MODULE 3

ENZYME KINETICS

Metabolism (All of the chemical reactions that occur in a cell.) refers to all of the
organized chemical reactions in a cell. Reactions in which chemical compounds are
broken down are called catabolic reactions (Reactions in which chemical compounds
are broken down.) while reactions in which chemical compounds are synthesized are
termed anabolic reactions(Reactions in which chemical compounds are synthesized.) All
of these reactions are under the control of enzymes.

To live, grow, and reproduce, microorganisms undergo a variety of chemical changes.

They alter nutrients so they can enter the cell and they change them once they enter in
order to synthesize cell parts and obtain energy.

Enzymes are the proteins with catalytic activity. Very few biochemical reactions take
place without enzymes. The key to enzyme function rests in the ability of these proteins
to make otherwise slow and unfavorable reactions proceed at rates greater than 1 million-
fold faster compared to no enzyme at all. Enzymes are substances present in the cell in
small amounts which speed up or catalyze chemical reactions. Enzymes are the biological
catalysts and specialised proteins with catalytic power, regulation, and enzyme
specificity.

Enzymes are globular proteins usually have tertiary and quaternary structure. Enzymes
are a linear chain of amino acids, which give rise to a three-dimensional structure. They
are synthesized by ribosomes that are attached to endoplasmic reticulum Information for
the synthesis of enzymes are carried by DNA. Amino acids are bonded together to form
specific enzyme according to the DNA’s codes.

Structure of enzymes:

On the surface of the enzyme is usually a small crevice that functions as an active site . It
is the site on the surface of an enzyme to which a specific substrate binds or catalytic site
to which one or two specific substrates are able to bind.

Active site of an enzyme is the region that binds with the substrates, co factors and
prosthetic groups and contains residue that helps to hold the substrate. They occupy less
than 5% of the total surface area of the enzyme. It has specific shape due to tertiary
structure of the enzyme. Change in the shape of the protein affects the shape of the active
site and function of the enzyme.

There must be atleast three different points of interaction between the enzyme and the
substrate. These interactions can have either a binding or catalytic function

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The reacting groups of each substrate are brought together in the vicinity of one or more
catalytic sites.

Active site are divided into two types

• Binding site- chooses the substrate and binds to the active site
• Catalytic site- performs the catalytic action of the enzyme

Thus ensuring that the enzyme and the substrate molecules are held in a fixed orientation
w.r.t each other , with the reacting group or groups in the vicinity of catalytic sites

In X-ray diffraction studies it is clearly revealed that a defined pocket or cleft in the
enzyme molecule into which the whole or the part of the substrate can fit.
In 3-D structure, it shows that it contains a portions of a polypeptide chain. The amino
acid residues involved may be widely separated in the primary structures, are brought
together in space because of the twists and turns within the molecule, the binding and the
catalytic sites must be either amino acid side chains. Substrate binding may involve a
variety of linkages, but the bonds formed are usually relatively weak ( non covalent).

Amino acid residues in the active site which do not have a binding or catalytic function
may not contribute to the specificity of the enzyme but their side chains must be of
suitable size, shape and character not to interfere with the binding of the substrate, but
they might interfere with the binding of other, chemically similar substances. Also, the
active sites often includes the polar and the non polar amino acid residues creating an
hydrophilic and hydrophobic microenvironment and it is not found else where on an
enzyme molecule. In general, functions of enzymes not only depend on the binding and
the catalytic sites but also on the environment on which these sites occur

Co factors:

All enzymes are proteins. However, without the presence of a non-protein component
called a cofactor, many enzyme protein lack catalytic activity.

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Cofactors are non-proteinous substances that associate with enzymes to assist them in
their reaction. A cofactor is essential for the functioning of an enzyme. Cofactor can be
organic molecules or ions .

They are two types:

Organic co factors
Inorganic co factors

Inorganic co factors:

• These are inorganic molecules required for the proper activity of enzymes
• Enzyme carbonic anhydrase requires Zn+2 for its activity
Example: Hexokinase has co factor Mg+2

Oranic co facotors
• These are the organic molecules required for the proper activity of enzymes
Example: Glycogen phosphorylase requires the small organic molecule pyridoxal
phosphate

Organic polymers are further classified as

Prosthetic group and co enzyme

Prosthetic group

• It is tightly bound organic co factor

• Examples: Flavins, heme groups, biotin

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Co enzyme:

• It is loosely bound organic co factor

• Examples: NAD+

Metal ions: For the catalysis of certain enzymes, a metal ion is required at the active site
to form coordinate bonds.

Example: Zinc is a metal ion cofactor used by a number of enzymes.

