Enzymes

Enzymes are biological catalysts made up of proteins that speed up chemical reactions
without being used up themselves.

Enzymes are globular proteins. Hence, they are soluble in water.

If sliced apples are dipped in hot water of about 70 to 80°C for a minute as soon as they
are cut, they will not turn brown even after several minutes of being cut.

The heat of the hot water denatures the enzymes.

This stops the reaction that would otherwise make the apples turn brown.

Apples have compounds called polyphenols, which act as substrates or keys that bind
with oxidising enzymes.

These enzymes act as locks to finally

produce products mar appear brown

In Color

Lock and key hypothesis

Enzymes have an active site resembling a lock to which a substrate can fit and bind like
a key.

When a substrate binds with an enzyme, the resulting structure is called an enzyme-
substrate complex.

It is within the enzyme-substrate complex that the

Products

reaction takes place, at the end of which new molecules known as products are formed.

After the reaction is complete, the enzyme is free to work on another substrate again.

The active site of an enzyme is complementary in shape to its substrate. Therefore, a

specific type of enzyme can work on only one particular substrate.

For example, amylase works only on starch, protease works only on proteins, and
lipase works only on fats.
How do enzymes speed up the chemical reactions they catalyse?

In many chemical reactions that do not involve enzymes, the substrate will not be
converted to a product unless it is temporarily given some extra energy.

This extra energy is called activation energy.

For example, Benedict's test for reducing sugar involves a heating step.

In the heating step, Benedict's reagent is heated with a sugar solution to make them
react.

Here, activation energy is provided in the form of heat energy.

In case of enzyme-catalysed reactions, enzymes decrease the activation energy of the

reactions they catalyse by holding the substrates in such a way that their molecules
react more easily.

By lowering the activation energy, enzymes speed up the chemical reactions.

The enzyme-catalysed reactions take place rapidly at a much lower temperature than
they otherwise would.
There is no difference in the energy released after product formation, whether the
reaction occurs with or without enzymes.

The enzymes do not change the energy yield of the reaction.

How are the active sites of enzymes formed?

The tertiary structure of proteins have foldings that form a 3-D shape that is unique for
an enzyme.

Such a 3-D shape results in the formation of pockets or grooves called active sites.

What makes the active site so specific towards a particular substrate?

The overall 3-D shape of the enzymes provide their active site a special arrangement of
amino acids within them that specifically bind only with a particular substrate and
catalyse a particular reaction.
So, the 3-D shape of the enzyme is responsible for the formation of the active site as
well as its specificity. Or, the level of protein structure that an enzyme has is responsible
for the formation of the active site and its specificity.

The level of protein structure is what gives enzymes their particular 3-D shape.

Do active sites always have a fixed shape?

The shape of the active site is not always fixed, but it is dynamic, as suggested by a
hypothesis known as the induced fit hypothesis.

Induced fit hypothesis

The induced fit hypothesis is basically the same as the lock and key hypothesis. but it
emphasises on the enzyme molecules to be more flexible, unlike a rigid lock. In this
example, the shape of the binding site of the enzyme and the substrate are not
complementary.

This hypothesis states that an enzyme is a flexible structure and its active site can
change its shape as the substrate approaches it.

This flexibility ensures that their shapes are complementary to each other and fit
perfectly.

This makes catalysis even more efficient.

Temporary bonds hold the substrate in place to

form an enzyme-substrate complex.

This complex lowers the activation energy and allows the reaction to proceed at a faster
rate.

The substrate is converted to a product by specific R-groups in the active site, just like
in the lock and key mechanism.
After the reaction is completed, the products move away from the enzyme and the
active site returns to its initial shape.

A simple analogy to this model would be a person wearing a glove. The glove, like the
enzyme, changes shape to fit the hand.
An example of enzyme that catalyse reactions by induced fit hypothesis is lysozyme.

Lysozyme is found in tears, saliva, and other secretions.

Lysozyme has a tertiary level of protein structure and changes its shape to fit with its
substrate.

Can we measure the rate of a reaction?

There are two methods to measure the rate of a reaction.

Method 1: Measurement of rate of formation of

product

Method 2: Measurement of rate of disappearance

of substrate

This is because a large number of substrate molecules are present that react with
enzymes at their active sites.

