Module 2: Foundations in Biology

Enzymes
Enzyme function and structure

Enzymes are biological catalysts - they speed up the rate of chemical reactions happening inside
our body. They work by reducing the activation energy of a reaction. Activation energy is defined as
the minimum amount of energy needed for a reaction to happen. If less energy is needed, then
reactions can take place as lower temperatures than would be needed without an enzyme. Without
the enzymes in our bodies, the reactions that happen inside of us would not be possible at normal
body temperature. Remember that enzymes are unchanged at the end of a reaction which means
they can be reused.

Enzymes can be classed as either intracellular if they catalyse reactions inside cells e.g. RNA
polymerase, or extracellular if they catalyse reactions outside of cells e.g. amylase. All enzymes are
globular proteins and have regions called active sites. The active site of an enzyme has a specific
shape and allows the substrate to bind. Other enzymes may have regulatory regions where an
inhibitor can bind, which we refer to as the allosteric site.

Mechanisms of enzyme action

Scientists have two ideas to explain the way in which enzymes work: the ‘lock-and-key’ model and
the ‘induced-fit’ model. They are models because they are our best-accepted theories based on the
evidence we have available.

Lock and Key model

The lock and key model is the simpler of the two theories of enzyme action. This model suggests
that the substrate fits into the enzyme’s active site in the same way in which a key fits into a lock. The
shape of the substrate and the active site are perfectly complementary to each other. Catalysis
happens in the following stages:

1. The substrate binds to the enzyme’s active site, forming an enzyme-substrate complex (ES
complex).
2. The enzyme converts the substrate into product, forming an enzyme-product complex (EP
complex).
3. The product is released from the enzyme’s active site.
The Induced Fit model

The induced fit model suggests that the shapes of the enzyme’s active site and its substrate are not
exactly complementary, but when the substrate enters the active site, a conformational change
(change of shape) occurs which induces catalysis. The induced fit model can be broken down into
the following stages:

1. The substrate enters the enzyme’s active site, forming an ES complex.

2. The enzyme undergoes a conformational change which causes the conversion of substrate into
product, forming an EP complex.
3. The product is released from the enzymes active site.
Comparing the two models of enzyme action

The advantage of the lock-and-key model is that it explains why most enzymes display such high
specificity to their substrates. Each enzyme will catalyse only a certain type of reaction and will only
bind to a single specific substrate out of the millions of different molecules that are floating around our
bodies. However, not all enzymes catalyse a single chemical reaction. For example, lipase exhibits
broader specificity and can bind to a variety of lipids, which only the induced fit model is able to
explain. In addition, the induced fit model is better able to explain how catalysis actually occurs. A
conformational change, which would place stress on the bonds within the substrate, can explain
how bonds would break in order for the products to form. This makes the induced fit model the
more widely accepted model of the two.

Factors which affect rate of reaction

Enzyme concentration

As enzyme concentration increases, the rate of

reaction increases since more active sites will be
available to bind to substrate molecules. This means that
there will be more frequent collisions between the
enzyme and substrate, so there will be more formation of
enzyme-substrate complexes. However, a point will be
reached when increasing enzyme concentration does not result in further increases in reaction rate.
At this point, something else has become a limiting factor, such as the availability of substrate.

Substrate concentration

As substrate concentration increases, the rate of

reaction increases since there are more substrate
molecules to fill the enzyme’s active sites. There will
be more frequent collisions so more formation of
ES complexes. At some point a ‘saturation’
point is reached where all of the enzyme’s active
sites are occupied with substrate molecules, so
the addition of more substrate molecules will have
no effect on the rate of reaction. At this point, the
reaction is proceeding as fast as possible, which is
referred to as Vmax. The only way the reaction can go any faster is by increasing enzyme
concentration.
Temperature

At low temperatures, the rate of reaction will be

slow because the enzyme and substrate have low
amounts of kinetic energy. This means that there
won’t be many collisions so there will be reduced
formation of ES complexes. As the temperature is
increased, the number of collisions increases,
increasing the formation of ES complexes and
increasing the rate of reaction. If the temperature
becomes really high, hydrogen bonds will begin to
break within the protein, causing it to unravel and become denatured. If enzymes are denatured, they
lose the shape of their active sites which means they cannot bind to their substrate, decreasing the
rate of reaction.

pH

Each enzyme has its own optimum pH at which it

works best. Pepsin, the enzyme which digests protein
in the stomach, works best in acidic environments
whereas the enzymes responsible for the digestion of
carbohydrates work better at a more neutral pH.
Deviations from the optimum pH change the charge on
the enzyme, which affects ionic bonding within its
structure. Deviations in pH also break hydrogen
bonds. This causes it to change shape and become
denatured, decreasing the rate of reaction as pH deviates from the enzyme’s optimum conditions.

Temperature coefficient Q10

The temperature coefficient, Q10, tells you how much reaction rate changes when the temperature
is raised by 10 degrees. It is calculated using the equation:

When Q10 = 2, the rate is doubling for every 10oC increase in temperature. When Q10 = 3, the rate
trebles for every ten degree increase. Most enzyme-catalysed reactions have a Q10 of around 2.
Investigating the rate of an enzyme-controlled reaction

There are two ways of measuring the rate of an enzyme controlled reaction: By measuring the speed
at which product is formed or reactant is removed.

