M.

SC CHEMISTRY -IV

Unit 3

Enzymes
Enzymes are protein molecules in cells which work as catalysts.[1] Enzymes speed up chemical
reactions in the body, but do not get used up in the process.

An enzyme is a catalyst or a chemical produced by cells to speed up a biochemical reaction. It is

typically a protein that acts on certain organic substances by promoting chemical changes through
catalysis. For example, pepsin is an enzyme that aids in digestion.

Enzyme is usually a protein molecule with a characteristic sequence of amino acids that fold to
produce a specific three-dimensional structure, which gives the molecule unique properties. Other
molecule with catalytic activity is ribozyme, an enzyme made of RNA rather than protein.

Almost all biochemical reactions in living things need enzymes. With an enzyme, chemical
reactions go much faster than they would without the enzyme.

The substances at the start of the reaction are called substrates. The substances at the end of the
reaction are the products. Enzymes work on the substrates, and turn them into products.

The first enzyme was found in 1833, by Anselme Payen.

Enzymes

Enzymes are biological catalysts, which activate various biochemical reactions of a living
cell in a highly specific and precise manner.

Enzymology is the study of enzymes.

The name enzyme was coined by Kuhne in 1878.

Pasteur recognised that some microorganisms like yeasts have got a capacity to cause
fermentation in wine. In 1897, Buchner discovered that yeast extract could bring about
fermentation of grape juice, like the living yeast cells. He also observed that the extract has
lost its catalytic activity on boiling. He coined the word zymase for the active principle
involved in the fermentation. The substance on which the enzyme acts is called substrate.

Enzymes are essentially proteins but all proteins are not enzymes.

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Structure of an enzyme

Many enzymes consist of a protein and non-protein component. They are called holoenzymes.
The protein component of a holoenzyme is called apoenzyme and the non-protein component may
be inorganic ions such as Mg2+ and Mn2+ . They are tightly attached to apoenzyme. They are
called activators. The non-protein components may be organic substances such as NAD+,
NADP+and FAD+. They are loosely attached to the apoenzyme. They are called coenzyme or
cofactors. An enzyme will not function without its prosthetic group.

The specific region of the enzyme, which is involved in biochemical reaction, is termed active
site. It is at the active site where the enzyme combines with its substrates to bring about
biochemical reactions. An enzyme may have more than one active site. They are required only in
very small quantities and yet capable of bringing about changes in large number of substrate
molecules.

Nomenclature and Classification of Enzymes:-

 Enzymes are classified according to the reactions they catalyze.

 Except for some enzymes such as pepsin, rennin, and trypsin, most of enzymes are
commonly named by adding a suffix’’ase’’to the particular name of the substrate molecule
it is acting upon. For example:-Lipase catalysis hydrolysis of lipid triglyceroids. Sucrase=
act on sucrose to form glucose and fructose.
 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.
 The International Enzyme commission (IEC) system has divided the enzymes into six
major groups based on type of reaction catalyzed.
 Each enzyme is assigned a code number or EC (enzyme commission number), four-digit
classification number and a systematic name, which identifies the reaction catalyzed.

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Classification of enzymes according to International Enzyme commission (IEC)

EC (enzyme
Name of common enzymes
commission Enzyme group Reaction catalysed
(examples)
number)
Enzymes of this
group add or remove
hydrogen and
oxygen atoms or
electrons from one
substance to another
during the catalysis.
Dehyrogenases, Oxidases and
EC-1 Oxidoreductases(N) Any enzyme which
Oxygenases
catalyzes a reduction
has to also catalyze
the reverse
(oxidation) reaction,
thus the double-
barreled name
"oxidoreductase." .
These enzymes
catalyze the transfer
of a functional group
(methyl-,acyl-
,amino- or Aminotransferase or transaminases -
phosphate) of atoms Amino-ketogroup,Kinases that
EC-2 Transferases. from one molecule regulate metabolism by transferring
to another. A phosphate from ATP to other
common example moleculs e.g. Hexokinase:
involves transfer of a
phosphate between
ATP and a sugar
molecule.
Enzymes, which add Lipases e.g. Glycerol ester hydrolase
water to the substrate Phosphatases e.g.Glucose-6-
and hydrolyze or Phosphatase Choline esterase-
EC-3 Hydrolases.
decompose it to give hydrolysases acetylcholine 4.
products is Peptidases---hydrolyses peptides 5.
hydrolysis . Nucleases e.g.nucleotidase,

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nucleosidase 6. Carbohydrases e.g.

