Enzymes.

CHAPTER 3:
ENZYMES
DR. GOMAA
Enzymes
• Enzymes are globular proteins that serve as biological catalysts.
• They speed up or slow down metabolic reactions but remain
unchanged at the end.
• Both intracellular and extracellular.
• They may facilitate the breaking of an existing bond or the
formation of a new bond.
Mode of Action of Enzymes

• Substrates = the molecules that bind to the enzyme
• Products = new substances formed.
1
Active Site:
Active site = area in enzyme's molecule where the substrate bind to
enzyme --> enzyme-substrate complex.
2
• 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.
Enzyme Specificity
I. Lock and Key Hypothesis
II. Induced Fit Hypothesis
I. Lock and Key Hypothesis
 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.
3
II. Induced Fit Hypothesis
 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 one‘s hand or clothing on a person.
 This hypothesis is more acceptable.
4
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.
The Course of an Enzyme-Catalyzed Reaction

• Measurement of the rate of formation of the product or the rate
of disappearance of the substrate.
5
Following the course of reaction
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.
6
2. Measurement of the rate of disappearance of starch in the
reaction:
• 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 colour 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.
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• 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, then measure the light absorbance.
• The darker the blue-black colour, the greater the absorbance, and
the greater the concentration of starch.
Factors affecting the rate of enzyme-catalyzed reactions

These factors are:
1. Temperature
2. pH
3. Enzyme concentration
4. Substrate concentration
5. Inhibitor concentration
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1. Effect of temperature on enzymatic reaction
 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.
Optimum temperature for enzymatic reaction
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.
9
Effect of temperature on enzymatic reaction
2. Effect of pH on enzymatic reaction:

 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.
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• 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 trypsin:
• Pepsin (active in the stomach) - optimum pH of 1.5
• Trypsin (active in the small intestine) - optimum pH of 8.0.
3. Effect of enzyme concentration on rate of reaction

• 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.
11
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. Effect of substrate concentration on the rate of reacion
• When there are 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.
12
5. Effect of inhibitor concentration on the rate of reaction
Inhibitor = a substance that slows down the rate at which an enzyme
works.
 Competitive inhibitor
 Non-competitive inhibitor
Competitive Inhibitor
• 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.
13
Non-competitive Inhibitor
• 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 can not bind with
enzyme.
• Relative concentrations 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).
14
Enzymatic Reation Velocity Calculation
Michaelis - Menten Equation
Michaelis-Menten equation describes the velocity of enzymatic
reactions (V) by relating it to [S] - concentration of a substrate S.
• Vmax represents the maximum rate achieved by the system, at

maximum (saturating) substrate concentrations.
• The Michael’s 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.
15
Enzyme Immobilization
• 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.
16
Immobilizing enzyme in alginate
• 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.
• 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.
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CHAPTER 3:

Mode of Action of Enzymes

I. Lock and Key Hypothesis

The Course of an Enzyme-Catalyzed Reaction

• Mash up some biological material like potato tuber or celery

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

To obtain quantitative results, use a colorimeter.

Factors affecting the rate of enzyme-catalyzed reactions

 As to ↑, kinetic energy of reacting molecules ↑--> ↑ successful

Optimum temperature for enzymatic reaction

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

2. Effect of pH on enzymatic reaction:

3. Effect of enzyme concentration on rate of reaction

• The greater the proportion inhibitor: substrate, the more inhibitor

• Relative concentrations of the inhibitor and the substrate does not

• Vmax represents the maximum rate achieved by the system, at

• One way of immobilizing enzymes: mixing the enzyme with

• The substrate that the enzyme acts upon is able to diffuse

• Immobilized enzymes are widely used in industry because it allows

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