The protein portion of the enzyme, called an apoenzyme . It is the protein portion of an
enzyme requiring a cofactor for its reaction.

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When the enzyme combines with the cofactor to form the whole enzyme or
haloenzyme(An enzyme composed of an apoenzyme and its cofactor.)

Haloenzyme

If the cofactor is an organic molecule, it is called a coenzyme. Some cofactors are ions
such as Ca++, Mg++, and K+; other cofactors are organic molecules called coenzymes (An
organic molecule, such as NAD, FAD, or NADPH, functioning as a cofactor in an
enzyme reaction. ) which serve as carriers for chemical groups or electrons. NAD +,
NADP+, FAD, and coenzyme A (CoA) are examples of coenzymes.

Some enzymes bind tightly that it is difficult to remove without damaging the enzyme it
is sometimes called a prosthetic group. Both protein and the cofactor components may be
directly involved in the catalytic processes taking place.

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Enzyme-Substrate Reaction

In the reaction, enzyme combines with the reactant known as substrate. The binding of
the substrate to the enzyme causes the flexible enzyme to change its shape slightly
through a process to form a temporary intermediate called an enzyme-substrate complex

Enzyme-Substrate Reaction

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They increase the rate of chemical reactions taking place within living cells without
themselves suffering any overall change by lowering the energy of activation, the energy
that must be supplied in order for molecules to react with one another. Enzymes lower the
energy of activation by forming an enzyme-substrate complex allowing products of the
enzyme reaction to be formed and released

Activation Energy and Enzymes

Enzymes work based on the energy changes during a chemical reaction . In a reaction
where the product has a lower energy than the substrate, the substrate naturally turns into
product ( i.e., equilibrium lies in the direction of the product).

In the product formation, substrate must overcome an energy barrier called the activation
energy. Larger the activation energy, i.e., slower the reaction will be. This is because only
a few substrate molecules will have sufficient energy to overcome the activation energy
barrier.

Most biological reactions have large activation energies, so in the absence of enzyme,
progress is slow.

Example: 2H2O2---- 2H2O +O2

For catalase reaction, activation energy is -86kJ/mol with no catalyst.

In the presence of inorganic catalyst, activation energy is 62kJ/mol

And in the presence of enzyme catalyse, it is 1kJ/mol

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Type of enzymes and its classification

Intracellular enzymes:

• They are synthesized and retained in the cell for the use of cell itself
• They are found in cytoplasm, nucleus, mitochondria and chloroplast
• Example:Oxydoreductase catalyses biological oxidation
• Enzymes involved in reduction in the mitochondria

Extracellular enzymes:

• Enzymes are synthesis in the cell but are secreted from the cell to work externally
• Example: Digestive enzyme produced by the pancreas are not used by the cells
in the pancreas but are transported to the duodenum

Classification of enzymes:

According to the International Union of Biochemists (I U B), enzymes are divided into
six functional classes and are classified based on the type of reaction in which they are
used to catalyze.

The six kinds of enzymes are

• hydrolases,
• oxidoreductases,

• lyases,

• transferases,

• ligases and

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 • isomerases.

Oxidoreductases

:The enzyme Oxidoreductase catalyzes the oxidation reaction where the electrons tend to
travel from one form of a molecule to the other.These catalyze oxidation and reduction
reactions,

• e.g. pyruvate dehydrogenase, catalysing the oxidation of pyruvate to acetyl

coenzyme A.

Transferases:

• These catalyze transferring of the chemical group from one to another compound.
• The Transferases enzymes help in the transportation of the functional group
among acceptors and donor molecules.

• An example is a transaminase, which transfers an amino group from one molecule

to another.

Hydrolases:

• They catalyze the hydrolysis of a bond.

• Hydrolases are hydrolytic enzymes, which catalyze the hydrolysis reaction by
adding water to cleave the bond and hydrolyze it.
• . For example, the enzyme pepsin hydrolyzes peptide bonds in proteins.

Lyases:
• These catalyze the breakage of bonds without catalysis,
• Adds water, carbon dioxide or ammonia across double bonds or eliminate these to
create double bonds.
• e.g. aldolase (an enzyme in glycolysis) catalyzes the splitting of fructose-1, 6-
bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.

Isomerases:

• They catalyze the formation of an isomer of a compound.

• The Isomerases enzymes catalyze the structural shifts present in a molecule, thus
causing the change in the shape of the molecule.
• Example: phosphoglucomutase catalyzes the conversion of glucose-1-phosphate
to glucose-6-phosphate (phosphate group is transferred from one to another
position in the same compound) in glycogenolysis (glycogen is converted to
glucose for energy to be released quickly).