Amylase act on amylose Lactase,
Maltase 7. Enzymes acting on C—N
linkage Urease converts urea into
ammonia, Asparginase. Glutaminase,
Arginase
Non-hydrolytic
addition or removal
Decarboxylase removes CO2 from a
of group from the
EC-4 Lyases. orb keto acids or aminoacids.
substrates. C-C,C-
,Fumrase, aldolase.
N,C-O or C-S bonds
may be split
Change of a
substrate into
isomeric forms (a
Phosphohexoseisomerase.Epimerases
EC-5 Isomerases related form )by
or RacemasesCis, Trans isomerase
intra molecular
rearrangement e.g.
isomerasition
Liagate means to
join together .
Formation of C-C,
C-S, C-O& C-N
bonds by
condensation
reactions. These
Pyruvate carboxylase Acetyl Co A
EC-6 Ligases enzymes carry out
synthetase (acting on fatty acids).
synthetic reactions
where two
molecules joined at
the utilization of a
“high energy
phosphate bond of
ATP. ”

Chemical nature of enzymes :-

i. Enzymes are typically proteins, but certain types of RNA can also serve as catalysts. These
RNA molecules are called ribozymes.
ii. There are 20 common amino acids which make up the building blocks of all known
enzymes. The sequence and number of the 20 amino acid varies in different enzymes.
iii. This sequence is specific for a particular enzyme and determines the properties of the
enzyme. The amino acids are covalently joined together by peptide bonds. Enzymes may
also consist of more than a single polypeptide chain. Each polypeptide chain is called a
subunit, and may have a separate catalytic function.

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iv. Many enzymes have non-protein groups which are necessary for enzymatic activity.
v. Metal ions and organic molecules called coenzymes or cofactors are components of many
enzymes. Coenzymes which are tightly or covalently attached to enzymes are termed
prosthetic groups.
vi. The complete active enzyme; i.e., apoenzyme (protein portion) plus the cofactor
(coenzyme, prosthetic group or metal-ion activator) is called a holoenzyme, while just the
protein part without its cofactor is called the apoenzyme.

image1103

vii. Enzymes composed wholly of protein are known as simple enzymes in contrast to complex
enzymes, which are composed of protein plus a relatively small organic molecule.
viii. According to Holum, the cofactor may be::-
o A coenzyme or cofactor - a non-protein organic substance which is dialyzable,
thermo stable and loosely attached to the protein part.
o A prosthetic group - an organic substance which is dialyzable and thermostable
which is firmly attached to the protein or apoenzyme portion.
o A metal-ion-activator -The substrate forms, a complex with the metal ion and then
reacts with the enzyme. Examples of metal ions which function as cofactors are
Na+, K+, Ca++ Co++, Mg++, Mn++, Cd++, Fe++, Cr+++ and Al+++. .The
separation of enzyme from its metal component generally results in complete loss
of activity. The metal cofactors required for enzyme activity, are called
metalloenzymes e.g. cytochrome oxidase
ix. Sometime terms 'prosthetic group' and 'coenzyme' have been used synonymously.But some
authors, however, distinguish between a prosthetic group and a coenzyme.
x. The prosthetic group remains attached to the apoenzyme while undergoing oxidation and
reduction.The coenzyme on the other hand may undergo reduction while attached to one
apoenzyme, and then migrate to another apoenzyme where it can be oxidized.
xi. Thus NAD, NADP and CoA are considered to be coenzymes while hemes, flavins and
biotin are considered to be prosthetic groups.
xii. Coenzymes act as donor or acceptor of functional groups or atoms removed from or added
to a substrate by an enzyme. They are not consumed during reaction but like enzymes,
coenzymes also remain unchanged at the end of a reaction.
xiii. An enzyme may function independently of a coenzyme but a coenzyme cannot function
without an enzyme.
xiv. Example of coenzymes are :- Nicotinamide adenine dinucleotide (NAD), Nicotinamide
adenine dinucleotide phosphate (NADP), Adenosine triphosphate( ATP), Flavin
mononucleotide(FMN), Flavin adenine dinucleotide(FAD) are hydrogen
carriers,Adenosine triphosphate( ATP) transfers the phosphate group and Coenzyme
A(CoA) the acyl group.
xv. Many coenzymes are closely related to vitamins and are the derivation of vitamins .FAD
and FMN contain Riboflavin (vitamin B2) as a component.Riboflavin is the hydrogen
accepting part of FAD or FMN.

