Enzymes as biological catalysts;

i. Concept of active site and enzyme specificity;

ii. Enzyme activity in relation to pH,
iii. temperature,
iv. substrate and
v. enzyme concentration;
vi. Active site directed and non-active site directed inhibition;
vii. end product inhibition;
viii. Commercial uses of enzymes-pectinases and proteases.

Introduction
Enzymes are proteins that help speed up metabolism, i.e. the chemical reactions in our
bodies. Remember that metabolism is the sum total of all chemical reactions in our bodies.
The enzymes, therefore, build some substances and break others down. All living things
have enzymes. Our bodies naturally produce enzymes.

All enzymes are protein in nature, except ribozymes. All proteins, including enzymes, are
made of chains of amino acids chemically bonded to one another. These bonds give each
enzyme a unique structure, which determines its function.

Enzyme nomenclature.

Enzymes are generally named after the substrate or chemical group on which they act, and
the name takes the suffix -ase. Thus, the enzyme that hydrolyses maltose is named maltase.
There are, however, exclusions to this rule. Examples of these exclusions to this terminology
are trypsin, pepsin, and papain, which are trivial names.

Concept of active site and enzyme specificity;

The active site is a groove or pocket formed by the folding pattern of the protein. This three-
dimensional structure, together with the chemical and electrical properties of the amino
acids and cofactors within the active site, allows only a specific substrate to bind to the site,
thus determining the enzyme’s specificity.

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In the lock and key hypothesis, the shape of the active site matches the shape of its
substrate molecules. This makes enzymes highly specific. Each type of enzyme can usually
catalyse only one type of reaction (some may catalyse a few types of reactions).

How enzymes work?

Enzymes work by binding to reactant molecules and holding them in such a way that the
chemical bond-breaking and bond-forming processes take place more readily.

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Reaction coordinate diagram showing the course of a reaction with and without a catalyst.

The graph below shows the activation energy in a reaction that is

i. not catalysed by an enzyme and (purple line)
ii. one that is catalysed by an enzyme (green line)

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 iii. Enzyme activity in relation to pH,
Enzymes are affected by changes in pH. The most favourable pH value - the point
where the enzyme is most active - is known as the optimum pH. This is
graphically illustrated in Figure 14.

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Extremely high or low pH values generally result in complete loss of activity
for most enzymes. pH is also a factor in the stability of enzymes. As with
activity, for each enzyme there is also a region of pH optimal stability.
The optimum pH value will vary greatly from one enzyme to another as
shown in the table below.

ENZYME OPTIMUM pH
1 Lipase (pancreas) 8.0

2 Lipase (castor oil) 4.7

3 Pepsin 1.5 - 1.6

4 Trypsin 7.8 - 8.7

5 Urease 7.0
6 Maltase 6.1 - 6.8
7 Amylase (pancreas) 6.7 - 7.0
8 Amylase (malt) 4.6 - 5.2
9 Catalase 7.0

In addition to temperature and pH there are other factors, such as ionic

strength, which can affect the enzymatic reaction. Each of these physical and
chemical parameters must be considered and optimized in order for an
enzymatic reaction to be accurate and reproducible.

iv. Temperature,
As the temperature increases so does the rate of enzyme activity. An
optimum activity is reached at the enzyme's optimum temperature. A
continued increase in temperature results in a sharp decrease in activity as
the enzyme's active site changes shape. It is now denatured.

As temperature increases so do the rate of enzyme reactions. A ten-degree

centigrade rise in temperature will increase the activity of most enzymes by
50% to 100%. Variations in reaction temperature as small as 1 or 2 degrees
may introduce changes of 10% to 20% in the results. This increase is only up
to a certain point until the elevated temperature breaks the structure of the
enzyme. Once the enzyme is denatured, it cannot be repaired. As each
enzyme is different in its structure and bonds between amino acids and
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peptides, the temperature for denaturing is specific for each enzyme.
Because most animal enzymes rapidly become denatured at temperatures
above 40°C, most enzyme determinations are carried out somewhat below
that temperature.

Figur
e 1. Effect of temperature on reaction rate.

Over a period of time, enzymes will be deactivated at even moderate

temperatures. Storage of enzymes at 5°C or below is generally the most
suitable. Lower temperatures lead to slower chemical reactions. Enzymes will
eventually become inactive at freezing temperatures but will restore most of
their enzyme activity when temperatures increase again, while some
enzymes lose their activity when frozen.

Kinetic Energy and Internal Energy

The temperature of a system is to some extent a measure of the kinetic
energy of the molecules in the system. Collisions between all molecules
increase as temperature increases. This is due to the increase in velocity and
kinetic energy that follows temperature increases. With faster velocities,
there will be less time between collisions. This results in more molecules
reaching the activation energy, which increases the rate of the reactions.
Since the molecules are also moving faster, collisions between enzymes and
substrates also increase. Thus the lower the kinetic energy, the lower the

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temperature of the system and, likewise, the higher the kinetic energy, the
greater the temperature of the system.

As the temperature of the system is increased, the internal energy of the

molecules in the system will increase. The internal energy of the molecules
may include the translational energy, vibrational energy and rotational
energy of the molecules, the energy involved in chemical bonding of the
molecules as well as the energy involved in nonbonding interactions. Some of
this heat may be converted into chemical potential energy. If this chemical
potential energy increase is great enough some of the weak bonds that
determine the three-dimensional shape of the active proteins may be
broken. This could lead to thermal denaturation of the protein and thus
inactivate the protein. Thus too much heat can cause the rate of an enzyme-
catalysed reaction to decrease because the enzyme or substrate becomes
denatured and inactive.

