Enzymes

Active Site, Activation Energy & Enzyme Specificity

Enzymes are globular proteins that serve as biological catalysts.

They speed up or slow down metabolic reaction, but remain unchanged.
They may facilitate the breaking of an existing bond or the formation of a
new bond.
Substrates = the molecules that bind to the enzyme
Products = new substances formed.
1. Active Sites
Active site = area in enzyme's molecule where the substrate bind to
enzyme --> enzyme-substrate complex.

The R groups of amino acids at the active site form temporary bonds with
the substrate molecule. This pulls the substrate slightly out of shape, causing it
to react and form products.
By; Dr. Mufaddal

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2. Activation energy

Activation energy = energy the substrates need for changing themselves

into products. Heating provides activation energy.

Enzymes reduce activation energy needed ---> reaction take place

at low to. They do this by distort the shape of the substrate when it binds
at the enzyme's active site.

By; Dr. Mufaddal

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3. Enzyme specificity
Lock and Key hypothesis (Emil Fisher, 1894)

The shape of the active site of the enzyme and the substrate molecules
are complementary.

They possess specific 3-D shapes that fit exactly into one another.

Like a key into a lock, only the correct size and shape of the substrate (the
key) would fit into the active site of the enzyme (the lock).

This shows the high specificity of enzymes, however it is too rigid.

The active site has a complementary shape to the substrate.

Induced fit hypothesis (Koshland, 1958)

The shape of the active site of the enzyme and the substrate molecules
are NOT complementary.

In the presence of the substrate, the active site continually reshapes by its
interactions with the substrate, until the substrate is completely fit into it.

The enzyme is flexible and molds to fit the substrate molecule like gloves
fitting ones hand or clothing on a person.

This hypothesis is more acceptable.

By; Dr. Mufaddal

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The active site forms a complementary shape to the substrate only after
binding.

Following the course of an enzyme-catalyzed reaction

Measurement of the rate of formation of the product or the rate of

disappearance of the substrate.
1. Measurement of the rate of formation of O2 in the reaction:

Mash up some biological material like potato tuber or celery stalks, mix them
with water and filter the mixture to obtain a solution containing catalases.
Add the mixture to H2O2 (hydrogen peroxide) in a test tube.
Use small tubes --> not too much gas in the tube above the liquid.
Collect the gas in a gas syringe and recording the volume every minute until
the reaction stops.
Note - You can replace the gas syringe by an inverted measuring cylinder over
water.

By; Dr. Mufaddal

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2. Measurement of the rate of disappearance of starch in the reaction:

By; Dr. Mufaddal

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Add amylase solution to starch suspension in a test-tube.

Take samples of the reacting mixture at regular time intervals, and test for
the presence of starch using iodine in KI solution.
When starch is present, iodine is dark blue.
If the blue color lightens, starch is breaking down.
When there is no starch, the iodine solution will remain orange-brown.

To obtain quantitative results, use a colorimeter.

Put some of the iodine solution into one of the colorimeter tubes, place it in
the colorimeter and adjust the dial to give a reading of 0. This is your
standard, with no starch.
Every minute, take a sample of the liquid from the starch-amylase mixture
and add it to a clean colorimeter tube containing iodine solution. Mix
thoroughly, and then measure the light absorbance.
The darker the blue-black color, the greater the absorbance, and the greater
the concentration of starch.

By; Dr. Mufaddal

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Factors affecting the rate of enzyme-catalyzed reactions

These factors are:

- Temperature
- pH
- Enzyme concentration
- Substrate concentration
- Inhibitor concentration
When an enzyme solution is added to a solution of its substrate, the
molecules collide.
With time, the quantity of substrate (changed into product) --> frequency of
collisions --> rate of reaction gradually . The reaction rate is fastest at the
start of the reaction (substrate concentration is greatest). --> When comparing
reaction rates of an enzyme in different circumstances, we should measure
the initial rate of reaction.
1. Temperature

As to , kinetic energy of reacting molecules --> successful collision -> rate of reaction.
At optimal to enzyme's activity is maximal --> rate is maximal.
Above this temperature, H bonds holding enzyme molecule in shape begin to
break --> change tertiary structure of the enzyme (denaturation) --> active
site is deformed ---> binding of substrate with enzyme --> rate of
reaction.