Ligases:

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 • Ligases catalyze the association of two molecules.
• The Ligases enzymes are known to charge the catalysis of a ligation process.
• For example, DNA ligase catalyzes the joining of two fragments of DNA by
forming a phosphodiester bond.

A. Characteristics of Enzymes

a. Chemically, enzymes are generally globular proteins. (Some RNA molecules called
ribozymes(An enzyme composed of RNA. Usually found in the nuclear region of a cell.)
can also be enzymes. These are usually found in the nuclear region of cells.) Their
molecular weight generally ranges from 14000 to 400000 daltons

b. Enzymes are catalysts (A substance which speeds up a chemical reaction.) that

breakdown or synthesize more complex chemical compounds. They allow chemical
reactions to occur fast enough to support life. Enzymes speed up the rate of chemical
reactions because they lower the energy of activation, the energy that must be supplied in
order for molecules to react with one another and do not undergo any chemical change
during the complete reaction. Enzymes are very efficient. An enzyme generally can
typically catalyze between 1 and 10,000 molecules of substrate per second.

c. Enzymes are only present in small amounts in the cell since they are not altered
during their reactions. A small amount of enzyme added to a reaction also can speed up
the process/ accelerate the chemical reactions

d. Enzymes are highly specific for their substrate in the reaction. Generally there is one
specific enzyme for each specific chemical reaction.

e. Enzymes cannot change the free energy of a reaction, which means they do not change
whether the reaction is absorbing energy or releasing overall.

f. It will not affect the nature and properties of the end product.

g. They are colloidal in nature.

h. Enzymes are sensitive to change in pH, temperature and substrate concentration.

****Enzymes get affected by temperature, which means they can degenerate at high or
low temperatures and work efficiently at optimum temperature.

****Enzymes are also pH sensitive, which means highly acidic or highly basic nature is
not suitable for the enzymes to survive and perform efficiently

B. Enzyme Activity

Enzyme activity is affected by a number of factors including:

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a. the concentration of enzyme

Assuming a sufficient concentration of substrate is available, increasing enzyme

concentration will increase the enzyme reaction rate, because there are more enzyme
molecules available to catalyze the reaction. Therefore, more enzyme substrate complex
form.

b. the concentration of substrate

At a constant enzyme concentration and at lower concentrations of substrates, the

substrate concentration is the limiting factor. As the substrate concentration increases, the
enzme reaction rate increases. However, at very high substrate concentrations, the
enzymes become saturated with substrate and a higher concentration of substrate does not
increase the reaction rate.

c. the temperature

Each enzyme has an optimum temperature at which it works best. A higher temperature
generally results in an increase in enzyme activity. As the temperature increases,
molecular motion increases resulting in more molecular collisions. If, however, the
temperature rises above a certain point, the heat will denature (When an enzyme loses its
functional, three-dimensional shape and can no longer react with its substrate. ) the
enzyme, causing it to lose its three-dimensional functional shape by denaturing its
hydrogen bonds. Cold temperature, on the other hand, slows down enzyme activity by
decreasing molecular motion.

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d. the pH

Each enzyme has an optimal pH that helps maintain its three-dimensional shape. Changes
in pH may denature enzymes by altering the enzyme's charge. This alters the ionic bonds
of the enzyme that contribute to its functional shape.

For most enzymes, optimum pH is about 7 to 8, but a few enzymes can work at extreme
pH, such as gastric protease ( pepsin) in stomach, with an optimum pH of 1.

e. salt concentration

Each enzyme has an optimal salt concentration. Changes in the salt concentration may
also denature enzymes.

f. Product concentration

Accumulation of reaction products generally decreases the enzyme velocity. For certain
enzyme the products combine with the active site of enzyme, thus inhibit the enzyme
activity.

g. Effect of light and radiation

Exposure of enzymes to ultraviolet , beta, gamma and x-rays inactivates certain enzymes
due to the formation of peroxidases.

The reactants of the enzyme-catalyzed reactions are termed as substrates are each enzyme
is quite specific in character acting on a particular substrate or substrates to produce a
particular product or products.

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Types of Enzyme specificities:

Enzymes derive tremendous catalytic power from dramatic acceleration of normally slow
biochemical reactions. Enzymes are also usually highly selective in terms of their
substrates, be it another protein or other compound such as lipids, sugars, or nucleic
acids.

However, degrees of specificity exist within particular classes of enzymes. Let's consider
proteolytic enzymes. These are enzymes that cleave peptide bonds and, thus, function by
breaking down proteins.

Some of these enzymes, such as subtilisin, will cleave peptide bonds regardless of the
specific amino acids that are joined by that particular bond. Trypsin, on the other hand,
only cleaves peptide bonds on the carboxyl-side of lysine and arginine residues.

