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3.1 Mode of action of enzymes:

There are many different enzymes, each one specific to a particular
reaction. This specificity is the key to understanding the efficient
functioning of cells and living organisms.

a) explain that enzymes are globular proteins that catalyse

metabolic reactions


Enzymes are protein molecules which can be defined as biological catalysts. A

catalyst is a substance which increases the rate of chemical reaction without
itself being used up and remains unchanged at the end of the reaction.

Enzymes are globular proteins, like all globular proteins enzyme molecules are
coiled into a precise three-dimensional shape with hydrophilic R -groups (side
chains) on the outside of the molecule ensuring that they are soluble.

There are about 40,000 different enzymes in human cells, each controlling a
different chemical reaction. They increase the rate of reactions by a factor of
between 106 to 1012 times, allowing the chemical reactions that make life
possible to take place at normal temperatures. They were discovered in
fermenting yeast in 1900 by Buchner, and the name enzyme means "in yeast".
As well as catalysing all the metabolic reactions of cells (such as respiration,
photosynthesis and digestion), they also act as motors (ATP synthase enzyme),
membrane protein pumps and hormone receptors.

b) state that enzymes function inside cells (intracellular enzymes)

and outside cells (extracellular enzymes)

Intracellular enzymes are found inside the cell and perform catalytic
activity inside the cell.
Extracellular enzymes act outside the cell (e.g. digestive enzymes).

Notes by Adeel Ahmad 0092-3334224391

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c) explain the mode of action of enzymes in terms of an active site,
enzyme/substrate complex, lowering of activation energy and
enzyme specificity

How do enzymes work?

There are three ways of thinking about enzyme catalysis. They all describe the
same process, though in different ways.

1. Enzymes Distort the Substrate in the Active Site

Enzymes are proteins, and their function is determined by their complex
structure. The reaction takes place in a small part of the enzyme called the active
site, while the rest of the protein acts as "scaffolding". The substrate molecule fits
into the active site. The amino acids around the active site bind to the substrate
molecule (usually by weak hydrogen and ionic bonds), so these amino acids make
the enzyme specific for one reaction only, as other molecules won't bind in the
active site.

Many enzymes also have small non-protein molecules called coenzymes at their
active sites to help bind to the substrate. These coenzymes include metal ions
(such as Fe2+, Mg2+, Cu2+) and small organic molecules (such as haem, biotin,
FAD, NAD or coenzyme A) and they form an essential part of the catalytic process.
Many of these are derived from dietary vitamins, which is why vitamins are so
The active site actually catalyses the reaction by changing shape slightly after the
substrate has bound. This change distorts the substrate molecule in the active site,
making it more likely to change into the product. For example if a bond in the
substrate is to be broken, that bond might be stretched by the enzyme, making it
more likely to break. Alternatively if a bond is to be made between two molecules,

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the two molecules can be held in exactly the right position and orientation and
pushed together, making the bond more likely to form. The enzyme can also
make the local conditions inside the active site quite different from those outside
(such as pH, water concentration, charge), so that the reaction is more likely to

Mode of enzyme action

1. Lock and key model
The shape of the enzyme's active site exactly mirrors the part of the substrate
molecule which combines with it. The precise shape of the active site is important;
it must be complementary to the shape of the substrate. This configuration has
led to the name of this 'lock and key' model of enzyme action, where the active
site of the enzyme acts like the lock and the substrate fits in to it like a key.

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2. Induced fit model
The induced fit model of
enzyme action proposes
that the structure of the
active site, the lock does
not fit the substrate
initially but once a suitable
substrate combines with
it, a conformational
change occurs so that the
enzyme closes up to enfold
the substrate. The lock is
therefore fluid wrapping itself around a key of the correct type to fit it more

2. Enzymes Take an Alternative Reaction Pathway

In any chemical reaction, a substrate (S) is converted into a product (P):
In an enzyme-catalysed reaction, the substrate first binds to the active site of the
enzyme to form an enzyme-substrate (ES) complex, then the substrate is
converted into product while attached to the enzyme, and finally the product is
released. This mechanism can be shown as:
The enzyme is then free to start again.

