2020-21

Enzymes: Introduction,
history and nomenclature

Dr. Sanjay Yadav

 Catalyst Enzymes
Function Increase/ decrease the rate of -are proteins that increase rate of
a chemical reaction but remain chemical reactions converting substrate
unchanged. into product
Mol Wt Low High
Nature simple inorganic complex proteins
Reaction Rates Slower Faster
Specificity Not Very Specific Very Specific
Condition of Rx. High temp, pressure Mild conditions, physiological pH &
temperature
 Brief History

Until the nineteenth century, it was considered that processes

such as the souring of milk and the fermentation of sugar to
alcohol could only take place through the action of only living
organism.

French chemist Anselme Payen was the first to

discover an enzyme, diastase (now known as
amylase; hydrolyses starch to sugar) in 1833.
He also discovered and named Cellulose.

The beginning of biochemistry is correlated to the discovery of

the first enzyme diastase and Eduard Buchner's first
demonstration of a complex biochemical process alcoholic
fermentation in cell-free extracts.
A few decades later, when studying the
fermentation of sugar to alcohol by yeast,
Louis Pasteur concluded that this fermentation
was caused by a vital force contained within
the yeast cells called "ferments", which were
thought to function only within living
organisms (1876).

In 1877, German physiologist Wilhelm

Kühne (1837–1900) first used the term
enzyme, which comes from Greek meaning
"leavened" or "in yeast", to describe this
process.
James Batcheller Sumner was an American
chemist. He discovered that enzymes can
be crystallized, for which he shared the
Nobel Prize in Chemistry in 1946 with John
Howard Northrop and Wendell Meredith
Stanley.

In 1926, James Sumner crystallized urease

from jack-bean extracts. He was also the
first to prove that enzymes are proteins. It
took almost ten years, to crystalize first
protein.
 ENZYME
-is a substance that acts as a catalyst in living organisms, regulating the
rate at which chemical reactions proceed without itself being altered in
the process.

-catalyze all aspects of cell metabolism, including includes the digestion

of food, in which large nutrient molecules (such as proteins,
carbohydrates, and fats) are broken down into smaller molecules; and
the construction of cellular macromolecules from smaller precursors.

-blood coagulation, healing, diseases, breathing, digestion,

reproduction, and many other biological activities are also carried out
by Enzymes

-many inherited human diseases, such as albinism, phenylketonuria,

hemophilia result from a deficiency/ mutations in a particular enzyme.
 Enzyme Characteristics
Higher reaction rates. The rates of enzymatically catalyzed reactions
are typically 106 to 1012 times greater than those of the
corresponding uncatalyzed reactions) and are at least several orders
of magnitude greater than those of the corresponding chemically
catalyzed reactions.

Milder reaction conditions. Enzymatically catalyzed reactions occur

under relatively mild conditions: temperatures below 100°C,
atmospheric pressure, and nearly neutral pH.

Greater reaction specificity. Enzymes have a vastly greater degree of

specificity with respect to the identities of both their substrates
(reactants)
All known enzymes are proteins. They are high molecular weight compounds
made up principally of chains of amino acids linked together by peptide bonds.
Enzymes can be denatured and precipitated with salts, solvents and other
reagents.

Exceptions: Ribozymes
Ribozymes are enzymes made of RNA that are sometimes also associated with
auxiliary proteins. After their discovery in the early 1980s, ribozymes have been
found in the genomes of many species from all kingdoms of life. Ribozymes
catalyze reactions such as RNA splicing and RNA cleavage.
Enzymes are biological molecules (typically proteins)
that dramatically and significantly speeds up the rate of
the chemical reactions inside the cells by reducing the
required activation energy.
 Activation Energy

For two molecules to react they must collide with one

another. They must collide in the right direction
(orientation) and with sufficient energy.

Sufficient energy means that between them they have

enough energy to overcome the energy barrier to
reaction. This is called the activation energy.