Another example of a highly specific enzyme is DNA polymerase I which synthesizes

DNA from a single stranded DNA template. This enzyme is very good in generating an
accurate nucleotide sequence because it contains an editing function that corrects any
misincorporated base into the sequence. Thus, the wrong nucleotide is inserted into the
newly synthesized strand less than once in a million times (nucleotides).

Hence ,the characteristic feature of enzymes is that they are specific in action. Enzyme
specificity is based on the ability to choose a particular substrate, and it is a molecular
regulation mechanism.
This can be attained only with the similar shapes between the substrate and the enzyme,
which is the structural complementarity. One of the properties of enzymes that makes
them so important as diagnostic and research tools is the specificity they exhibit relative
to the reactions they catalyze. A few enzymes exhibit absolute specificity; that is, they
will catalyze only one particular reaction. Other enzymes will be specific for a particular
type of chemical bond or functional group

Enzyme specificity can be categorized into groups:

1. Bond specificity
2. Group specificity
3. Substrate specificity
4. Optical or stereospecificity
5. Geometrical specificity
6. Cofactor specificity

Bond Specificity: Specific to substrate having similar bonds and similar structures
Specificity is less. Known as relative specificity.

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Examples
• Amylase can hydrolyze alpha-1,4 glycosidic linkage in starch and glycogen
• Lipase can hydrolyze ester bond between glycerol and fatty acid in fats

Some enzymes exhibit group specificity- they may act on several different though closely
related, substrates to catalyze a reaction involving a particular chemical group.

Group specificity: Enzymes are specific to the type of bond and the groups that surround
it. This category shows more specificity than bond specificity. Also known as moderate
specificity and structural specificity.
Catalyse same type of reaction for similar substrates. Their action is group specific.
the enzyme will act only on molecules that have specific functional groups, such as
amino, phosphate and methyl groups.

Example: Endopeptidases and exopeptidases.

Hexokinases transfer phosphates to hexoses
Pepsin hydrolyze a peptide bond in which the amino group is contributed by an aromatic
amino acid ( phenyl alanine, tyrosine and tryptophan).

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Alcohol dehydrogenase- will catalyze the oxidation of a variety of alcohols
Hexokinase – will assist the transfer of phosphate from ATP to several different
hexose sugars.

Substrate specificity ( Absolute specificity): Enzymes show specificity towards one

substrate and one reaction in this type. For this, specificity is high. Also known as
absolute specificity.

the enzyme will catalyze only one reaction i.e., enzyme act only on one substrate

Example: Lactose, sucrose, maltose have a specific function and do not perform any
other reaction.

Maltase acts on maltose

Sucrase acts on sucrose

glucokinase catalyses the transfer of phosphate from ATP to glucose and to no other
sugar (although the specificity of the so-called ‘glucokinase’ found in liver is less clear-
cut, in contrast to the true glucokinases of bacteria and invertebrates).

Uncatalyzed reactions often give rise to a wide range of products , but enzyme- catalysed
reactions are product- specific as well as being substrate-specific.

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Optical or stereospecificity: In addition to showing chemical specificity, enzymes
exhibit stereo chemical specificity – if a substrate can exist in two stereo chemical forms,
chemically identical but with a different arrangement of atoms in 3-D space, then only
one of the isomers will undergo reaction as a result of catalysis by a particular enzyme.

Enzymes are specific to the substrates and their optical configuration. Stereo is the
arrangement of atoms in space, and the specificity is said to be very high. the enzyme will
act on a particular steric or optical isomer

Example:
• Starch can be digested with alpha glycosidase but cellulose cannot be digested by
the same enzyme. As the sugars in cellulose are in beta orientation to the cellulose
digestion needs beta glycosidase.
• L- amino acid oxidase can mediates the oxidation of L-amino acids to oxoacids
but not D- amino acid. a separate enzyme , D- amino acid oxidase , is required for
the corresponding oxidation of D-amino acids.

Even greater specificity is shown by the fungal enzyme glucose oxidase, which catalyses
the reaction.

No other naturally occurring sugar, including -D- glucose & -D- galactose , can
be acted upon to any appreciable extent.

The only enzymes which act on both stereoisomeric forms of the substrate are those
whose function is to interconvert L- & D- isomers.

Ex: alanine racemase, which catalyses the reaction:

L-alanine D-alanine

Enzyme catalysed reactions may yield stereospecific prkoducts even when the substrate
posseses no asymmetric carbon atom.

