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1.20102 cellularprocesses

UNIT 4: ENZYMES
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Unit 4: Enzymes

Objectives

After your revision of this topic you should be able to:

1. Understand the meaning and use of the following terms: substrate, product, metabolite,
metabolic pathway, enzymes, catalysis, specificity, activation energy, inhibitor and enzyme-
substrate complex.
2. Discuss the structure of enzymes and the properties of enzymes which follow from their protein
nature.
3. Give a simple explanation of how enzymes work and the effects of temperature, pH and
inhibitors on enzymes activity.
4. Illustrate the above by reference to the enzymes involved in digestion.

Introduction

Complex food molecules obtained during food capture must be broken down to smaller, simpler
molecules in the alimentary canal before they can be absorbed into the body. Once in the body,
they can be distributed to cells and made into molecules needed for cell function. These
chemicals in the cell are also constantly turning over - they are being synthesised, degraded and
resynthesised (Figure 1). As we have seen this involves complex metabolic pathways. Enzymes
that catalyze specific chemical steps allow for cellular control of these metabolic pathways.
Enzymes are organized into teams in metabolic pathways. Enzymes often work in sequences.
The cell regulates enzymatic activity. The amount of enzyme produced can control the rate of a
reaction; this is typically accomplished by feedback mechanisms.

Figure 1: Interrelationships in cell metabolism.

How chemicals react

Chemical reactions involve the making and breaking of chemical bonds. Collision theory
provides a simple model of how a chemical reaction might take place. If molecules called the
reactants collide with sufficient force they can combine to form a transition state. This is unstable
and the chemical bonds break and reform to make up the products of the reaction. The energy
required to form the transition state is called the energy of activation (EA), see Figure 2. In
normal situations a large amount of activation energy is often required for a chemical reaction to
take place. However, substances called catalysts can lower the activation energy for a chemical
reaction and hence speed up the process. Many substances can act as catalysts, for example
iron, nickel and platinum. In cells, enzymes act as the catalysts for chemical reactions and thus
lower the activation energy needed to initiate a chemical reaction. However, enzymatic action has
no effect on the overall free energy change.
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Figure 2: Activation energy and the effect of a catalyst.

Enzymes are protein molecules which have the ability to catalyse specific chemical reactions of
metabolic pathways in living cells by lowering the activation energy of the chemical reactions.
Note the following properties of enzymes:
• they work very rapidly i.e. they are efficient
• they are not destroyed in the reaction
• they shorten the time taken to reach equilibrium of the reaction
• they act in low concentrations

Nearly all enzymes are protein catalysts that speed the rate of chemical reactions. They are
usually very specific i.e. they will only act on one or a very few chemically related substances.
Lactase, for example, will only act on lactose, converting it to glucose and galactose.

Most enzyme names end in –ase. Sucrase is an enzyme that reacts with sucrose. Other enzymes
have older names ending in –zyme. Enzyme names such as pepsin and trypsin give no clues as
to their function. Not all enzymes are specific (e.g. lipases react with a variety of fats). Note that
not all organic catalysts are enzymes; some nucleotide-based molecules function as enzymes as
well.

Enzyme structure

Enzymes are high molecular weight molecules of protein. Hence they are much larger than the
substrate molecules. They have a complex three dimensional shape. Disruption of this shape
results in loss of activity. All enzymes have an active site - a small part of the molecule where
the substrate binds to the enzyme and where the reaction takes place, see Figure 3. The active
site is not rigid, but binding of the substrate to it involves conformational changes in both the
enzyme and (typically) the substrate.

Many enzymes are composed of a protein component (an apoenzyme) and a non-protein co-
factor before they become active. Inorganic cofactors include elements such as Mg, Ca, Fe, Cu,
Zn, and Mn. In some cases the co-factor may be a small metal ion derived from mineral nutrients
in the diet. For example, some peptidases require zinc for their activity. Organic non-protein
cofactors bind with the enzyme, forming a coenzyme. Coenzymes are typically transfer agents.
ADP, NADPH, and FADH2 are coenzymes. ATP is a coenzyme. Coenzyme A is important in the
transfer of groups derived from organic acids. Most vitamins are coenzymes or are parts of
coenzymes. Lack of adequate amounts of minerals and vitamins in the diet of animals can result
in deficiency diseases.
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Figure 3: Comparative dimensions of the enzyme and substrate molecules.

Enzyme action

An enzyme works by forming an enzyme-substrate (ES) complex.

The making and breaking of chemical bonds by an enzyme is preceded by the formation of an
enzyme-substrate complex: the substrate must bind to the active site, thus (see also Figure 4):

E + S ===== ES ===== EP ===== E + P

enzyme substrate enzyme- enzyme- enzyme product

substrate product
complex complex

After binding to the substrate, the product is released, and the enzyme can be reused.

Figure 4: The basic mechanism of enzyme action.

A simple model for this process - called the lock and key model for enzyme action - pictures the
enzyme’s active site as having a complementary shape to the substrate. The substrate fits into
the active site rather like a key must fit a lock if it were to work (see Figure 4). This provides an
explanation for the observed specificity of enzyme action.