Enzyme can only alter the rate of reaction, not the position of
the equilibrium (E: enzyme; S: Substrate; P: Product)

E + S ⇄ ES ⇄ EP ⇄ E + P
The molecules upon which enzymes may act are called substrates,
and the molecules formed by enzyme action are called products.
Substrate
Products

Enzyme Enzyme

Like all catalysts, enzymes take part in the reaction - that is how
they provide an alternative reaction pathway. But they do not
undergo permanent changes and so remain unchanged at the end
of the reaction.
 Enzyme Mechanism
Lock and Key: proposed in 1890 by Emil Fischer to explain
binding between the active site of an enzyme and a substrate
molecule. The active site was thought to have a fixed
structure (the lock), which exactly matched the structure of a
specific substrate (the key). Thus, the enzyme and substrate
interact to form an enzyme–substrate complex.
 Observations made
by X-ray diffraction
studies have shown
that the active site
of an enzyme is
more flexible than
the lock-and-key
Proposed by Koshland in 1958 to theory would
explain the protein conformational suggest
changes in the binding process.
This model suggests that an enzyme,
when binding with its substrate,
optimizes the interface through
physical interactions to form the final
complex structure.
Enzymes have an active site.
This is part of the molecule that
has just the right shape and
functional groups to bind to one
of the reacting molecules.
All enzymes are proteins. However, without the presence
of a non-protein component called a cofactor, many
enzyme proteins lack catalytic activity. When this is the
case, the inactive protein component of an enzyme is
termed the apoenzyme, and the active enzyme, including
cofactor, the holoenzyme.
 Cofactor

Some cofactors/ coenzymes are only transiently associated with

a given enzyme molecule, so that they function as Co-substrates.

Nicotinamide adenine dinucleotide (NAD+) and nicotinamide

adenine dinucleotide phosphate (NADP+) are examples of
cosubstrates.
 § are non-protein but essential
enzyme partners like simple metal
ions (Cu2+, Fe3+, or Zn2+). Many trace
elements are essential for us, because
COFACTORS they are cofactors (iron, magnesium,
manganese, cobalt, copper, zinc, and
(METAL IONS) molybdenum).

§ Italso explains, in part, the toxic

effects of certain heavy metals. For
example, Cd2+ and Hg2+ can replace
Zn2+ (all are in the same group of the
periodic table) in the active sites of
certain enzymes and thereby render
these enzymes inactive.
The roughly one-third of all enzymes that contain tightly
bound metal ions are termed metalloenzymes. Metal ions
that participate in redox reactions generally are complexed to
prosthetic groups such as heme or iron sulfur clusters.
 § is an organic, non-protein
compound that binds with an
enzyme to catalyze a reaction.
§ are often broadly included in
Coenzymes cofactors, but they are
chemically different.
§ cannot function alone, but can
be reused several times when
paired with an enzyme
 Examples of coenzymes:

1. Nicotineamideadenine dinucleotide (NAD),

2. Nicotineamide adenine dinucelotide phosphate (NADP), and
3. Flavin adenine dinucleotide (FAD).
4. Coenzyme A (CoA) that is involved in the transfer of acyl
groups.

NAD+ is an obligatory oxidizing agent in the alcohol dehydrogenase (ADH)

reaction:

The product NADH dissociates from the enzyme for eventual

reoxidation to NAD+ in an independent enzymatic reaction.
 Prosthetic group

When a cofactor is bound permanently (covalent or non-

covalent bond) to the enzyme and it is difficult to remove
it without damaging the enzyme, it is called a Prosthetic
group.

Ex: A heme prosthetic group is tightly bound to proteins

known as cytochromes through extensive hydrophobic
and hydrogen-bonding interactions together with
covalent bonds.
Three-dimensional structures of c-type cytochromes. The
polypeptide backbones (blue) are shown in analogous
orientations such that their heme groups (red) are viewed
edge-on. The Cys, Met, and His side chains that covalently
link the heme to the protein.
 Examples of Prosthatic groups
Pyridoxal phosphate: is active form of vitamin B6, and
is coenzyme in a variety of enzymatic reactions of amino
acid metaboloism.

Flavin mononucleotide (FMN): FMN is

from riboflavin (vitamin B2) by the enzyme riboflavin
kinase and functions as the prosthetic group of
various oxidoreductases, including NADH dehydrogenase.

Thiamin pyrophosphate: is a thiamine (vitamin

B1) derivative which is produced by the enzyme thiamine
diphosphokinase.
CLASSIFICATIONS
OF ENZYMES
16/04/2021
 CLASSIFICATIONS OF ENZYMES
(CLASSIFIED BASED ON TYPE OF REACTION THEY CATALYZE)
§ Enzymes are commonly named by appending the suffix –
ase to the substrate name or to a phrase describing the
enzyme’s catalytic action.

§ Like,
Urease catalyzes the hydrolysis of urea, and alcohol
dehydrogenase catalyzes the oxidation of primary and
secondary alcohols to their corresponding aldehydes and
ketones by removing hydrogen.