Ex: the action of glycerol kinase on glycerol always results in the production of L-
glycerol-3-phosphate(sn-glycerol-3-phosphate)

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Substrates are bound to enzymes by multiple weak interactions: interactions between an
enzyme and its substrate involve relatively weak, non-covalent bonding. These
interactions typically include electrostatic bonds, hydrogen bonds, van der Waals forces,
and hydrophobic interactions. The enzyme and the substrate often have complementary
shapes that allow for these interactions to take place.

 Catalytic sites form clefts or crevices: substrate molecules are often bound within
a cleft or crevice of an enzyme. Water is, in many instances, excluded from the
cleft (unless it is involved in the reaction) and the overall nonpolar character of
the cleft can enhance binding of the substrate.

However, the cleft can also contain polar residues that create a microenvironment with
these amino acids taking on catalytic properties. This is an important exception to the rule
that the core of a protein is primarily made up of hydrophobic residues.

 The specificity of binding depends on the precisely defined arrangement of atoms

in an active site: to fit into a catalytic site a substrate must have a matching shape.

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 • Any two molecules have to collide for the reaction to occur along with the right
orientation and a sufficient amount of energy.
• The energy between these molecules needs to overcome the barrier in the
reaction. This energy is called activation energy.
• Enzymes are said to possess an active site. The active site is a part of the molecule
that has a definite shape and the functional group for the binding of reactant
molecules.
• The molecule that binds to the enzyme is referred to as the substrate group. Active
site of the enzyme molecule will be specific for the binding of the substrate
molecules
• The substrate and the enzyme form an intermediate reaction with low activation
energy without any catalysts.
• Each enzyme molecule has an active site for specific binding of substrate
molecules. The enzyme works by altering the activation energy of the reaction.

The catalytic site of an enzyme can be described as follows

(i) The substrate process to the active site of the enzyme, fitting into it.
(ii) Binding of the substrate induces the enzymes to alter its shape leading to the
formation of the Enzyme substrate (ES) complex.
(iii) The active site of the enzyme now is near the substrate and breaks its chemical bonds
and a new enzyme product complex is formed.
(iv) The enzyme releases the products of the reaction and the free enzyme is ready to bind
to another molecule of the substrate and run through the catalytic cycle once again.

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 • Enzymes are biocatalysts, which are high molecular weight
proteinous compounds.
• It enhances the reactions which occur in the body.
• It helps the substrate by providing the surface for the reaction to
occur.
• The enzyme comprises hollow spaces occupying groups such as -
SH, -COOH, and others on the outer surface.

FISCHER “LOCK-AND-KEY” Hypothesis:

Emil Fischer in early 1890’s suggested that enzyme specificity impilies the presence kof
comjkplementary structural features between the enzyme and the substrate : a substrate
might fit into its complementary site on the enzyme as a key fits into a lock . This is
entirely consistent with the more detailed aspects of active site structure .

According to the lock-and-key model, all structures remain fixed throughout the binding
process.

Representatiion of the interaction between an enzyme and its substrate, according to the
lock-and –key model.
The single substrate is bound at the two points, bringing the reacting group into the
vicinity of two different catalytic sites .

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KOSHLAND “INDUCED –FIT” Hypothesis:

The lock-and-key hypothesis explains many features of enzymes specificity, but takes no
acaount of the known flexibility of proteins.

X-ray diffraction analysis and data from several forms of spectroscopy, including
magnetic resonance(nmr), have revealed differences in structure between free and
substrate- bound enzymes. Thus, the binding of a substrate to an enzyme may bring about
a conformational change, i.e., a change in 3-D structure but not in primary structure. The
bonds formed between a substrate and its binding sites may have replaced previously
existing linkages between each binding site and neighbouring groups on the enzyme .
Also, the presence of a substrate at the active site may exclude water molecules and thus
make the region more non-polar.Both of these factors could be responsible for some
degree of change in tertiary structure takng place.

A more refined model has been proposed, called the "induced fit" model by Koshland
in1958. Essentially, an enzyme assumes a complementary shape to that of its substrate
only after the substrate binds to the enzyme. i.e., the structure of a substrate may be
complementary to that of the active site in the enzyme-substrate complex, but not in the
free enzyme: a conformational changes takes place in the enzyme during the binding of
substrate which results in the required matching of structures

This is a more dynamic scenario compared to the lock and key hypothesis.

• The substrate which has an opposite charge of the enzyme fits into these spaces,
just like a key fits into a lock.
• This substrate binding site is called the active site of an enzyme (E).

• The favourable model of enzyme-substrate interaction is called the induced-fit

model.

• This model states that the interaction between substrate and enzyme is weak, and
these weak interactions induce conformational changes rapidly and strengthen
binding and bring catalytic sites close enough to substrate bonds.