So, the rate of reaction depends on the number of substrate molecules present.
It also depends on the speed at which an enzyme can convert a substrate into a product
and move on to bind with other substrate molecules.

Method 1: Measurement of rate of formation of product

As the reaction proceeds, more and more substrates are converted to products and
there are fewer and fewer substrate molecules to bind with the enzymes.

As fewer substrate molecules are left, the reaction gets slower and slower, until it
eventually stops.

This initial rate of reaction can be measured by calculating the slope of a tangent to the
curve, as close to time zero as possible.

The oxygen produced should not escape from leaks in the rubber bung. Only then can
we measure the true volume of oxygen produced

The oxygen bubbles are given off almost instantly as soon as the reaction starts.
Measure only the initial rate of reaction from the graph.

Enzyme-substrate complex concentration

The gradual increment phase in the graph is due to more and more substrates reacting
with the enzyme to form enzyme-substrate complexes.

The decline phase is when the substrates get used up and become fewer and fewer.
Enzyme concentration

The enzymes are never used up in a reaction.

So their concentration does not change at all.

Method 2: Measurement of rate of disappearance of substrate

Steps for measuring starch concentration using a colorimeter:

• Prepare a series of known concentrations of starch solution in test tubes.

Then, add two drops of iodine solution in each test tube.

• Measure their respective absorbance values in the colorimeter.

• Plot a graph of absorbance against concentration keeping concentration on the x-axis.
We call this graph the standard curve.

• Based on the absorbance value of the sample, find out the corresponding
concentration of starch from the standard curve.

Starch, when mixed with an amylase solution, forms maltose as a product.

When samples of this reaction mixture are regularly taken and mixed with some iodine
solution, the undegraded starch forms a blue-black colour with the iodine solution.
As the reaction progresses, the intensity of the blue-black colour of successive samples
gradually decreases. This can be detected by using a colorimeter.

When a graph of the concentration of starch against time is plotted, the starch
concentration is observed to gradually decline in every successive sample.

The slope of the tangent to the curve right at the start of the reaction gives the initial rate
of disappearance of starch.

Similarly, if a tangent is drawn at any point in the curve, its slope gives the rate of
reaction at that particular point in time.
How can enzymes be controlled to regulate the rate of reactions
they catalyse?
Enzymes can be controlled by controlling the factors that affect their actions:

Factors affecting enzyme action:

1. Temperature
2. рн
3. Enzyme concentration
4. Substrate concentration
5. Inhibitor concentration

Three different ranges of temperatures affect enzymes. They are low, optimum, and
high temperatures.

At low temperature, both the enzyme and the substrate molecules have low kinetic
energy. So, their speed of movement is also low.

The chance of product formation is less at a low speed of movement of enzymes, which
results in a rare binding between the substrate and the enzyme molecule.
However, the enzyme and the substrate molecules gain more kinetic energy with a rise
in temperature.

This increases the speed of movement between the enzyme and the substrate
molecules, which result in effective collision between them.

Effective collisions between enzyme and substrate molecules cause more substrate
molecules to react with the enzyme molecules, thereby resulting in more product
formation.

The most effective collisions resulting in maximum amount of product formations occur
at the optimum temperature, which is the most favourable temperature.

Very high temperature is not favourable for enzymes as the hydrogen bonds and
hydrophobic interactions, which stabilise the shape and structure of the enzymes, get
easily broken.

When such hydrogen bonds and hydrophilic interactions within the enzyme are
destabilised, the enzyme as a whole loses its shape and activity and thereby it gets
denatured.

Both low and high temperatures are not favourable, whereas optimum temperature is
the most favourable for enzymes and the reaction they catalyse.

Effect of pH on enzymes

Most enzymes prefer a pH where they have their highest rate of activity. This pH is
known as the optimum pH.
The pH alters the active site to make it non-complementary to the extent that the
enzyme may get denatured.

How does pH affect the active site of the enzyme?

Does it affect the structure of enzymes which is interconnected to the function of the
enzyme?

pH affects the active site of enzymes by influencing the bonds that stabilise the whole
enzyme structure.

The high concentration of hydrogen ions present in the low pH of the surroundings
interacts with the R-groups of the amino acids of the active site.

