme.
They are often similar in shape to the substrate
molecules.
They can bind with the active site and prevent
substrate molecules from binding.
The binding is only temporary and does not
permanently damage the enzymes.
Cont’d
 Cont’d
The overall effect on the rate of reaction depends on
the relative concentrations of substrate and inhibitor
molecules.
 Examples: If there were 99 substrate molecules for every
inhibitor molecule then:
 99% of the collisions would be between E and S
 The reaction would proceed at 99% of the maximum
rate.
If the ratio were 90 substrate molecules to ten inhibitor
molecules, there would be:
 10% inhibition
 The reaction rate would fall to 90% of maximum.
Cont’d
Increasing substrate concentration effectively
‘dilutes’ the effect of the inhibitor.
If enough substrate is added, the inhibitor is
unlikely to collide with the enzyme.

Fig. 3.18 Effect of substrate concentration on inhibition by a

competitive inhibitor
 Cont’d
Examples:
*The painkiller ibuprofen act on the enzyme cyclo-
oxidase-2, competing with the precursors of
prostaglandins.
 *Cyanide (metabolic poison) acts as a competitive
inhibitor of the enzyme cytochrome oxidase, an
important enzyme in the release of energy in
respiration.
The action of sulphonamides(the first antibiotics).
 Inhibiting an enzyme needed for the synthesis of folic acid
 Non-competitive inhibitors

Non-competitive inhibitor is a molecule that alters

the conformation (the shape or structure of
something) of the active site by binding with the
allosteric site of the enzyme; it prevents the
substrate from binding and inhibits enzyme activity.
An allosteric site is a site other than the enzyme’s
active site.
The term allostric means another space from the
Greek , allos, other, + steros, shape.
 Cont’d
Non-competitive inhibitors :
 Do not compete with the substrate for the active
site . Instead, they bind to the allosteric site.
 Have no structural similarity to substrate.
 Produces a conformational change in the enzyme
& its active site
 The active site can no longer bind with the
substrate to catalyze the reaction.
 
 Cont’d
The effectiveness of non-competitive inhibitors is
not affected by the concentration of the substrate.
Suppose there are enough inhibitor molecules to
bind with the allosteric sites of 80% of the enzyme
molecules, the non-competitive inhibition bind with
their allosteric sites.
80% of the enzyme will be inhibited irrespective of
the number of substrate molecules and
The reaction rate will drop to 20% of maximum.
Non-competitive inhibitors- are particularly
important in regulating metabolic pathways in cells
 Cont’d

Fig. 3.20 The effect of substrate concentration on non-

competitive inhibitor
 Cont’d
Table 3.7 Enzyme inhibition
Substrate Inhibitors Enzymes Inhibition Products

Precursor of Aspirin Cyclo-oxidase- Irreversible inhibition Prostaglandins

prostaglandins 2

Precursor of Ibuprofen Cyclo-oxidase- Competitive inhibition Prostaglandins

prostaglandins 2

Intermediate-A Cyanide Cytochrome Competitive inhibition Intermediate-A

oxidase (ATP is released)

Threonine Isoleucine Threonine Non-competitive Isoleucine

deaminase inhibition(allosteric
inhibition)
The effects of inhibitors on enzyme kinetics.
Enzymes vary greatly in their ability to catalyze
reactions.
The catalytic activity of an enzyme is revealed by
studying its kinetics, that is, the rate at which it
catalyzes a reaction under various experimental
conditions.
In 1913, Leonor Michaelis and Maud Menten reported
on the mathematical relationship between substrate
concentration and the velocity of enzyme reactions as
measured by the amount of product formed (or
substrate consumed) in a given amount of time.
Cont’d
According to the equation, when the substrate
concentration [S] is set at a value equivalent to KM,
then the velocity of the reaction (V) becomes equal
to Vmax/2, or one-half the maximal velocity. Thus,
KM = [S], when V = Vmax/2.
Cont’d
Competitive inhibitor
The affinity of the substrate appears to be decreased
when inhibitor is present
(Km,app >Km)
Competitive inhibitors alter the apparent Km, not the
Vmax
prevents substrate from binding, so increases Km
and no effect on Vmax
   Vmax,app = Vmax
Km,app > Km
 Cont’d
R e a c t io n R a t e
- Inhibitor
Vmax