§ Lack of nomenclature rules resulted in two different

names being used for the same enzyme or, conversely, in
the same name being used for two different enzymes.
The International Union of
Biochemistry and Molecular
Biology (IUBMB) formerly
known as International Union
of Biochemistry (I.U.B), in
1961 divided enzymes in to
6 classes.

In August 2018, the IUBMB

added a new enzyme class
EC 7 and named it
Translocases.
v Enzymes are classified and named according to the nature
of the chemical reactions they catalyze. There are seven
major classes of enzymatic reactions as well as subclasses
and sub-subclasses.

v Each enzyme is assigned two names (accepted and

systematic) and a four-part classification number.

v Its accepted or recommended name is convenient for

everyday use and is often an enzyme’s previously used
name.
Its systematic name is used when
ambiguity must be minimized.

Systematic name is

-name of its substrate(s)

-followed by a word ending in -ase
specifying the type of reaction the enzyme
catalyzes according to its major group
classification.
Example
Aconitase: recommended name
Aconitate hydratase: systematic name and
EC 4.2.1.3 : the classification number
(“EC” stands for Enzyme Commission).

(Aconitase catalyzes the stereospecific isomerization of

citrate to isocitrate via cis-aconitate in the tricarboxylic acid
cycle).

Alcohol Dehydrogenase: recommended name

alcohol: NAD+ oxidoreductase: systematic name and
EC 1.1.1.1: the classification number

(The enzyme catalyzes the oxidation of primary aliphatic

alcohols).
For our purposes, the recommended
name of an enzyme is usually adequate.
However, EC classification numbers are
increasingly used in various Internet-
accessible databases and EC classification
numbers can be obtained via the Internet
(http://expasy.org/enzyme/).
 Four-digit nomenclature
IUBMB have recommended four-digit naming of enzymes,
which is preceded by alphabets, EC and separated by dots

First digit: Represents enzyme class like 1 for

Oxidoreductase.

Second and Third digit: describes the kind of reactions

enzyme is catalysing.

Fourth digit: distinguish between them by defining actual

substrate.

EC 1.1.1.1 is alcohol dehydrogenase

EC 1.1.3.4 is Glucose oxidase
mm
 (EC.1.X.X.X)

§ Oxidoreductases are a class of enzymes that catalyze

oxidoreduction reaction.
A– + B → A + B–
where A is the oxidant and B is the reductant.

§ They catalyzes the transfer of electrons from one

molecule, the reductant (electron donor) to another,
molecule the oxidant (electron acceptor).

§ Nicotinamide adenine dinucleotide Phosphate

(NADP) or NAD+ are commonly used cofactors of
oxidoreductase.
Oxidoreductases are of different types

1) Oxidases
2) Dehydrogenases
3) Peroxidases
4) Hydroxylases
5) Oxygenases.
1) Oxidases use Oxygen as electron acceptor but do not
incorporate it into the substrate.

2) Dehydrogenases are enzymes that use other than

oxygen as electron acceptor like NAD+ (Ex: Alcohol
dehydrogenase).

3) Peroxidases catalyzes the reduction of hydrogen

peroxide and uses H2O2 as electron acceptor
(horseradish peroxidase).

4) Hydroxylases add hydroxyl groups to its substrates

(Tyrosine hydroxylase).
 Oxidoreductase…

5. Oxygenases directly incorporates oxygen from

molecular oxygen into organic substrates. Oxygenase can
be

-Mono-oxygenase or
-Di-oxygenase

Monooxygenases, or mixed function oxidase, transfer one

oxygen atom to the substrate, and reduce the other oxygen
atom to water. (Ex: Cytochrome P450 oxidases)

Dioxygenases, or oxygen transferases, incorporate both

atoms of molecular oxygen (O2) into the product(s) of the
reaction (tryptophan 2,3-dioxygenase).
 Transferases (EC.2.X.X.X)
Transferases are enzymes that catalyze the transfer of a
group of atoms, such as amine, carboxyl, carbonyl, methyl,
acyl, glycosyl, and phosphoryl from a donor substrate to an
acceptor compound.

These include methyltransferases and formyltransferases

for transferring single-carbon groups – methyl (CH3) and
formyl (CHO) groups respectively and transaldolases for
transferring a three carbon ketol group.