Under optimal pH, the R-groups of the amino acids of the active site are well ionised to
allow ionic bonding between the substrate and the enzyme.
At low pH, the high concentration of hydrogen ions affects the ionisation of the R-groups
that ends up deforming such ionic bondings in the active sites

orine enzvmes

Hence, at low pH, no binding between the active site and substrate can happen.

The deformation in the ionic bonding makes the active site non-complementary in
shape, thereby making the enzyme non functional or denatured.

In an alkaline environment, the high concentration of hydroxyl ions also affects the
ionisation of the R-group and deforms ionic bonding between the active site and the
substrate, just as in the acidic environment.
Graph: Volume of oxygen collected against time after reacting hydrogen peroxide with
different concentrations of catalase enzyme.

How do we calculate the initial rate of reaction between catalase and hydrogen peroxide
for each concentration of enzyme?

It is calculated by determining the slope of a tangent to the curve in the graph as close
to time zero as possible.
A plot for the initial rate of reaction against enzyme concentration yields a curve, which
is nearly a straight line.

The rate of reaction is directly proportional to the enzyme concentration.

The more the enzymes are present, the faster is the rate of the reaction as there are
more active sites available for the substrates to slot into.

Keep the concentration of enzymes fixed but change the concentration of substrates

Effect of various concentrations of hydrogen peroxide on the action of catalase

Steps involved to determine the effect of the substrate, hydrogen peroxide on the action
of catalase enzyme:

Step 1: Plot a graph for volume of oxygen collected against time.

Step 2: Calculate the slopes of the tangent to the curves in the graph to find out the
initial rate of reaction for the various substrate concentrations.

Step 3: Plot a graph for the initial rates of

reaction against various substrate concentrations.

The initial rate of reaction increases with the increase in the substrate concentration
until the active sites of the enzymes are fully occupied by the substrates.

After that, there is no further increase in the rate of reaction even though the
concentration of the substrate has increased.

When the active sites of the enzyme are fully occupied by the substrates, the enzyme is
said to be 'saturated'.
A saturated enzyme works at its maximum possible rate known as the maximum
velocity (V max), beyond which it cannot work any faster.

Do all enzymes have the same affinity towards their substrates?

All enzymes have varying affinity towards their substrates.

Does an increase in the substrate concentration increase the rate of reaction even when
there is less affinity between the enzyme and the substrate?

If there is not enough affinity between the enzyme and its substrate, an increase in the
substrate concentration, even up to infinity, cannot increase the rate of reaction.

If the enzyme has enough affinity for its substrate, one can use the right concentration
of substrates to increase the rate of reaction.

Michaelis-Menten constant (K m)

It is used to know and compare the affinity of different enzymes for their substrates.

Michaelis-Menten constant or Km refers to the

substrate concentration at which an enzyme works at half its maximum rate.

At the stage of (max), the enzymes are fully occupied in their active site.

So, at the stage of the ' max . half of all

the active sites of the enzymes are occupied by the substrates.

The higher the affinity of enzymes towards their substrate, the lower the Km (Michaelis-
Menten constant).
The lysozyme enzyme has the highest affinity for its substrate as it has the lowest
Kvalue, while the carbonic anhydrase has the least among the others.

Inhibitors are biomolecules that can greatly slow down enzyme action.

Inhibitors lower the rate of reaction, unlike enzymes.

Inhibitors are molecules that compete with the substrates to bind with the enzyme.

Types of inhibitors:

1. Competitive reversible inhibitors

2. Non-competitive reversible inhibitors

Under normal enzyme-substrate binding, the substrate binds with the enzyme at its
active sites, and the enzyme converts the substrate into products.

Competitive reversible inhibitors compete with substrates to bind with the enzyme at its
active site.
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It depends on whose concentration is higher.

If the concentration of substrate is higher, then the substrate wins.

If the concentration of the inhibitor is higher, the inhibitor wins.

When a competitive reversible inhibitor binds with the enzyme at its active site, the
function of the enzyme is inhibited. Such inhibition is reversible and not permanent.

The rate of reaction also decreases in the presence of a competitive inhibitor.

Non-competitive inhibitors bind to a site other than the active site.

How do non-competitive inhibitors inhibit the substrate from binding with the enzyme at
the active site?