3. Enzymes Lower the Activation Energy

The way enzymes work can also be
shown by considering the energy
changes that take place during a
chemical reaction. We shall consider a
reaction where the product has a lower
energy than the substrate, so the
substrate naturally turns into product
(in other words the equilibrium lies in
the direction of the product). Before it
can change into product, the substrate
must overcome an "energy barrier"
called the activation energy (EA). The
larger the activation energy, the slower
the reaction will be because only a few substrate molecules will by chance have
sufficient energy to overcome the activation energy barrier. Imagine pushing

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boulders over a hump before they can roll down hill, and you have the idea. Most
physiological reactions have large activation energies, so they simply don't happen
on a useful time scale. Enzymes dramatically reduce the activation energy of a
reaction, so that most molecules can easily get over the activation energy barrier
and quickly turn into product.

For example for the breakdown of hydrogen peroxide

(2H2O2 2H2O + O2):
EA = 86 kJ mol with no catalyst

EA = 62 kJ mol-1 with an inorganic catalyst of iron filings

EA = 1 kJ mol-1 in the presence of the enzyme peroxidase (catalase).

d) investigate the progress of an enzyme-catalysed reaction by

measuring rates of formation of products (for example, using
catalase) or rates of disappearance of substrate (for example, using

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Measuring the Rate of Enzyme Reactions:
1. Firstly you need a signal to measure that shows the progress of the reaction. The
signal should change with either substrate or product concentration, and it should
preferably be something that can be measured continuously. Typical signals
include colour changes, pH changes, mass changes, gas production, volume
changes or turbidity changes. If the reaction has none of these properties, it can
sometimes be linked to a second reaction that does generate one of these changes.

2. If you mix the substrate with enzyme and measure the

signal, you will obtain a time-course. If the signal is
proportional to substrate concentration it will start high
and decrease, while if the signal is proportional to
product it will start low and increase. In both cases the
time-course will be curved (actually an exponential

3. How do you obtain a rate from this time-course? One

thing that is not a good idea is to measure the time taken
for the reaction, for as the time-course shows it is very
difficult to say when the reaction actually ends: it just
gradually approaches the end-point.
The rate is in fact the slope (or gradient) of the
time-course, so we can see that the rate (and
slope) decreases as the reaction proceeds.
The best measurement is the initial rate - that is
the initial slope of the time-course. This also
means you don't need to record the whole time-
course, but simply take one measurement a short
time after mixing.

4. Repeat this initial rate measurement under

different conditions (such as different
temperatures or substrate concentrations) and
then plot a graph of rate vs. the factor. Each point
on this second graph is taken from a separate
initial rate measurement (or better still is an
average of several initial rate measurements
under the same conditions). Draw a smooth curve
through the points.

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Be careful not to confuse the two kinds of graph (the time-course and
rate graphs) when interpreting data.
Starch Agar Plates
Agar plates can be used to assay (or test) a sample (e.g. animal or plant tissue, or
microbes) for the presence of an enzyme. The substrate is dissolved in the agar,
so a starch agar plate is used to test for amylase. If a source of amylase is placed
in the agar plate, the amylase will diffuse out through the agar, turning the starch
into maltose as it goes. If the plate is the flooded with iodine the reaction will then
show up as a clear ring where the starch has been digested around the enzyme
source. The higher the concentration of enzyme in the sample, the higher the
diffusion gradient, so the faster the enzyme diffuses through the agar, so the larger
the ring in a given time. The diameter of the ring is therefore proportional to the
enzyme concentration.

This technique is useful to measure the concentration of enzyme, but it does not
measure rate of reaction, since the size of the clear zone is limited by the rate of
diffusion through agar, which is generally much slower than the reaction rate.
This technique can also be used for other enzymes, e.g. a milk agar plate can be
used to test for a protease enzyme.