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Properties of enzymes
Enzyme reactions are carried out under mild conditions, they are highly specific, involve very fast
reaction rates, and are carried out by numerous enzymes with different roles.

1. Proteinous nature:

Nearly all enzymes are proteins although some catalytically active RNA molecules have been
identified.

2. Colloidal nature:

In the protoplasm, enzymes exist as hydrophilic colloids. Due to colloidal nature, they are isolated
by dialysis.

3. Substrate specificity:

A given enzyme only catalyzes one reaction or a similar type of reaction. For example, maltase
acts only on maltose while pancreatic lipase acts in a variety of fats. Sometimes, different
enzymes may act on the same substrate to produce different end products. The substrate
specificity of enzyme is based on amino acids sequence in the catalytic site as well as the optical
isomeric form of the substrate.

4. Required in small amounts:

Only a small amount of enzyme is required to carry out chemical reactions. The number of
moles of substrate (starting chemical of a reaction) transformed into one or more products
(into another chemical) per minute by 1 mole of enzyme is known as the turnover number
(or catalytic center activity) of the enzyme. The turnover number varies with the different
kinds of enzymes. The enzyme carbonic anhydrase required for excretion of CO2 from the
body has the highest turnover number of any known enzyme: the formation of 36 million
molecules of H2CO3 per minute by one molecule of enzyme. A new unit for measurement
of enzyme activity in SI is katal (Symbol, “kat”) Onekatal is that catalytic activity in which
the number of moles of substrate transformed into products, in one second.

5. CATALYTIC PROPERTY:

(i) Enzyme require in small concentration for any chemical change,

(ii) They don’t initiate the catalysis but accelerate the rate of catalysis by lowering the activation
energy,

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(iii) They remain unchanged at the end of reaction,

(iv) Their presence doesn’t alter the properties of end products,

(v) Enzymes accelerate the forward or reverse reactions to attain the equilibrium but don’t shift
the equilibrium,

(vi) Usually enzyme catalysed reactions are reversible, but not always,

(vii) They require hydration for activity.

Enzymes speed up the rate of a chemical reaction without being used up in the reaction
they catalyse. So remain unchanged at the end of the reaction. They can be used over and
over again. Enzymes do not alter the amount of product formed. However, the substrate
(S)first binds to the active site of the enzyme to form an enzyme-substrate (ES) complex.

image1104

The ES complex is broken and enzyme is released as soon as the substrate is converted to
the product, thus allowing the enzyme to start all over again.

a. Enzymes bind their reactants (substrates) at special folds and clefts in their structures
called "active sites. Each active site is specifically shaped so that only certain, very
specific substrate molecules can fit into it. The active site on enzyme sucrase for
instance, is shaped so that sucrose molecules fit nearly perfectly inside. In 1890 Emil
Fischer compared the enzyme-substrate relationship to a "lock and-key." This model
was extended by Daniel Koshland Jr. in 1958 by his "induced fit" model, In the lock-
and-key method, the substrate which act as a key fits into an enzyme's active site which
act as keyhole and forms

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image1109

an enzyme-substrate complex.

b. In the induced fit model, the shape of the enzyme changes after binding with the
substrate to form an enzyme-substrate complex the shape change brings about a closer
fit between enzyme and substrate.