Measuring product formation using catalase

A good example of a reaction in which product formation is measured is the reaction catalysed by
catalase, which breaks down hydrogen peroxide into oxygen and water.

Method:

1. Add a set volume/concentration of hydrogen peroxide to a test tube along with a set volume
of buffer solution to control pH.
2. Place a bung on top of the test tube and connect to an inverted measuring cylinder using
delivery tubing.
3. Using a pipette, add catalase the test tube. Quickly re-insert the bung and delivery tube.
4. Measure the volume of oxygen produced in the measuring cylinder every ten seconds for the
first minute of the reaction (using a stopwatch to record the time).
5. Repeat two more times. Calculate the mean volume of oxygen produced at each ten second
time interval.

Control variables: volume of catalase, volume and concentration of hydrogen peroxide solution, pH,
temperature.

Plot a graph with time on the x-axis and volume of oxygen on the y-axis. You can determine the initial
reaction rate by drawing a tangent to the curve at t=0. Draw a straight line against the curve right at
the beginning of the reaction and calculate the gradient by dividing the change in y by the change in x.
You can repeat the experiment with different concentrations of catalase to determine the effect of
changing enzyme concentration on the initial rate of reaction.

Measuring reactant removal using amylase

Amylase is the enzyme which hydrolyses starch into maltose. You can measure decreasing starch
concentration as the reaction progresses by adding iodine, which turns blue/black in the presence of
starch.

Method:

1. Place a cuvette containing iodine solution in a colorimeter with a red filter. Zero the machine.
2. In a separate cuvette, pipette a given volume (e.g. 0.5 ml) of starch solution and a set volume
of iodine solution. Mix together – the solution should turn blue/black. Place in the colorimeter
and record the absorbance.
3. Add amylase solution of a set concentration/volume into the same cuvette and starch the
stopwatch.
4. Record the absorbance of the solution every ten seconds.
5. Repeat the whole process two more times and calculate a mean absorbance for each ten
second interval.

Control variables: concentration of iodine solution, volume of starch, pH, temperature.

Plot a graph with time on the x-axis and absorbance on the y-axis. You can repeat the experiment
with difference concentrations of starch to determine the effect of changing substrate
concentration on initial reaction rate.

Cofactors and coenzymes

Cofactors are non-protein molecules that need to be bound to an enzyme in order for it to work.
Some cofactors are inorganic and work by helping the enzyme bind to its substrate. They aren’t
involved in the reaction so they are chemically unchanged. For example, magnesium ions are
cofactors for the enzyme DNA polymerase.

Other cofactors are organic molecules and are called coenzymes. These work by directly
participating in the reaction so are chemically altered by the end of the reaction. They often carry
chemical groups between different enzymes. For example, NAD+ is a coenzyme which transfers
hydrogen (and electrons) between the enzymes involved in respiration. Many coenzymes are derived
from vitamins.
When a cofactor is tightly bound to its enzyme, it’s called a prosthetic group. For example, the iron-
containing heme group is a prosthetic group found in haemoglobin, forming a permanent part of its
structure.

Competitive vs non-competitive inhibitors

Competitive inhibitors are ones which bind to

the active site of the enzyme, blocking the
substrate from binding - they compete with the
substrate for access to the enzyme’s active site.
The effect of a competitive inhibitor can be
reduced by increasing substrate concentration.
If there is way more substrate compared to
inhibitor then the substrate is much more likely to
collide with the enzyme’s active site.

Non-competitive inhibitors are ones which bind

to a site on the enzyme away from its active site.
This region is known as its allosteric site. The effect of a non-competitive inhibitor cannot be
reduced by increasing substrate concentration.

Inhibitors can also be classed as reversible or irreversible. Reversible inhibitors form weak bonds
(e.g. hydrogen, ionic bonds) with the enzyme and are easily removed. Irreversible inhibitors form
strong covalent bonds with the enzyme and can’t be removed easily.

Some drugs and metabolic poisons act as enzyme inhibitors:

• Antibiotics inhibit bacterial enzymes, such as transpeptidase, an enzyme involved in

forming the cell wall. This weakens the cell wall, causing it to burst when water enters the
bacterial cell by osmosis.
• Cyanide is an irreversible inhibitor of cytochrome c oxidase, an enzyme involved in
respiration. Organisms that have ingested cyanide will die due to a lack of cellular respiration.

Inhibiting enzymes is an important way of protecting cells. For example, digestive enzymes such as
proteases are made in the rough ER and golgi before being secreted. To prevent proteases from
degrading proteins inside the cell, they are first synthesised in an inactive precursor form. Part of the
precursor molecule inhibits its activity. Once the proteases are secreted, a chemical reaction
removes the precursor molecule and the enzyme becomes active.
End-product inhibition

End-product inhibition is when the final product in a metabolic pathway inhibits an enzyme that
acts earlier on in the pathway. For example, glycolysis is the first stage of respiration and is made up
of a series of reactions which converts glucose into energy (ATP). Phosphofructokinase is an enzyme
which catalyses one of these reactions and is inhibited by ATP, preventing further glycolysis if plenty
of energy is already available.

End-product inhibition is a good way of regulating metabolic pathways and controlling the amount of
product produced. It is reversible, so when the product begins to drop, the enzymes will be able to
function again to make more of the product.