Ex: Glutathione transferases; peptidyl transferase & DNA

methyltransferases
 Hydrolases (EC.3.X.X.X )
v Hydrolases constitute a very complex ensemble of enzymes
many of which are involved in xenobiotic metabolism.

v They are hydrolytic enzymes and use water to cleave a

chemical bond.

v Activity of hydrolases results in generation of smaller

(monomers) molecules from large (polymers) molecules. Ex.
Lipases hydrolyze lipids and proteases convert protein to
amino acids).

v Ex: Esterases (carboxyleasterases), Proteases (cysteine

endopeptidases), Glycosidases (cellulase), Nucleosidases
(purine nucleosidase), and Lipases.
Acetylcholine esterase (cholinesterase) is an important
example of hydrolases. Acetylcholine is a potent
neurotransmitter for voluntary muscle. After the nerve
impulse moves on, the action of the neurotransmitter
molecules must be stopped by cholinesterase, which
hydrolyzes acetylcholine to choline and acetic acid.

Common Hydrolase Rx.

 Lyases (EC.4.X.X.X )
Lyase is an enzyme catalytically aiding in breaking
various chemical bonds by means of an "elimination"
reaction, other than hydrolysis and oxidation. This
reaction often results in the formation of a new cyclic
structure or a new double bond.

To obtain either a double bond or a new ring, lyase acts

upon the single substrate and a molecule is eliminated.

Lyases are different from other enzymes for only one

substrate is required for the reaction in one direction.
Ex: Enzymes of Citric Acid Cycle (Krebs cycle) and
glycolysis.
In glycolysis, aldolase is example of Lyases, which readily
and reversibly degrade fructose 1,6-bisphosphate into the
products glyceraldehyde 3-phosphate and
dihydroxyacetone phosphate, which is an example of a
lyase cleaving carbon-carbon bonds.
 Isomerase (EC.5.X.X.X )

Isomerases are a general class of enzymes that

convert a molecule from one isomer to another.
Isomerases facilitate intramolecular rearrangements
in which bonds are broken and formed.

The substrate and product of isomerase have same

molecular formula and only one substrate are involved
in isomerase reactions.
Examples

In the second step of glycolysis, an isomerase

converts glucose-6-phosphate (aldose) into one of its
isomers, fructose-6-phosphate (ketose).
 Ligase (EC.5.X.X.X )
Ligase also called Synthetase catalyze the joining of two
molecules, deriving the needed energy from the cleavage of an
energy-rich phosphate bond (ATP to ADP).

Used mostly in molecular biology reactions for Cloning.

Ex: T4 DNA Ligase

 EC7.X.X.X Translocases
Translocases are added as Class 7 in enzyme classification
by IUBMB in August 2018.

Translocases catalyse the movement of ions or molecules

across membranes or their separation within membranes.

First six classes of enzymes did not describe the important

group of enzymes, which catalyze the movement of ions or
molecules across membranes or their separation within
membranes.

Few of these enzymes show hydrolysis activity and

previously classified as ATPases (EC 3.6.3.-), however, the
hydrolytic reaction is not their primary function.
 Examples
ADP/ATP translocase (ANT) imports ADP) from the cytosol and
exports ATP from the mitochondrial matrix, which are key transport
steps for oxidative phosphorylation in eukaryotic organisms.

ADP from the cytosol is

transported back into the
mitochondrion for ATP
synthesis and the
synthesized ATP, produced
from oxidative
phosphorylation, is
exported out of the
mitochondrion for use in
the cytosol, providing the
cells with its main energy
currency.
 Mitochondrial Membrane Proteins

Several proteins encoded by the nuclear genes are

required for mitochondrial functions and they must be
imported into the mitochondria.

Translocase of the outer membrane (TOM)

and translocase of the inner membrane (TIM) mediate the
import of proteins into the mitochondrion.
 Enzyme Kinetics
(Enzymes are highly effective catalysts, commonly enhancing
reaction rates by a factor of 105 to 1017 )
 Enzyme Kinetics: Basics

Active sites (Substrate binding pocket of enzymes)

To catalyze a chemical reaction, the

enzyme forms a complex with
substrate in its active catalytic site.

The active site is usually a three-

dimensional cleft or crevice in the
enzyme formed by one or more amino
acids of the polypeptide chain which
can be located far apart in sequence.
 Active Sites may include distant residues as
shown by different colors

In lysozyme, an enzyme
that degrades the cell
walls of some bacteria,
the important groups in
the active site are
contributed by residues
numbered 35, 52, 62, 63,
101, and 108 in the
sequence of 129 amino
acids
Active Site of RNase A with RNA
Cofactors are also part of active site, which in
combination with functional groups participate in
transforming the substrate into products.