image1110

The transition state is the name given to the distorted shape of the active site and
substrate. This sudden change in shape can lead to the breaking of bonds within a
single substrate molecule, forming two new molecules. Conversely, it can also
bring two-substrate molecules close enough together for them to bond with each
other, forming one new molecule. After a new product is formed, the enzyme
releases it, goes back to its original shape, and is able to bind new substrate
molecules and start the reaction once again.

c. Enzymes are highly Specific, each enzyme generally works with only particular kinds
of molecules called the substrate.This specificity is due to the shapes of the enzyme
molecules.This specificity is dictated by the presence of the active site. The molecules

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that the enzymes attract and hold onto are known as the substrate for that particular
enzyme.

image1113

For example, starch is the substrate for the enzyme amylase but maltose is the
substrate for the enzyme maltase. Both of these enzymes are important for
digestion.

d. Enzyme increase the rate of the reaction without changing the equilibrium constant of
a reaction. Enzymes can speed up the same chemical reaction going in opposite
directions.Enzymes can catalyze the breakdown of larger macromolecules into smaller
building blocks (known as a catabolic reaction) but they are equally important in
catalyzing the joining together of building blocks into larger macromolecules (known
as an anabolic reaction).
e. In the absence of enzyme higher activation energy is needed for the conversion of
substrate A to product B. As the concentration of substrate A is increased, the rate of
product B formation increases. In the presence of aenzyme , the reaction rate is
accelerated. All chemical reactions require some amount of energy to get them started.
This energy is known as the activation energy (Ea ).

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image1105

A enzyme speeds up a chemical process by lowering the activation energy which

the substrate must reach before being converted to product. It does this by allowing
a different chemical mechanism or alternative reaction pathway which has a lower
activation energy. Catalysts do not change the energy balance between reactants
and products; catalysts do lower the energy barrier between reactants and products.
By bringing the reactants closer together, chemical bonds may be weakened and
reactions will proceed faster than without the catalyst.

Factors affecting catalytic activity of enzymes

Several factors affect the rate at which enzymatic reactions proceed - temperature, pH, enzyme
concentration, substrate concentration, and the presence of any inhibitors or activators.

A. Temperature: -The rate of an enzyme-catalysed reaction increases steadily with an

increase in temperature, but only to a point. Because every enzyme has a specific optimum
temperature. Most enzyme show maximum activity in a temperature range of 25-40
degrees Celsius.

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image1111

B. Effects of pH: -Every enzyme has its own optimum pH-the most favourable pH value -
the point where the enzyme is most active. However, most enzymes show maximum
activity in a pH range 6.0-7.5. A pH below 7 indicates acidic conditions whereas a pH
above 7 indicates basic conditions. Any shift towards alkaline or acidic side results in a
decrease in enzyme activity because it denatures the enzyme molecule (change it shape).

image1112

Example: - Pepsin (enzyme) of gastric juice has optimum at pH 2.0, while trypsin shows
maximum activity at pH 8.8.

C. Concentration of enzyme :-As the enzyme concentration increases the rate of the reaction
also increases, because there are more enzyme molecules (and so more active sites),
available to catalyse the reaction therefore more enzyme-substrate complexes form.

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image1114
D. Substrate concentration:-The rate of an enzyme-catalysed reaction is also affected by
substrate concentration. As the substrate concentration increases, the rate increases because
more substrate molecules can collide with active sites, so more enzyme-substrate

complexes form.
image1115

For a given enzyme concentration, the rate of reaction increases with increasing substrate
concentration up to a point, above which any further increase in substrate concentration
produces no significant change in reaction rate. Each enzyme has a saturation point. The
enzyme/substrate complex has to dissociate before the active sites are free to accommodate
more substrate. Provided that the substrate concentration is high and that temperature and
pH are kept constant, the rate of reaction is proportional to the enzyme concentration.

Inhibition of enzyme activity by enzyme inhibitors: -Some substances reduce or even stop the
catalytic activity of enzymes in biochemical reactions. They block or distort the active site. These
chemicals are called inhibitors, because they inhibit reaction.