The induced-fit model states a substrate binds to an active site and both change shape
slightly, creating an ideal fit for catalysis.

Enzymes promote chemical reactions by bringing substrates together in an optimal

orientation, thus creating an ideal chemical environment for the reaction to occur.

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The induced –fit hypothesis essentially requires the active site to be floppy i.e., flexible
and not rigid and the substrate to be rigid, allowing the enzymes to wrap itself around the
substrate, in this way bringing together the corresponding catalytic sites and reacting
groups. In some respects, the relationship between a substrate and an active site is similar
to that between a hand and a woolen glove: in each interaction the structure of one
component ( substrate or hand) remains fixed and the shape of the second
component( active site or glove) changes to become complementary to that of the first.

Such a mechanism helps to achieve a high degree of specificity for the enzyme. Hence,
there is a greater range of substrate specificity. Model is consistent with a wider range of
enzyme

Advantages
• Support enzymes which acts on different substrates of different conformation
• Enhance fidelity of molecular recognition in the presence of competitor via
conformational proof reading
• Much accepted as enzymes are not rigid and different conditions promotes
differential interactions, if it was rigid all the actions were same as always.

In the lock-and –key mechanism , the active site is always structurally intact, with the
catalytic sites aligned and freely accessible. Thus a suitable reacting group, whether part
of an appropriately bound substrate or not, can come into contact with the region of
catalytic activity and some degree of reaction takes place.

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The lock-and-key theory of enzyme action proposes that the enzyme's active site and the
shape of the substrate molecule are complementary to one another. This allows the
substrate to fit into the enzyme, like how a key would fit into a lock. If the substrate
doesn't fit, then the enzyme will not act on it.

• Active site has a rigid shape

• Only substrates with the matching shape can fit.
• Substrate is a key that fits the lock of the active site of the enzyme
• Ex: Amino acid R- groups of enzyme helps to mediate the interaction of active
site and substrate
• This is old model, does not work for all enzymes

Limitations:
• Generally applicable to enzymes that work on single type of substrate
• Indicates the active site as a rigid shape but in actual it is flexible
• Rigid shape is insensitive to environment modification for substrate binding

In the induced-fit mechanism, on the other hand, different catalytic components might be
separated by a considerable margin in the free enzyme, minimizing the risk of a chance
collision of a reactive group with both of them; it is also possible that access to the
catalytic groups of the free enzyme might be blocked. Only when a binding group of the
substrate is recognized by the corresponding site of the enzyme and the binding process
proceeds does the conformational change take place which results in all the relevant
groups in the substrate and the enzyme coming together.

A similar binding group in a substance other than the substrate might trigger off a
conformational change but , in general, this would not result in catalytic groups being

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brought together in the vicinity of an appropriate reacting group, so no reaction would
take place . this would be termed non –productive binding.

Units of Enzyme activity:

Enzymes are expressed as activities. Activities of the enzymes are

expressed as (1) katal (2) International Unit (3) Arbitrary unit (4) Specific
activity

• One unit of enzyme‘s catalytic activity is defined with enzyme unit, or

international unit for enzyme (symbol U or IU)

1 U (μmol/min) is defined as the amount of the enzyme that catalyzes the

conversion of one micromole (1 μmol ) of substrate per minute under the specified
conditions of the assay method such as pH and temperature ( usually 25 0C) and
substrate concentration.

• The specific activity of an enzyme is expressed as the number of units per

milligram of protein.

Specific activity of enzyme = ( total activity units/ total protein in mg)

• The enzyme unit was adopted by the International Union of Biochemistry in

1964.
• IN 1978 , katal was favored as the unit for enzyme activity based on time as
seconds. One katal is the enzyme activity that converts one mole of substrate
per second under specified assay conditions

( OR)

It is the amount of the enzyme which catalyze the conversion of one mole of
substrate into product in one second under the experimental conditions.

One katal is abbreviated as kat.

1 U = 1 μmol/min = 1/60 μmol/s ≈ 16.67 nmol/s;

16.67 nkat = 16.67 nmol/s;

Therefore, 1 U = 16.67 nkat

Arbitrary unit is defined as the amount of enzyme which transforms one

micromole of substrate into product per minute under assay conditions.

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 • Turn over number (kcat)

It is defined as the limiting number of chemical conversions

of substrate molecules per second that a single active site will execute for a
given enzyme concentration [ET] for enzymes with two or more active sites.

For enzymes with a single active site, kcat is referred to as the catalytic constant.

limiting reaction rate Vmax and catalyst site concentration e0 as follows

• It is the number of substrate molecules transformed per minute by one enzyme

molecule.

