Chapter 2: Molecular Biology

2.5 Enzymes
p. 80-85
AIMS:
1. Describe what an enzyme is.
2. Explain how enzymes work and the importance of their
specificity.
3. Describe and explain the factors which affect enzyme activity.
4. Explain how denaturation occurs.
5. Describe how to design experiments using enzymes.
6. Explain how immobilised enzyme work.
7. Describe how to produce lactose-free milk and its advantages.
 Globular proteins.
Catalysts: they speed up chemical reactions without being chemically
altered themselves (they can be re-used many times).
o Biological catalysts: are enzymes made inside the cells of living organisms.

Enzymes work on:

Substrates: the substances that enzymes convert into products.
(Enzyme)
Substrate Product
o Enzymes are typically named after the molecules they react with (called the
substrate) and end with the suffix -ase
For example: lipids are broken down by the enzyme lipase
 Activation Energy
In order for a chemical reaction to occur,
molecules must have enough energy to
break the bonds which hold them together.
Molecules can gain the energy needed by
raising the temperature, however in living
organisms, temperature cannot be raised
that high as the organism will not survive.
Enzymes will catalyse the reaction by
choosing/providing a pathway with the
lowest activation energy/by lowering the
activation energy.
o Activation energy is the energy required by a
chemical reaction in order to begin (to be
carried out).
 Enzyme-substrate specificity:
There are thousands of biological
enzymes, but each only catalyses one
type of reaction.
This is due to the active site of an
enzyme:
o This is a special region on the surface of an
enzyme which has a shape and chemical
properties that match that of a substrate.
o The substrate will bind to the active site, but
other substances will not a specific
substrate is capable of binding to a particular
site.
o Products are released from the active site
and the enzyme is free to move to another
substance.
 A proposed model used to describe enzyme
specificity
-and- a model that was proposed to
demonstrate specificity and that a substrate fits into a specific
enzyme just as a key fits into a lock.
 -
oversimplified and a new hypothesis has
been proposed:
Evidence from research (X-ray crystallography)
shows that the active site is not a rigid shape
like a lock.
The is the current
model used to describe enzyme action.
The model proposes that although the active
site has a distinct shape, it is flexible.
The substrate enters the active site, which
modifies its shape to surround it, like a hand
fits into a glove.
Once the products are formed, they do not
have the same shape/properties as the active
site so they will be released. The enzymes is
free to move to another reaction.
 Enzyme activity:
The three stages of enzyme catalysis:
I. The substrate binds to the active site of the enzyme (some enzymes have two
substrates that bind to two different parts of the enzyme).
II. Substrates change to different chemical substances the products.
III. The products separate from the active site the enzyme is free to convert
another substrate.
The substrate and enzyme must collide for a reaction to occur.
o Most reactions occur in water.
o Particles dissolved in water are in contact with each other and are in continuous
motion.
o The direction of motion of each particle changes repeatedly and is random
(diffusion of particles in liquids).
o Collisions between substrates and enzymes occur because of the random motion
successful collisions occur when a substrate and the active site are correctly aligned.
Enzyme-substrate collisions: substrates at the correct
orientation as the active site can bind to form:
Enzyme-Substrate complexes
 Factors affecting enzyme activity:
Temperature:
o Liquid particles are in continual random
motion.
o Heating of a liquid - molecules gain more
kinetic energy.
o Substrates and enzymes move faster at higher
temperatures and increases the chances of
successful collisions.
Enzymes heated above their optimum
temperature:
o The enzymes vibrate more and more as
temperature rises causing intramolecular
forces to weaken.
o Eventually bonds break causing the structure
of the enzyme and its active site to change.
o The change is permanent: the enzyme is
denatured.
o Enzyme activity falls as enzymes become
denatured until it comes to an end.
Low temperatures result in insufficient thermal energy for the activation
of an enzyme-catalysed reaction to proceed.
Increasing the temperature will increase the speed and motion (kinetic
energy) of both enzyme and substrate, resulting in higher enzyme activity.
This is because a higher kinetic energy will result in more frequent,
successful collisions between the enzymes and substrates.
At an optimum temperature (which varies for different enzymes), the rate
of enzyme activity will be at its peak.
Higher temperatures will cause enzyme stability to decrease, as the
hydrogen bonds.
This causes the enzyme (particularly the active site) to lose its shape,
resulting in the loss of activity. The enzyme denatures.
 Factors affecting enzyme activity:
pH:
o Acidity is due to the presence of Hydrogen ions the lower
the pH the higher the concentration of H ions.
o Reducing the pH by one unit means that a solution is 10
times more acidic.
o Alkalinity is due to the presence of hydroxide ions.
o Enzymes have an optimum pH at which their activity is
highest.
o If the pH changes, activity decreases and stops altogether.
o When the H concentration is higher/lower than optimum
the structure of the enzyme is altered until it becomes
irreversible The enzymes denature.
Enzymes vary in optimum pH
o Changing the pH will alter the charge of the enzyme, which
in turn will alter protein solubility and overall shape.
o Changing the shape or charge of the active site will diminish
its ability to bind the substrate, reducing enzyme function.
 Factors affecting enzyme activity:
Substrate Concentration:
If the concentration of substrates is
increased, substrate successful
collisions between the enzyme and
substrate will occur more frequently
and the rate increases.