Substrates are bound to enzymes by multiple weak

attractions. The noncovalent interactions in ES
complexes are much weaker than covalent bonds. These
weak reversible contacts are mediated by electrostatic
interactions, hydrogen bonds, and van der Waals forces.
The enzyme and substrate have complementary
shapes. The directional character of hydrogen bonds
between enzyme and substrate often enforces a high
degree of specificity.
ES Complex: Transient complex formed between
substrate and enzyme also known as Michaelis Complex.
The ES complex is a transient very short living complex of enzyme
and substrate, which is formed when enzyme comes in perfect
contact with its substrate.

The substrate causes

a conformational
change, or shape
change, when it
enters the active
site.
Activation energy: Energy required for a molecule to form
an activated complex which is in the transition of making or
breaking a chemical bond. In an enzyme-catalyzed reaction,
this corresponds to the formation of the activated enzyme-
substrate (ES) complex.

An enzyme is able to
reduce the activation
energy by forming a
transition state in a
more favourable
manner.

Enzyme, by nature, create a more "comfortable" fit for the

substrate of a reaction to progress to a transition state.
 Enzymes lower the activation energy, but where does the energy
to lower the activation energy come from?

The binding energy between enzyme and substrate is important

for catalysis. Free energy is released by the formation of a large
number of weak interactions between a complementary enzyme
and its substrate.

Only the correct substrate can participate in most or all of the

interactions with the enzyme and thus maximize binding energy,
accounting for the exquisite substrate specificity exhibited by
many enzymes.

The energy released by the interaction between the enzyme and

the substrate can be thought of as lowering the activation energy.
 Enzyme Activity Vs. Specific Activity

Enzyme activity is a measure of the catalytic ability

and there are two methods to measure enzyme
activity: one of them is to measure the decrease in
substrate concentration in a period of time, and the
other is to measure the increase in concentration of a
product after a period of time.

Enzyme activity = moles of substrate converted

per unit time = rate × reaction volume.
 The specific activity is the
activity of an enzyme per
milligram of total protein
Specific (expressed in μmol min−1
mg−1).
Activity
Specific activity gives a
measurement of enzyme
purity in the mixture.
 Specific Activity of Hepatic Enzyme (ex:
CYP450) will be same or different in different
tissues with same substrate and time and
temperature of reaction ?
Homeostasis

Steady States

Equilibria
Homeostasis refers to the entire internal environment
of the cell, tissue or organism. Like proteostasis refers to
protein homeostasis of cell.

Equilibrium or chemical equilibrium is state of chemical

solutions, which has reached at low energy level and
maintaining it.
Steady state (or dynamic equilibrium) can be
restricted to describing specific mechanisms or
reactions in which amounts of few or all
intermediates remain unchanged while reaction
is going on.

Ex: A cell is in homeostasis because every

mechanism that keeps it alive is in a steady
state. ... Potassium/ Sodium concentration can
be said to be in a steady state.
 Rate of a Reaction
(is the concentration of substrate disappearing or product
produced per unit time)

At constant temperature, the rate of an elementary reaction is

proportional to the frequency with which the reacting molecules
come together.

The proportionality constant is known as a rate constant and is

symbolized k.

For the elementary reaction A → P, the instantaneous rate of

appearance of product or disappearance of reactant, which is
called the velocity (v) of the reaction, is

A=Reactant;
P=Product
 Michaelis-Menten Equation
Michaelis–Menten kinetics is one of the best-known models
of enzyme kinetics.

Named after German biochemist Leonor Michaelis and Canadian

physician Maud Menten.

Michaelis-Menten kinetics, a
general explanation of the
velocity and gross
mechanism of enzyme-
catalyzed reactions. First
stated in 1913, it assumes
the rapid reversible
formation of a complex
between an enzyme and
its substrate (ES).
 Enzyme Kinetics Beginning

In 1902, Adrian Brown was studying Hydrolysis of Sucrose

by enzyme Invertase (b-fructofuranosidase)

Sucrose + H2O Glucose + Fructose

Brown observed that

When concentration of Sucrose >>>>>> Enzyme

The rate of reaction becomes independent of Sucrose

concentration (Zeroth Order Reaction).
 Brown proposed that the overall reaction is composed of
two elementary reactions in which the substrate forms a
complex with the enzyme that subsequently
decomposes to products, regenerating enzyme:

(k= rate constant)

When concentration of Sucrose >>>>>> Enzyme

Second step of the reaction becomes rate limiting (for overall

rate) and the overall reaction rate becomes insensitive to further
increases in substrate concentration.