By; Dr. Mufaddal

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Enzymes in the human body generally have an optimum to of about 37oC,

organisms that have evolved to live in much higher or lower temperatures may
have much higher or lower optimum to.
2. pH

Most enzyme molecules only maintain their correct tertiary structure (exhibit
maximum activity) within a very narrow pH range.
Optimum pH - is the pH at which an enzyme has maximum activity.
Biological buffers help maintain the optimum pH for an enzyme.
Changes in pH can make and break intra- and intermolecular bonds,
changing the shape of the enzyme and, therefore, its effectiveness.
Most enzymes have an optimum pH that falls within the physiological range
of 7.0-7.5.
Notable exceptions are the digestive enzymes pepsin and trysin:
pepsin (active in the stomach) - optimum pH of 1.5
trypsin (active in the small intestine) - optimum pH of 8.0.

By; Dr. Mufaddal

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3. Enzyme concentration
When there are more substrate than enzyme:

Limiting factor is Enzyme concentration

concentration of enzyme --> collisions between enzyme and substrate -> rate of the reaction (directly proportional to the enzyme concentration )
Increasing the enzyme concentration beyond a certain point does not
change the rate of reaction BECAUSE when there are less substrate than
enzyme:
limiting factor is Substrate concentration
Concentration of enzyme does NOT rate of reaction.

Limiting factor:

Factor that directly affects the rate of reaction at which a process occurs if its
quantity is changed.
Its value has to be in order to the rate.

4. Substrate concentration
When there is more enzyme than substrate:

Limiting factor is Substrate concentration

concentration of substrate --> collisions between enzyme and
substrate --> rate of the reaction
Increasing the substrate concentration beyond a certain point does not
change the rate of reaction BECAUSE when there are less enzyme than
substrate:
limiting factor is Enzyme concentration
Concentration of substrate does NOT rate of reaction.

By; Dr. Mufaddal

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5. Inhibitor concentration
Inhibitor = a substance that slows down the rate at which an enzyme works.
Competitive inhibitors
Have similar shape to the enzyme's normal substrate.
Can fit into the enzyme's active site, preventing the substrate from
binding.
The greater the proportion inhibitor: substrate, the more inhibitor
molecules (not substrate molecules) will bump into an active site.
Relative concentrations of the inhibitor and the substrate will affect the
degree to which a competitive inhibitor slows down a reaction.
Non-competitive inhibitors
Have different shape than the substrate.
Do not bind to the active site.
Bind to a different part of the enzyme --> changes the enzyme's shape
(including the active site) --> substrate cannot bind with enzyme.
A relative concentration of the inhibitor and the substrate does not
affect the degree to which a non-competitive inhibitor slows down a reaction
(if you add more substrate, it still won't be able to bind).

By; Dr. Mufaddal

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By; Dr. Mufaddal

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Investigating factors affecting the rate of enzyme-catalyzed reactions

1. Temperature
The effect of to on enzyme activity
You can use almost any enzyme reaction for this, such as the
action of catalase on H2O2. You could use the same method of collecting the
gas that is described there, but here is another possible method:

Set up several conical flasks containing H2O2 (the same volume

and concentration). Stand each one in a water bath at a particular to. Use at
least 5 to over a good range (0-90o C). Better to set up 3 sets of tubes at
each to --> mean result for each to.
Take a set of test tubes and add the same volume of catalase solution to
each one. Stand these in the same set of water baths.
Leave all the flasks and tubes to come to the correct to. Check with a
thermometer.
Take the first flask with H2O2, dry its base and sides and stand it on a
sensitive top-pan balance. Pour in the catalase solution (same to) and
immediately take the balance reading.
Record the new balance readings every 30 seconds for about 3 minutes. The
readings will as O2 is given off.
Repeat with the solutions kept at other temperatures.
Work out the initial rate of each reaction, either taken directly from your
readings, or by drawing a graph of mass lost (mass of O2) against time for
each to, and then working out the gradient of the graph over the first 30
seconds or 60 seconds of the reaction.

Use your results to plot a graph of initial rate of reaction (y-axis) against to.

By; Dr. Mufaddal

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2. pH

The effect of pH on enzyme activity

You can adapt the method described earlier for investigating the effect
of to on the rate of breakdown of H2O2 by catalase.
Vary pH by using different buffer solutions added to each enzyme solution.
Keep to, enzyme concentration, substrate concentration and total volume of
reactants the same for all the tubes.
Record, process and display results.

3. Enzyme concentration

The effect of enzyme concentration on rate of reaction

You could use the following method to investigate the effect of enzyme
concentration on the rate at which the enzyme catalase converts
its substrate H2O2 to H2O and O2.

Prepare a catalase solution as described earlier.

Prepare different dilutions of this solution:

By; Dr. Mufaddal

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And so on. The final 'solution' prepared should be 10 cm3 of distilled

water.