Optimum Temperature
Each enzyme has a temperature range in which a maximal rate of reaction is
achieved. This maximum is known as the temperature optimum of the
enzyme. The optimum temperature for most enzymes is about 37 degrees
Celsius. There are also enzymes that work well at lower and higher
temperatures. For example, Arctic animals have enzymes adapted to lower
optimal temperatures; animals in desert climates have enzymes adapted to
higher temperatures. However, enzymes are still proteins, and like all
proteins, they begin to break down at temperatures above 104 degrees
Fahrenheit. Therefore, the range of enzyme activity is determined by the
temperature at which the enzyme begins to activate and the temperature at
which the protein begins to decompose.

v. Substrate and

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Substrate concentration

Enzymes will work best if there is plenty of substrate. As the concentration of

the substrate increases, so does the rate of enzyme activity. However, the
rate of enzyme activity does not increase forever. This is because a point will
be reached when the enzymes become saturated and no more substrates can
fit at any one time even though there is plenty of substrate available.

As the substrate concentration increases so does the rate of enzyme activity.

An optimum rate is reached at the enzyme’s optimum substrate
concentration. A continued increase in substrate concentration results in the
same activity as there are not enough enzyme molecules available to break
down the excess substrate molecules.

vi. Enzyme concentration;

Increasing the amount of enzyme also increases the frequency of with which
the enzyme and substrate collide. As a result, enzyme-substrate complexes
form more quickly and the rate of reaction increases. However, there is a
limit as eventually there will be more enzyme molecules than substrate.
Some enzymes become redundant as they won't have any substrate to bind.
Any further increase in enzyme concentration has no further effect on the
reaction rate.

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 ENZYME INHIBITORS

An enzyme inhibitor is a molecule that binds to an enzyme and decreases its activity. ... The
binding of an inhibitor can stop a substrate from entering the enzyme's active site and/or
hinder the enzyme from catalysing its reaction.
An enzyme inhibitor is a molecule that disrupts the normal reaction pathway between
an enzyme and a substrate
 Enzyme inhibitors can be either competitive or non-competitive depending on their
mechanism of action

Types of Enzyme Inhibition

Enzyme inhibitors prevent the formation of an enzyme-substrate complex and hence
prevent the formation of product
 Inhibition of enzymes may be either reversible or irreversible depending on the
specific effect of the inhibitor being used

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 Normal Enzyme Reaction
 In a normal reaction, a substrate binds to an enzyme (via the active site) to form an
enzyme-substrate complex
 The shape and properties of the substrate and active site are complementary,
resulting in enzyme-substrate specificity
 When binding occurs, the active site undergoes a conformational change to optimally
interact with the substrate (induced fit)
 This conformational change destabilises chemical bonds within the substrate,
lowering the activation energy
 As a consequence of enzyme interaction, the substrate is converted into product at
an accelerated rate

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 Competitive Inhibition
 Competitive inhibition involves a molecule, other than the substrate, binding to the
enzyme’s active site
 The molecule (inhibitor) is structurally and chemically similar to the substrate (hence
able to bind to the active site)
 The competitive inhibitor blocks the active site and thus prevents substrate binding
 As the inhibitor is in competition with the substrate, its effects can be reduced by
increasing substrate concentration

Non-competitive Inhibition
 Non-competitive inhibition involves a molecule binding to a site other than the active
site (an allosteric site)
 The binding of the inhibitor to the allosteric site causes a conformational change to
the enzyme’s active site
 As a result of this change, the active site and substrate no longer share specificity,
meaning the substrate cannot bind
 As the inhibitor is not in direct competition with the substrate, increasing substrate
levels cannot mitigate the inhibitor’s effect

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 Examples of Enzyme Inhibition
Enzyme inhibitors can serve a variety of purposes, including in medicine (to treat
disease) and agriculture (as pesticides)
 An example of a use for a competitive inhibitor is in the treatment of influenza via the
neuraminidase inhibitor, RelenzaTM
 An example of a use for a non-competitive inhibitor is in the use of cyanide as a
poison (prevents aerobic respiration)

Relenza (Competitive Inhibitor)

 Relenza is a synthetic drug designed by Australian scientists to treat individuals
infected with the influenza virus
 Virions are released from infected cells when the viral enzyme neuraminidase
cleaves a docking protein (haemagglutinin)
 Relenza competitively binds to the neuraminidase active site and prevents the
cleavage of the docking protein
 Consequently, virions are not released from infected cells, preventing the spread of
the influenza virus

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 Host Status: Normal Infection Treatment
with Relenza

Cyanide (Non-competitive Inhibitor)

 Cyanide is a poison which prevents ATP production via aerobic respiration, leading
to eventual death
 It binds to an allosteric site on cytochrome oxidase – a carrier molecule that forms
part of the electron transport chain
 By changing the shape of the active site, cytochrome oxidase can no longer pass
electrons to the final acceptor (oxygen)
 Consequently, the electron transport chain cannot continue to function and ATP is
not produced via aerobic respiration

THE END

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