It has long been known that both the enzyme and substrate(s) change shape slightly when they
combine, a phenomenon known as ‘induced fit’. The most flexible part of the enzyme is the
active site; this flexibility enables the active site to mould itself around the substrate. This provides
another model for how some enzymes work. The current model of enzymatic action is the
induced-fit model.
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Factors affecting enzyme activity

Note that some enzymes are first produced in an inactive form. This is the case for many
digestion enzymes. For example, pepsin is produced in the inactive form pepsinogen. On
reaching the acidic environment of the stomach, the hydrogen ions cause small parts of the
pepsinogen molecule to split off giving the active pepsin molecule. Similar things happen with
trypsinogen to form trypsin and various pro-peptidases to give peptidases. Why do you think this
might be a common pattern for digestive enzymes?

Enzyme activity is also affected by factors such as temperature, pH and the presence of
inhibitors.

• Temperature - chemical reactions are faster at higher temperatures: the rate is

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approximately doubled for each 10 C rise in temperature. The same is true for enzyme
catalysed reactions up to an optimal temperature, the point at which the enzyme works
best. However, above this temperature, activity drops off. This is due to the enzyme
being denatured: its complex three dimensional shape is affected and the substrate can
no longer bind to the active site.

See Figure 5 which shows the effect of temperature on the rate of an enzyme-catalysed reaction.

• pH - or hydrogen ion concentration - extremes in pH, like high temperatures,

deactivate enzymes. Less extreme changes favour the enzyme such that it has an
optimal pH - a pH at which it works best. For example, pepsin works best at a pH
around 2. The changes in activity with pH are related to changes which take place to the
shape and structure of the active site and substrate and hence the ability to form the
enzyme-substrate complex, see Figure 4.
A B

Figure 5: The effect of A) temperature and B) pH on the rate of an enzyme-controlled reaction.

• Inhibitors - some chemicals can combine with the active site and stop the substrate from
forming an enzyme-substrate complex. Irreversible inhibitors permanently inactivate the
enzyme. Cyanide is a very powerful poison because it effectively inhibits some enzymes
involved in energy production in cells. Various nerve gases and heavy metals (Hg, Pb)
are other examples. Other substances which have a similar shape to the substrate,
compete with the substrate for access to the active site. They can form a reversible
enzyme-inhibitor complex, see Figure 6. Sulphonamide drugs and penicillin antibiotics,
which are used to combat bacterial infections, are examples of this type of reversible,
competitive inhibition.
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Normal (= reaction) Abnormal (= no reaction)

Figure 6: Competitive inhibition of an enzyme.

Reversible inhibition may be competitive or noncompetitive. Reversible competitive

inhibition involves a molecule that is structurally similar to the normal substrate. The
inhibitor binds to the active site temporarily. Another group of substances affect the enzyme
away form the active site - they are called reversible non-competitive inhibitors.
Reversible noncompetitive inhibition involves binding at a site other than the active site
temporarily (similar to allosteric inhibition). This type of inhibitors is very important in
controlling metabolic pathways by a process known as feedback inhibitions, see Figure 7.
Allosteric enzymes have a receptor site to which allosteric regulators bind. Some allosteric
regulators are inhibitors of the enzyme; others are activators of the enzyme.

A B

Figure 7: Non-competitive inhibition of an enzyme (A), and the regulation of enzyme pathways (B).
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 Activity 1
1. The table shows the rate of activity of an enzyme at different temperatures:
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TEMPERATURE C RATE mg of product.min
0 1.8
5 2.4
10 3.7
15 4.9
20 7.4
25 9.3
30 13.4
35 17.2
40 19.0
45 19.0
50 8.1
55 1.7
60 0
a. Draw a graph of these results
b. i) State the optimum temperature for this enzyme
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ii) Explain the rate of activity at 17 C
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iii) Explain the results at temperatures above 45 C.
c. Name two factors other than temperature which could affect the rate of
enzyme activity.
d. if the enzyme used in the experiment was amylase, name i) the substrate and ii) the
products.

2. The browning which occurs when many types of vegetable and fruit are peeled is caused by
enzymes called phenol oxidases. These catalyse the relatively slow conversion of naturally occurring
phenolic compounds in the fruit and vegetables into dark brown melanins.

phenols -----------> quinones -----------> melanins

(colourless) (yellow) (dark brown)

The results in the table below were obtained from an investigation into the browning of apple.
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Contents of tube (cm )
Tube Catechol Apple Buffer Dilute Dilute Appearance of tubes after
# extract Ph 7 acid alkali 10mins at rm temp.
1 2 - 5 - - Colourless
2 2 2 3 - - Dark brown
3 2 2 - 3 - Colourless
4 2 2 - - 3 Light brown
5 - 2 5 - - Light brown
6 2 2* 5 - - Colourless
*extract that had previously been boiled
a) Use the results of this investigation to i) suggest two ways in which apples, once peeled, can be
prevented from turning brown ii) state what the apple extract contains.

b) From the information given, what type of substance do you think catechol is, and what purpose
does it serve in this investigation?

c) Describe simple experiments which you might carry out to show that i) the contents of tube 1 were
effectively buffered; ii) oxygen is necessary for browning reactions to occur.

3. a) What is an enzyme?
b) Describe the effects on enzyme action of i) pH; and ii) temperature.
c) Explain the significance of enzymes in metabolic pathways.
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4. a) Using suitable examples, give an account of the properties of enzymes and describe the main
types of reactions carried out by them.
b) Discuss fully an hypothesis explaining how enzyme reactions are brought about.