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3.2 Factors that affect enzyme action:
Investigating the effects of factors on enzyme activity gives
opportunities for planning and carrying out experiments under
controlled conditions.

a) investigate and explain the effects of the following factors on the

rate of enzyme-catalysed reactions:
pH (using buffer solutions)
enzyme concentration
substrate concentration
inhibitor concentration

Factors that Affect the Rate of Enzyme Reactions

1. Temperature
All chemical reactions get faster as the temperature increases, but with enzyme
reactions this is only true up to a certain temperature, above which the rate slows
down again. This optimum temperature is about 37C for mammalian enzymes
but there are enzymes that work best at very different temperatures, e.g. enzymes
from the arctic snow flea work at -10C, and enzymes from thermophilic bacteria
work at 90C.
Up to the optimum temperature, the rate increases gradually with temperature
(usually in a curve, not a straight line). The rate increases because the enzyme and

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substrate molecules both have more kinetic energy, so they collide more often
resulting in the formation of large number of enzyme-substrate complex. The rate
does not drop down to zero at 0C for some enzymes and they are still functional
at 0C (rather very slow), so enzymes still work in the fridge (and food still goes
off). Some enzymes can even work in ice, though the rate is extremely slow due to
the very slow diffusion of enzyme and substrate molecules through the ice lattice.

This increase in the rate with temperature would continue indefinitely except that
the enzyme molecule itself is affected by temperature. Above about 40C there is
enough thermal energy to break the weak hydrogen bonds holding the secondary,
tertiary and quaternary structures of the enzyme together, so the enzyme (and
especially the active site) loses its specific shape to become a random coil. The
substrate can no longer bind, and the reaction is no longer catalysed. This
denaturation is usually irreversible. So the optimum temperature of enzymes is
usually about 40C (and mammals and birds maintain their body temperature at
around 40C) because that is the temperature at which hydrogen bonds break.
Remember that only the weak hydrogen bonds not peptide bonds are
broken at these mild temperatures; to break strong covalent bonds
you need to boil in concentrated acid.

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2. pH
Enzymes have an optimum pH at which they work fastest. For most enzymes this
is about pH 7-8 (physiological pH of most cells), but a few enzymes can work at
extreme pH, such as protease enzymes in animal stomachs, which have an
optimum of pH 1. The pH affects the charge of the R-groups of the amino acids at
the active site. For example carboxyl R-groups are uncharged (COOH) in acid pH
but negatively charged (COO) in alkali pH. Similarly amino R-groups are
positively charged (NH3+) in acidic pH but uncharged (NH2) in alkali pH. These
changes can affect the shape as well as the charge of the active site, so the substrate
can no longer bind and the reaction isn't catalysed.

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3. Enzyme concentration
As the enzyme concentration increases the rate of the reaction increases linearly,
because there are more enzyme molecules available to catalyse the reaction. At
very high enzyme concentration the substrate concentration may become rate-
limiting, so the rate stops increasing. Normally enzymes are present in cells in
rather low concentrations.

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b) explain that the maximum rate of reaction (Vmax) is used to
derive the Michaelis-Menten constant (Km) which is used to
compare the affinity of different enzymes for their substrates

Substrate concentration

The rate of an enzyme-catalysed reaction shows a curved dependence on substrate

concentration. As the substrate concentration increases, the rate increases
because more
substrate molecules
can collide with
enzyme molecules, so
more reactions will
take place.
At higher
concentrations the
enzyme active sites
become saturated
with substrate, so
there are few free
enzyme molecules, so
adding more substrate
doesn't make much
difference (though it
will increase the rate of E-S collisions).

How fast do enzymes work?

The speed at which an enzyme works is expressed as its turnover number. This
is usually defined as the number of substrate molecules turned into product in
one minute by one molecule of enzyme. Value ranges from less than a hundred to
many millions. Some examples are given in the table.