Example : Catalase turnover number = 6 X 106 / min

Enzymes can be usually categorized in two ways:

--positive and negative catalysts

• Reactions can be sometimes useful or harmful, or not useful. So, according to the
requirement, there are various catalysts.
• If the reaction is useful and slow, then a positive catalyst is used, which can
increase the reaction rate.
• If the reaction is not useful and it is either slow or fast, then a negative catalyst is
used, which can reduce the rate of reaction or stop the reaction.
• Positive catalysts are otherwise called promoters, and negative catalysts are
otherwise called inhibitors.

Promoters are the chemical substances that help in increasing the activity of enzymes.

Inhibitors are chemical substances that help in decreasing the activity of enzymes.

Linkage specificity: the enzyme will act on a particular type of chemical bond
regardless of the rest of the molecular structure.

Geometrical specificity: This has less specificity as the enzyme can act as a different
substrate having a similar size or geometry.

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Example: Alcohol dehydrogenase can act on any alcohol functional group like ethanol,
methanol, and propanol.

Co-factor specificity: This has greater specificity, and in this, enzymes act specifically
towards a specific substrate and co-factor. The reaction can be allowed if only the
substrate and the co-factor are the same and match, which is highly specific. If the co-
factor is absent, the enzyme remains inactive even though many substrate molecules are
present.

Enzymes work according to their specificity and perform various biological and
metabolic functions.

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Dual specificity:

26
Kinetics of unanalyzed chemical reactions:

Kinetics is the study of reaction rates and the factors influencing them. It is
not concerned with the chemical nature of the changes taking place.

Kinetics of the reactions are based on law of mass action proposed by

Guldberg and Waag in 1867. The law states that the rate of reaction is
proportional to the product of the activities of each of the reactant, each
activity being raised to the power of the number of molecules of that
reactant taking part, as indicated by the reaction equation.

For the reaction, aA + bB ------- products

Rate is proportional to the ( activity of A) a X ( activity of B) b

Rate of the reaction at time t = - d[A]/dt = -d[B]/dt= +d[P]/dt= k [A][B].

From the law of mass action, concept of order of the reaction was developed.

Initial velocity studies

• Concentration of the substrate [S] present will greatly influence the

rate of the product formation termed as the velocity (v) of the
reaction. Studying the effects of [S] on the velocity of the reaction is
complicated by the reversibility of enzyme reactions.

Example: conversion of product back to substrate.

• To overcome this , use of initial velocity (Vo) measurements are used.

At the start of the reaction, [S] is in large excess of [P], thus the initial
velocity of the reaction will be dependent on substrate concentration.
• Initial velocity is the initial linear portion of the enzyme reaction
when less than 10% of the substrate has been depleted or less than
10% of the product has formed.
• it allows to measure the rate of the reaction at the very beginning,
when the substrate concentration is relatively high and the enzyme-
substrate complex is forming at its maximum rate.
• The initial velocity is the rate of an enzymatic reaction prior to the
formation of product. This is measured because it allows the
biochemist to ignore the reverse rate of the enzymatic reaction which
makes collecting measurements and kinetic analysis easier

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Enzyme inhibition:

• Enzyme inhibition is a reaction between the molecule and the enzyme that
blocks the action of the enzyme either temporarily or permanently.
• Enzyme inhibitors tend to decrease the rate of an enzyme-catalyzed reaction
due to changes in pH, ionic strength and co factors.

There are two broad classes of enzyme inhibition:

Reversible inhibition and irreversible inhibition

Reversible inhibition- The inhibitor binds non covalently with the enzyme and the
enzyme inhibition can be reversed if the inhibitor is removed.

• They can be removed from [EI] complex by dialysis.

• Used to understand the enzyme mechanism and metabolic pathways.
• They obey M-M kinetics.

Reversible inhibition is further subdivided into

• Competitive
• Uncompetitive
• Non competitive

Examples: Succinylcholine

Irreversible inhibition- Inhibitors binds covalently with the enzymes and inactivate
them, which is irreversible.

• these inhibitors are usually toxic substances.

• They cannot be removed by [EI] complex.
• They are used to identify the amino acids present in the active site.
• They do not obey M-M kinetics
• Example: Aspirin; Penicillin

In competitive inhibition, an inhibitor molecule is similar enough to a substrate that it

can bind to the enzyme's active site to stop it from binding to the substrate.

• It “competes” with the substrate to bind to the enzyme.

• It closely resembles the chemical structure and molecular geometry of the
substrate.

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• If the inhibitor( I) occupies the active site of the enzyme ,it prevents the binding
of the substrate to the enzyme and no catalysis takes place.