Place each solution into a tube fitted with a gas syringe. Use small tubes -->
there is not too much gas in the tube above the liquid, but leave space to
add an equal volume of H2O2 solution at the next step. Label tube with a
waterproof marker. Better to prepare 3 sets of these solutions.
Place each tube in a water bath at 30oC.
Take another set of tubes and add 10 cm3 of H2O2 solution to each one. The
concentration of H2O2 must be the same in each tube. Stand these tubes in
the same water bath.
Leave all the tubes for 5 minutes --> correct to. Add the contents of 1 of
the H2O2 tubes to the first enzyme tube. Mix thoroughly.
Measure the volume of gas collected in the gas syringe after 2 minutes. If
you are using 3 sets, then repeat using the other 2 tubes containing the
same concentration of enzyme.
Do the same for each of the tubes of enzyme. Record the mean volume
of gas produced in 2 minutes for each enzyme concentration and plot a
line graph to display your results.
Note: if you find that you get measurable volumes of gas sooner than
2 minutes after mixing the enzyme and substrate --> take readings earlier.
The closer to the start of the reaction you make the measurements, the
better.

4. Substrate concentration

The effect of substrate concentration on enzyme activity

You can do this in the same way as described for investigating the effect
of enzyme concentration, but this time keep the concentration of catalase
the same and vary the concentration of hydrogen peroxide.

By; Dr. Mufaddal

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Michaelis - Menten Equation and Immobilizing an enzyme

Michaelis-Menten equation describes the velocity of enzymatic reactions

(v) by relating it to [S] - concentration of a substrate S.
Michaelis - Menten Equation

An example curve with parameters

Vmax = 3.4 and Km = 0.4
Here, Vmax represents the maximum rate achieved by the system, at maximum
(saturating) substrate concentrations.
The Michaelis constant Km is the substrate concentration at which the reaction
rate is half of Vmax.
Km is (roughly) an inverse measure of the affinity or strength of binding
between the enzyme and its substrate. The lower the Km, the greater the
affinity (so the lower the concentration of substrate needed to achieve a given
rate).
It permits prediction of whether or not the rate of formation of product will be
affected by the availability of substrate.

By; Dr. Mufaddal

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Immobilizing an enzyme in alginate

In industry enzymes are used on a large scale.
It is very costly to use enzymes only once, but most enzymes are only
commercially available in liquid or dehydrated forms and once they have been
used in solution it is very difficult and time consuming to separate them from
the product.
To allow their re-use, enzymes may be immobilized (attached to an inert,
insoluble material such as calcium alginate to form a gel capsule around
them). This way, the enzymes will be hold in place throughout the reaction,
easily separated from the products and may be used again.
One way of immobilizing enzymes:
mixing the enzyme with sodium alginate, then dropping the mixture
into calcium chloride solution. Sodium alginate will turn from liquid to solid when
immersed in calcium chloride. This produces small beads with the enzyme inside.

2 Na (Alginate) + Ca++ -------> Ca (Alginate)2 + 2 Na+

The substrate that the enzyme acts upon is able to diffuse through the gel,
although this may be quite slow.
Immobilized enzymes are widely used in industry because it allows the
reaction to flow continuously and the product will not be contaminated with the
enzyme so will not need to be purified.

By; Dr. Mufaddal

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Summary of Enzymes

1. Enzymes are globular proteins which catalyze metabolic reactions.

2. Each enzyme has an active site with a specific shape, into which the
substrate molecule or molecules fit precisely. This is the lock and key
hypothesis the substrate is compared with a key which fits precisely into
the lock of the enzyme.
3. The lock and key hypothesis has been modified. The modern hypothesis is
called the induced fit hypothesis. The active site is no longer regarded as a
rigid structure like a lock, but as a flexible structure which can change shape
slightly to fit precisely the substrate molecule.
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4. When the substrate enters the active site, an enzymesubstrate complex is