Enzyme Turnover Number

Carbonic Anhydrase 36000000
Catalase 5600000
Galactosidase 12000
Chymotrypsin 6000
Lysozyme 60
Some turnover numbers

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Michaelis-Menten constant:
V0 = Vmax ([S]/([S] + KM)

Michaelis and Menten Graph

The Michaelis-Menten equation arises from the general equation for an

enzymatic reaction: E + S ES E + P, where E is the enzyme, S is the
substrate, ES is the enzyme-substrate complex, and P is the product. Thus, the
enzyme combines with the substrate in order to form the ES complex, which in
turn converts to product while preserving the enzyme. The rate of the forward
reaction from E + S to ES may be termed k1, and the reverse reaction as k-1.
Likewise, for the reaction from the ES complex to E and P, the forward reaction
rate is k2, and the reverse is k-2. Therefore, the ES complex may dissolve back
into the enzyme and substrate, or move forward to form product.

At initial reaction time, when t 0, little product formation occurs, therefore the
backward reaction rate of k-2 may be neglected. The new reaction becomes:

E + S ES E + P

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The Michaelis-Menten equation is:

In this equation:

V0 is the velocity of the reaction.

Vmax is the maximal rate of the reaction.

[Substrate] is the concentration of the substrate.

Km is the Michaelis-Menten constant which shows the concentration of the

substrate when the reaction velocity is equal to one half of the maximal velocity
for the reaction. It can also be thought of as a measure of how well a substrate
complexes with a given enzyme, otherwise known as its binding affinity. An
equation with a low Km value indicates a large binding affinity, as the reaction will
approach Vmax more rapidly. An equation with a high Km indicates that the enzyme
does not bind as efficiently with the substrate, and Vmax will only be reached if the
substrate concentration is high enough to saturate the enzyme.

As the concentration of substrates increases at constant enzyme concentration,

the active sites on the protein will be occupied as the reaction is proceeding. When
all the active sites have been occupied, the reaction is complete, which means that
the enzyme is at its maximum capacity and increasing the concentration of
substrate will not increase the rate of turnover.

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c) explain the effects of reversible inhibitors, both competitive and
non-competitive, on the rate of enzyme activity

Enzyme inhibition
Inhibitors inhibit the activity of enzymes, reducing
the rate of their reactions. They are found naturally,
but are also used artificially as drugs, pesticides and
research tools. Inhibitors that bind fairly weakly and
can be washed out are called reversible inhibitors,
while those that bind tightly and cannot be washed
out are called irreversible inhibitors.
There are two kinds of inhibitors:

Competitive inhibitors
A competitive inhibitor molecule has a similar structure to the normal substrate
molecule, and it can fit into the active site of the enzyme. It therefore competes
with the substrate for the active site, so the reaction is slower. However, if the
substrate concentration is increased high enough the substrate will out-compete
the inhibitor and the rate can approach a normal rate.
The sulphonamide anti-bacterial drugs are competitive inhibitors.

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Non-competitive inhibitors
A non-competitive inhibitor molecule is quite
different in structure from the substrate
molecule and does not fit into the active site.
It binds to another part of the enzyme
molecule, changing the shape of the whole
enzyme, including the active site, so that it
can no longer bind substrate molecules. Non-
competitive inhibitors therefore simply
reduce the amount of active enzyme (just like
decreasing the enzyme concentration).
Poisons like cyanide, heavy metal ions and some insecticides are all non-
competitive inhibitors.

The two types of inhibitor can be distinguished experimentally by carrying out a

substrate vs. rate experiment in the presence and absence of the inhibitor. If the
inhibition is reduced at high substrate concentration then the inhibitor is a
competitive one.

d) investigate and explain the effect of immobilising an enzyme in

alginate on its activity as compared with its activity when free in

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Advantages of Using Immobilised Enzymes

The enzyme remains

fixed to the gel or other
materials, rather than
mixing freely with the
product(s). The product
is therefore enzyme free.
The enzymes can be
reused many times,
rather than being lost
with the product.
Immobilised enzymes
generally have a wide
range of pH and
temperature over which
they can act without
becoming denatured.