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 • In the presence of the Competitive inhibitor , Michaelis- Menton equation
becomes

Km value is increased by the factor . So the Km value increases whereas

Vmax remains unchanged.

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Degree of competitive inhibition depends on relative concentration of substrate and the
inhibitor.

Increase in the substrate concentration and keeping the inhibitor concentration constant,
the amount of inhibition decreases and conversely a decrease in substrate concentration
results in an increased inhibition. Thus the inhibition could be overcome by a high
substrate concentration.

Example for competitive inhibition:

 Malonate ( CO2-. CH2. CH2. CO2-) is a competitive inhibitor of the reaction

catalyzed by succinate dehydrogenase.

CO2-. CH2. CH2. CO2- CO2-. CH = CH. CO2

( succinate) fumerate

Malonate has two carbonyl groups like the substrate succinate and can fill the succinate
binding site on the enzyme. However, the subsequent reaction involves the formation of
a double bond and since malonate , unlike succinate has only one carbon atom between
the carbonyl groups it cannot react.

Non competitive inhibition

• In non-competitive inhibition, inhibitor binds with the enzyme at a place other

than the active site and alter the overall shape, so the active site cannot function
and the union of the substrate with the active site occurs less rapidly or not at all.
• inhibitor molecules bind to an enzyme at the allosteric site.

Allosteric site is the active site of an adjoining protein subunit. The binding of oxygen
to one subunit induces a conformational change in that subunit that interacts with the
remaining active sites to enhance their oxygen affinity.

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Divide by on RHS

33
 So, here for non competitive inhibition, the Km value is unchanged while Vmax is
lowered. Non competitive inhibition is reversible or irreversible. Non competitive
inhibition, in contrast to competitive inhibition cannot be overcome by increasing
substrate concentration because here there is no competition exist between inhibitor and
substrate for active site.

Example : Various heavy metals ions ( Ag+, Hg2+) inhibit the activity of many enzyme.

Urease is highly sensitive to any of these ions in traces. Heavy metals from mercaptides
with sulfhydryl [-SH] groups of enzymes.

Enz-SH + Ag+ Enz - SH – Ag + H+

The established equilibrium inactivities enzymes that require a -SH group for activity,
because of the reversibility of mercaptide formation, the inhibitor can be relived by the
removal of the heavy metal ions.

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35
• Uncompetitive inhibition- Uncompetitive inhibition, also known as anti-
competitive inhibition, takes place when an enzyme inhibitor binds only to the
complex formed between the enzyme and the substrate (the E-S complex).
• Uncompetitive inhibition typically occurs in reactions with two or more substrates
or products.

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 Multiply by on either sides

Where = and

37
=

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Enzyme Regulations

• In living systems, hundreds of different enzyme catalyzed

reactions occur simultaneously.
• These reactions must be regulated for the proper functioning
of a living system.
• Regulatory enzymes exhibit increased or decreased catalytic
activity in response to certain signals.
• An enzyme’s catalytic activity can be directly controlled
through structural alterations that influence the enzyme’s
substrate- binding affinity

Control of catalytic efficiency of enzymes is based on

• Allosteric enzyme regulation

• Feedback regulation

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Allosteric regulation

• Enzymatic activity can be activated or inhibited through non covalent interaction

of enzyme with metabolites other than the substrate.
• Allosteric proteins contains distinct regulatory sites and multiple functional sites.
• Allosteric enzyme is formed of more than one protein subunit. It has two sites; a
catalytic site for substrate binding and another site called allosteric site, where
effectors bind.
• Enzymatic activity can be controlled by positive and negative effectors/
metabolites through non-covalent interaction. This form of control is termed
Allosteric regulation.

• Positive effectors/allosteric activators -Binding of the effectors to the enzyme

increases its activity

e.g. ADP is allosteric activator for phosphofructokinase enzyme.

• Negative effector or allosteric inhibitor- Binding of the effectors to the enzyme

causes a decrease in its activity,

e.g. -ATP and citrate are allosteric inhibitors for phosphofructokinase enzyme.

-Glucose-6-phosphate is allosteric inhibitor for hexokinase enzyme

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Feedback regulation

• Feedback regulation of an enzyme occurs when a product of the reaction binds to

an allosteric site on the enzyme and affects its catalytic activity.
• Through feedback inhibition, the cell responds to the amount of reaction product
in order to regulate its further production.

• Feedback inhibition is usually performed by an “allosteric site” on an enzyme,

which alters the shape of the enzyme and, as a result, the behavior of the active
site
• The end product binding to the allosteric site delays or prevents the enzyme’s
activity, resulting in slight or no further end product being produced.
• The enzyme will encounter rarer particles of the end product as levels of the end
product decrease, and its activity will improve again.

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43