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image1116

Nonspecific Inhibitors -Denaturation: - Change in the spatial arrangement of polypeptide

chain within protein molecule so that its unique structure is change. Due to which physical or
biological properties are changed.
Specific Inhibitors: -Specific Inhibitors exert their effects upon a single enzyme.

1. Competitive inhibitors:-A substance which closely resembles the substrate in molecular

structure competes with the substrate for the active site.The inhibitor may interact with the
enzyme at the active site, but no reaction takes place. Characteristic for this mode of
inhibition is that increasing the concentration of substrate reduces the effect of the inhibitor,
and vice-versa i.e.reversible reaction.
2. Non competitiveInhibitors :-A noncompetitive inhibitor is a substance that interacts with
the enyzme, but usually not at the active site.Rather, the inhibitor alters the shape of the
enzyme in such a way that prevents the substrate from binding to the enzyme. In this mode
of inhibition, the activity of the enzyme is completely blocked by the inhibitor and
increasing the concentration of substrate does not restore enzyme activity.
3. Feedback Inhibition :-Enzyme activity is Suppressed by a product of the sequence of
reactions in which the enzyme is participating. It's like an artificial enzyme inhibitor, the
presence of the final product in a certain key concentration or greater slows down any
further pathway reactions.After the product has been used or broken down, inhibition is
relaxed and formation of the product resumes. Enzymes whose ability to catalyze a reaction
depends on molecules other than the substances on which they act directly are said to be
under allosteric control.

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Mechanism of Enzyme Action

Introduction - Enzyme Characteristics:

The basic mechanism by which enzymes catalyze chemical reactions begins with the binding of
the substrate (or substrates) to the active site on the enzyme. The active site is the specific
region of the enzyme which combines with the substrate. The binding of the substrate to the
enzyme causes changes in the distribution of electrons in the chemical bonds of the substrate
and ultimately causes the reactions that lead to the formation of products. The products are
released from the enzyme surface to regenerate the enzyme for another reaction cycle.

The active site has a unique geometric shape that is complementary to the geometric shape of a
substrate molecule, similar to the fit of puzzle pieces. This means that enzymes specifically
react with only one or a very few similar compounds.

Lock and Key Theory:

 Fischer’s lock and key hypothesis (Emil Fischer 1894)

This theory states that the substrate (key) is perfectly complementary to the active site (lock).
Only if the substrate fits perfectly would it be catalysed, showing that enzymes can only catalyse
specific substrates.

The specific action of an enzyme with a single substrate can be explained using a Lock and
Key analogy first postulated in 1894 by Emil Fischer. In this analogy, the lock is the enzyme
and the key is the substrate. Only the correctly sized key (substrate) fits into the key hole
(active site) of the lock (enzyme).

Smaller keys, larger keys, or incorrectly positioned teeth on keys (incorrectly shaped or sized
substrate molecules) do not fit into the lock (enzyme). Only the correctly shaped key opens a
particular lock. This is illustrated in graphic on the left.

QUES: Using a diagram and in your own words, describe the various lock and key theory of
enzyme action in relation to a correct and incorrect substrate.

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Induced Fit Theory:

 Kushland’s induced fit hypothesis

Although the both theories are recognized, the induced fit is the more accurate of the two.the
induced fit hypotheses explains that the shape of the enzyme molecule changes to fit the shape of
the substrate when they encounters , thereby enabling the substrate to bind more effectively.

Not all experimental evidence can be adequately explained by using the so-called
rigid enzyme model assumed by the lock and key theory. For this reason, a
modification called the induced-fit theory has been proposed.

The induced-fit theory assumes that the substrate plays a role in determining the final
shape of the enzyme and that the enzyme is partially flexible. This explains why
certain compounds can bind to the enzyme but do not react because the enzyme has
been distorted too much. Other molecules may be too small to induce the proper
alignment and therefore cannot react. Only the proper substrate is capable of inducing
the proper alignment of the active site.

In the graphic on the left, the substrate is represented by the magenta molecule, the
enzyme protein is represented by the green and cyan colors. The cyan colored protein
is used to more sharply define the active site. The protein chains are flexible and fit
around the substrate.