Non-competitive inhibitors can disrupt the hydrogen bonds and hydrophobic interactions
that hold the enzyme molecule in its 3-D shape.

Once the 3-D shape of the enzyme is distorted by the inhibitor, the active site of the
enzyme is also distorted, and the enzyme can no longer fit with the substrate.
Thus, non-competitive inhibitors inhibit substrate binding.

How strong is the inhibition by the non-competitive inhibitors?

Different inhibitors have different impacts on enzymes and on the rate of reaction.

The non-competitive inhibition is so strong and unfavourable that it sharply reduces the
rate of reaction and flattens the curve way below the normal V.

max of the reaction.

Inhibitors only reduce the rate of reaction.

They do not bring it down to zero.

Is inhibition of enzymes a good thing or a bad thing?

Inhibition of enzyme function can be lethal in cases where critical enzymes like the
cytochrome oxidase, responsible for cellular respiration, are inhibited by poisonous
compounds like potassium cyanide.

Cyanide disrupts cellular respiration by inhibiting this critical enzyme, which then leads
to the death of cells.

Thereby, major organs such as the lungs and heart, which are primarily made up of
muscle cells, cease to function.

Is inhibition of enzymes by inhibitors always bad?

Inhibition of enzymes by inhibitors is not always bad.

In many situations, inhibition is essential.

For instance, metabolic reactions in the human body must be finely controlled and
balanced. Otherwise, the enzymes would constantly churn out more and more products
uncontrollably.

How does our body control the enzymes to control the metabolic reactions?

One way of controlling metabolic reactions is to use the end-product of a chain of

metabolic reactions.

It is known as non-competitive reversible inhibition.

The substrate binds with enzyme 1 and converts it into the end-product through
intermediate products.

During this course, when the concentration of the end product increases, enzyme 1's
action is slowed down.

This is because the end-product binds to another part of the enzyme as a non-
competitive inhibitor.

This prevents substrates from binding with the enzyme.

The end-product can lose its attachment to the enzyme and go on to be used
elsewhere. This allows enzyme 1 to reform its active state.
Falling levels of the end-product allow increase in the function of the enzyme 1, as there
is no inhibition. So, the intermediates rise again, make the end-product, and the cycle
continues.

The end-product inhibition finely controls the levels of product between the narrow
upper and lower limits.

This is an example of a feedback mechanism.

Non-competitive inhibition of enzymes regulates the feedback mechanism.

Catalase-hydrogen peroxide

Enzymes never get used up in the reaction.

They remain in the reaction solution even after the product is formed.

Enzymes that are fixed on a matrix are not lost, unlike in solutions, where they are lost
when the enzyme solution is discarded.
A technique called enzyme immobilisation gives an advantage of re-using the enzyme
as well as yielding the end-product that is free from contamination by the enzyme.

Are immobilised enzymes inferior in performance than those in solution?

When small drops of a mixture of sodium alginate and lactase enzyme are passed
through a solution of calcium chloride, they form beads, which keep the lactase
enzymes immobilised.

When milk is run through a column of beads containing the immobilised lactase, the
lactase enzymes hydrolyse the lactose in the milk to glucose and galactose.

As a result, the milk becomes lactose-free.

The use of lactase solution to hydrolyse milk results in milk containing lactase enzyme
suspended in it as a contaminant, together with glucose and galactose.

How tolerant are the immobilised enzymes to changes in temperature and pH as

compared to the enzymes in solutions?
The immobilised papain enzyme is more tolerant to higher temperature than the free
papain in solution.

The immobilised enzyme functions at a wider range of pH conditions than it does in

solution.

The rate of reaction of immobilised enzyme is slightly lower than that of the enzyme in
solution (free state).

How do the immobilised enzymes tolerate the changes in temperature and pH, unlike
the enzymes in solution?
Once the structure of enzymes or their active site is distorted, the enzymes become
non-functional.

The immobilised enzymes are more tolerant to temperature and pH changes partly
because their molecules are held firmly in shape by the alginate beads.

Plus, the parts of the molecules that are embedded in the beads are not fully exposed to
the changes in temperature or pH.

Enzymes speed up reactions, and they can be controlled, protected, and re-used to
save time and cost of operations.