+ Inhibitor
Vmax
2

Km Km,app
[Substrate]
Cont’d
Non-competitive inhibitors :
The maximum velocity appears to be decreased in the presence
of the inhibitor (Vmax,app <Vmax)
Noncompetitive inhibitors decrease the apparent V max
proportionately to inhibitor concentration, but do not alter the Km
of the reaction since increasing substrate concentration is
ineffective.
Why does Km,app = Km for noncompetitive inhibition? Because:
The inhibitor binds equally well to free enzyme and the ES
complex, so it doesn’t alter apparent affinity of the enzyme for
the substrate.
Vmax,app < Vmax

Km,app = Km
Cont’d
R e a c tio n R a te
Vmax - Inhibitor

Vmax,app
1
V
+ Inhibitor
2 max
1
V
2 max,app

Km [Substrate]
Km,app
Cont’d

The noncompetitive inhibitor reduces Vmax without

affecting KM, whereas the competitive inhibitor increases
KM without affecting Vmax.
END PRODUCT INHIBITION(FEEDBACK
INHIBITION)

The first step (controlled by E1 is often controlled by the end product (E)
Therefore negative feedback is possible
 A→ B→ C → D→ E
The end products are controlling their own rate of production
If the requirement for substance E in the cell decreases, then the concentration of
E will increase. These potentially high concentration could be toxic.
Substance E acts as non-competitive inhibitor, which prevents enzymes catalysing
the reaction that converts A to B. As result, the entire reaction sequence is
halted/stop.
There is no build-up of intermediates (B, C, D)
no unnecessary accumulation of product
final product inhibits the enzyme controlling the first stage of a reaction
sequence
allosteric inhibitor of earlier enzyme
feedback inhibition
Cont’d

Synthesis of amino acid, isoleucine from amino acid, threonine.

isoleucine becomes the allosteric inhibitor of the first step in the pathway
as product accumulates it collides with enzyme more often than substrate does
End of unit questions : Answers

1. a)Non-protein substance that binds with

apoenzymes to form active enzyme
b)
Types of group Organic Protein Bind tightly

Apoenzyme √ √ √

coenzyme √ X X

iron X X X
Cont’d
Factor controlled How controlled Reason for controlling factor

Temperature Water bath Temperature

increases/decreases rate of
reaction.
High temperature may
denature enzymes

PH PH buffer Changes in pH can alter

charge on amino acids in the
active site

[s] Equal strength solution Different concentration s

influence rate of reaction by
affecting number of active site
in use
Cont’d
3. a) Enzyme A 36-38C0
 Enzyme B 76- 78 C0
b) enzyme B
 High optimum temperature
 Thermophilic means heat- loving
c) rate of reaction increases
Increased temperature gives more kinetic energy
Increased collisions between enzyme and substrate
More enzyme-substrate complexs formed
4. i) reaction velocity increases
 Increased number of substrate molecules
 Increased number of collisions with active site
Cont’d
ii)reaction velocity level off
All active sites occupied at any time
Extra substrate molecules have no effect
b)line increase to same maximum but maximum
reached at a lower concentration.
5. a) initial energy of reactants is the same
Final energy of products is the same
b) X and Y represents activation energies; higher fr
unanalyzed reaction.
Because enzymes allows transition state bonds in
reactants put under strain.
Cont’d
c)lower activation energy
So more reactant molecules have sufficient KE
To climb Ea hill so more reaction per second.
6. a) washing powder, leather industry, brewing, baking
b) reduce heat cost
 Reduce emission
c) lower temperatures due to lower energy requirements
reduce amount of fuel burned; reduce CO2 emission.
7.a) i)19C0
ii) may have been quicker just lower or jut higher than
this need more temperatures to be sure.
Cont’d

b) enzymes only speed up the reaction, des not

cause it to happen.
c) i) increased T0 increases KE- more collision
 ii) increased T0 causes increased vibration of
atoms in the enzyme molecule; begins to denature.
8. a)