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KINETICS OF ENZYME CATALYSIS;

MICHAELIS-MENTEN EQUATION

A reactant in an enzyme catalysed reaction is known as Substrate. According to the mechanism of

enzyme catalysis, the enzyme combines with the substrate to form an enzyme-substrate complex,
as suggested by Henri (1903). He also suggested that this complex remains in equilibrium with the
enzyme and the substrate. Later on in 1925, Briggs and Haldane showed that the steady state
treatment could be easily applied to the kinetics of enzymes.

Let us consider an enzyme catalysed reaction where S represents the substrate, E the enzyme and
ES is the enzyme-substrate complex and P is the products formed. The mechanism of the reaction
can be represented as

Where k1, k2 & k3 are the rate constants for the respective reactions.

The rate of formation of the complex ES is given by the following equation

𝒅 [𝑬𝑺]
= 0 = 𝑘1[E][S] - k2[ES] – k3[ES]
𝒅𝒕

= k1[E][S] – (k2+k3) [ES]

Where [E], [S] & [ES] represents the molar concentrations of the free enzyme, substrate and
the enzyme substrate complex respectively.
Now the [E] cannot be measured experimentally. The equilibrium between the free and the
bound enzyme is given by the enzyme conservation equation…
[E0] = [E] + [ES]
Where [E0] refers to the total enzyme concentration. So

[E] = [E0] - [ES]

Putting the value of [E] in equation we get

𝒅 [𝑬𝑺]
= 0 = 𝑘1{[𝐄𝟎] − [𝐄𝐒]}[S] - k2[ES] – k3[ES]
𝒅𝒕

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𝒅 [𝑬𝑺]
= 0 = 𝑘1{[𝐄0] − [𝐄𝐒]}[S] – (k2+k3) [ES]
𝒅𝒕

As the reaction proceeds, the intermediate complex formed in accordance with the suggested
mechanism decomposes instantaneously according to the same mechanism. On applying the
steady state mechanism, we have
𝒅 [𝑬𝑺]
=0
𝒅𝒕
At the stationary state, equation may be written as

𝑘1{[𝐄0] − [𝐄𝐒]}[S] = (k2+k3) [ES]

𝑘1[𝐸0][𝑆] = { ( k2 + k3) + k1 [S] } [ES]

𝒌𝟏[𝑬𝟎][𝑺]
[ES] = 𝒌𝟐+𝒌𝟑+𝒌𝟏[𝑺]

Dividing both sides by k1 we get

[𝑬𝟎][𝑺]
[ES] = 𝒌𝟐+𝒌𝟑
[ ]
𝒌𝟏 + 𝑺

The rate of formation of products, P is given by

𝑑[𝑃]
r= = k3[ES]
𝑑𝑡
substituting the value of [ES] in equation we get

𝑑[𝑃] 𝒌𝟑[𝑬𝟎][𝑺]
r= = 𝒌𝟐+𝒌𝟑+[𝑺]
𝑑𝑡
𝒌𝟏

𝑘2+𝑘3
where = 𝐾 M is known as Michaelis constant and so the equation may be
𝑘1
𝑑[𝑃] 𝒌𝟑[𝑬𝟎][𝑺]
r= =
𝑑𝑡 𝑲𝑴+[𝑺]
This equation is called Michaelis –Menten Equation.

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Further simplification of this equation can be made. If it is assumed that all the enzyme has
reacted with the substrate at high concentrations the reaction will be going at maximum rate.
There will be no free enzyme left. So that
[E0] = [ES]
So the equation will be

rmax =Vmax = k3 [E0]

where vmax refers to maximum rate. So, Michaelis-Menten Equation can be written as
𝑉𝑚𝑎𝑥[𝑆]
𝑟=
𝐾𝑚 + [𝑆]

If r = Vmax/2, i.e , if the rate of formation of product is equal to half of the maximum rate
at which the reaction proceeds at high concentrations of substrate, then

Km = [S]
Thus, Michaelis constant is equal to that concentration of substrate, S at which the rate of
formation of product is half the maximum rate obtained at a high concentration of substrate.

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