temporarily formed in which the R groups of the amino acids in the enzyme
hold the substrate in place.
5. Enzymes may be involved in reactions which break down molecules or join
molecules together.
6. Enzymes work by lowering the activation energy of the reactions they
catalyze.
7. The course of an enzyme reaction can be followed by measuring the rate at
which a product is formed or the rate at which a substrate disappears. A
progress curve, with time on the x-axis, can be plotted. The curve is
steepest at the beginning of the reaction, when substrate concentration is at
its highest. This rate is called the initial rate of reaction.
8. Various factors affect the rate of activity of enzymes. Four important factors
are enzyme concentration, substrate concentration, temperature and pH.
9. The greater the concentration of the enzyme, the faster the rate of reaction,
provided there are enough substrate molecules present. Similarly, the
greater the concentration of the substrate, the faster the rate of reaction,
provided enough enzyme molecules are present. During enzyme reactions,
rates slow down as substrate molecules are used up.
10. Each enzyme has an optimum temperature at which it works fastest. As
temperature increases above the optimum temperature, the enzyme
gradually denatures (loses its precise tertiary structure). When it is
completely denatured, it ceases to function. Denaturation is sometimes
reversible.
11. Each enzyme has an optimum pH. Some enzymes operate within a
narrow pH range; some have a broad pH range.
12. Enzymes are also affected by the presence of inhibitors, which slow down
their rate of reaction or stop it completely.
13. Competitive inhibitors are molecules which are similar in shape to the
normal substrate molecules. They compete for the active site of the enzyme.
This type of inhibition is reversible because the inhibitor can enter and leave
the active site.
14. Non-competitive inhibitors either bind permanently to the active site or
bind at a site elsewhere on the enzyme, causing a change in shape of the
active site. Such binding may or may not be reversible.

By; Dr. Mufaddal

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MCQs
1. Which of the following describes an enzyme?
A a catalyst with an active site which binds to the product of a reaction
B a fibrous protein with an active site which binds to a substrate
C a globular protein with hydrophilic groups on its surface
D an insoluble biological catalyst
2. The graph shows the energy changes during the progress of a chemical
reaction.

Which of the energy changes could be decreased by adding an enzyme?

A 1, 2 and 3
B 1 and 2 only
C 1 and 3 only
D 2 and 3 only
3. Which tests, carried out on samples taken at intervals during the course of
the reaction below, would enable the progress of the reaction to be followed?

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1
2
3
4

Benedicts test
Biuret test
Emulsion test
Iodine in potassium iodide solution test

A 1 or 2
B 1 or 4
C 2 or 3
D 3 or 4
4. Which of the following describes the effects of temperature on an enzymecontrolled reaction?
A At low temperatures, substrate molecules only rarely collide with an
enzymes active site.
B At low temperatures, the enzyme loses its shape and activity.
C At low temperatures, the enzyme becomes denatured.
D As the temperature increases, the number of collisions between enzyme and
substrate decrease.
5. Which statement does not describe the effect on an enzymes activity of
changing the pH?
A A pH that is very different from the enzymes optimum pH can denature the
enzyme.
B At low pH there are fewer hydrogen ions to interact with the R groups of the
amino acids that make up the enzyme.
C Changing the pH alters the interactions of the amino acids that make up the
enzyme.
D Changing the pH alters the enzymes three-dimensional shape.
6. The graph shows the effect of increasing the substrate concentration on the
rate of an enzyme-catalyzed reaction.

By; Dr. Mufaddal

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What explains why the rate of reaction levels off?

A All the enzyme active sites are saturated with substrate.
B All the substrate has been used up.
C The concentration of the enzyme is gradually decreasing.
D The rate at which enzyme and substrate collide is decreasing.
7. Curve X shows the progress of an enzyme-catalyzed reaction under
optimum conditions. Curve Y shows the same reaction with one factor changed
throughout the reaction.

Which factor was changed?

A A competitive inhibitor was added.
B A non-competitive inhibitor was added.
C The reaction took place at a different temperature.
D The reaction took place at a different pH.
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8. These statements are about enzyme inhibitors.

1 binds reversibly to an enzyme
2 binds to a site on the enzyme different from the active site
3 has a structural similarity to the enzymes normal substrate
4 can be displaced from the enzyme by a high concentration of the enzymes
normal substrate
Which statements describe a competitive inhibitor?
A 1, 2, 3 and 4
B 1, 3 and 4 only
C 2 and 3 only
D 3 and 4 only
9. Fibrous proteins from dead cells is difficult to remove from contact lenses.
Some cleaning solutions contain an enzyme to digest this protein to soluble
products.
What describes the enzyme and its activity?
A
B
C
D
it.

An active site on a fibrous protein binds to the enzyme and is hydrolyzed.

An active site on a soluble product binds to the enzyme and is digested.
An active site on a globular protein binds to a soluble product and digests it.
An active site on a globular protein binds to a fibrous protein and hydrolyses

10. Several different organisms produce lactase enzymes to hydrolyze lactose.

The different enzymes have different molecular sizes.
Which description of these different lactases is correct?
A Their active sites have the same shape.
B Their primary structure is the same.
C They each contain the same number of amino acids.
D They have exactly the same three-dimensional structure.

By; Dr. Mufaddal

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Answer for MCQs :

1 C
2 C
3 B
4 A
5 B
6 A
7 B
8 B
9 D
10 A

By; Dr. Mufaddal

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