Rate constant: k1 and k−1 are the forward and reverse rate
constants for formation of the ES complex (the first reaction), and
k2 is the rate constant for the decomposition of ES to P and E
(the second reaction).
 The Michaelis–Menten Equation Assumes that ES
Maintains a Steady State

The Michaelis–Menten equation describes the rate of the

enzymatic reaction as a function of substrate concentration.

The Michaelis–Menten model presumes a simple 2-step

reaction:
Step 1: Binding of substrate to enzymes
Step 2: Catalysis i.e., formation of product from substrates
The Michaelis–Menten Equation Assumption of
Equilibrium

i.e., amount of ES remains constant throughout the reaction

Here Ks is the dissociation constant of 1st step of the

enzyme reaction and ES complex is named as Michaelis
Constant for his contribution.
 The Steady State Assumption

The Michaelis–Menten Equation Assumes that

ES Maintains a Steady State in EQ:1

EQ:1

Under the physiological conditions, when amount of substrate is

too high than Enzyme
In this kinetic scheme, the formation of product from ES is a
first-order process.
The progress curve for the simple enzyme-catalyzed
reaction: With the exception of initial phase of the reaction
the slopes of progress curves for E and ES are essentially
zero as long as S>>E.

With the exception of the initial

stage of reaction, which is over in
milliseconds of mixing E and S, ES
(Michaelis Complex) remains
approximately constant until the
substrate is not exhausted. Hence
the rate of synthesis of ES equals to
its rate of consumption or ES
(Michaelis Complex) maintains a
steady state
Effect of ‘S’ concentration on initial velocity V0
Based on the graph Leonor Michaelis and Maud
Menten in 1913 postulated that the ‘E’ first combines
reversibly with S to form ES complex in a fast
reversible step-

The ES complex then breaks down in a slower second

step to yield the free enzyme and the reaction product
P:

Because the slower second reaction must limit the

rate of the overall reaction, the overall rate must
be proportional to the concentration of ES.
 Michaelis and Menten Equation

[S]: Substrate concentration

V0 : initial velocity
Vmax : maximal velocity
Km: Michaelis constant

All these terms are readily measured experimentally.

Derivation of MM Equation

(Keep in mind)
X

Initial Velocity (V0) = K2 (ES)

ES cannot be measured,
but at anytime free enzyme

Ef = Et-ES
 X

As, we cannot measure amount of ES, we have to convert

it to measurable forms--

Step 1:
The rates of formation and breakdown of ES are
determined by the steps governed by the rate
constants k1 and k-1 + k2 (breakdown)

free enzyme
Rate of ES formation k1([Et] - [ES])[S]
Rate of ES breakdown k-1[ES] + k2[ES]
 Step 2: Steady-state assumption
Initial rate of reaction reflects a steady state in
which [ES] is constant, which means -the rate of
formation of ES is equal to the rate of its
breakdown.
Rate of ES formation = Rate of ES breakdown

multiply ES as common factor

Adding the term k1 [ES][S] to both sides of
the equation
and simplifying gives

Solve the equation for ES

 (from last slide)

Both upper and lower values of

right side are divided by K1
 The term (k2+k-1 )/k1 is defined as the
Michaelis constant, Km.

(k2+k-1)/k1 is Michaelis Constant

(Ratio of ES breakdown to ES formation)

Substituting this into last Equation simplifies

the expression to
Recall the first equation
V0 = K2 (ES)

Substitute ES by last equation

 Further simplification of last equation

Considering that maximum velocity occurs

when the enzyme is saturated
(that is, with [ES]=[Et])
Vmax can be defined as
k2 [Et ]. Substituting this

Michaelis-Menten
Equation (or Rate Equation)
An important numerical relationship emerges
from the MM equation in the case when
V0 is exactly one-half Vmax
So,

when

V0 is exactly one-half Vmax

Practical definition of Km:

Km is equivalent to the substrate

concentration at which V0 is one-half
Vmax .
 Question

concentration is following (All concentrations

are in mM)
For Roll No. 1: Substrate Concentration is 0.10
For Roll No. 2: Substrate Concentration is 0.20
For Roll No. 3: Substrate Concentration is 0.30
For Roll No. 4: Substrate Concentration is 0.40
For Roll No. 5: Substrate Concentration is 0.50

Multiply your roll no with 0.1 to get the substrate

concentration