As substrate concentration rises

more and more, active sites are
occupied therefore the rate of the
reaction will eventually level off.
 Denaturation:
Enzymes are proteins and their
structure can be changed as they are
sensitive to change.
High temperature and extreme pH
changes, will irreversibly change
enzyme structure.
The substrate can no longer bind to
the active site and reactions are no
longer catalysed.
Enzymes dissolved in water may
become insoluble and form a
precipitate.
 A good time to mention variables:
Factors affecting a reaction can be investigated (temperature, pH,
concentration).
To design a reliable experiment the following information must be
considered:
o Independent variable: this is the factor we are investigating (changed by us - usually
on the X-axis). One independent variable is chosen at a time and a range of values are
chosen. In the method, a way to control the variable is described.
o Dependent variable: the measurement to be taken that will assess the rate of reaction
(usually on the y-axis). The measurement should be quantitative and as accurate as
possible. Results are repeated for reliability.
o The controlled variables: other factors that could affect the rate and should be kept
constant.
o Which enzyme / substrate reaction to use
o Example: If testing for effect of pH you would keep temperature constant.
o How will you measure the enzyme activity (i.e. the dependent variable)?

Choosing the Independent Variable
The main factors which will affect
the activity of an enzyme on a given
substrate are:
Temperature (use water baths to
minimise fluctuations)
pH (acidic or alkaline solutions)
Measuring Enzyme Activity
The method of data collection will depend on the
reaction occurring typically most reactions are
measured according to:
The mass/concentration/rate of substrate decomposition
(e.g. breakdown of starch).
The amount / rate of product formation (e.g. formation of
maltose).

Substrate/Enzyme concentration
(choose range to avoid saturation)
Presence of inhibitor (type of
inhibitor will be enzyme-specific)
Key things to consider when conducting an experimental investigation into a
factor affecting enzyme activity include:
What is an appropriate range of values to select for your independent
variable?
Have you chosen a sufficient time period for the reaction to proceed?
Have you identified, and controlled, all relevant extraneous variables?
Can you include a negative control condition (no enzyme) to establish
baseline readings?
Can you include a positive control condition to confirm enzyme activity?
Does the data collection method allow for sufficient precision in detecting
changes to levels of product / substrate?
Have all appropriate safety precautions been taken when handling relevant
substances?
 Immobilized enzymes:
Enzymes are widely used in industry:
Medicine
Food and nutrition
Biotechnology
Environment
Agriculture
Energy
They are usually immobilised by attachment/aggregation on
another material to restrict movement.
Methods of immobilisation:
Attachment to surfaces: glass beads (adsorption)
Entrapment in membrane/gel (alginate)
Aggregation by bonding enzymes together into particles
Benefits:
Catalysis controlled
Enzyme concentrations can be higher
Enzymes re-used saves money
Enzymes resistant to denaturation over a greater range of pH and
temperature
Products will not be contaminated with enzyme
 Lactose-free milk - Production and Advantages
Lactose is the disaccharide found in milk.
oIt is made of glucose and galactose.
oIt is broken down by the enzyme lactase.
oLactase is used in food processing to break down
lactose because:
Some people are lactose intolerant and cannot drink more than
250 ml of milk/day unless it is lactose reduced.
Galactose and glucose are sweeter than lactose so less sugar
needs to be added to foods containing milk.
Lactose may crystallise during the production of ice cream,
reducing its smooth texture. Glucose and galactose give a
smoother texture as they as soluble.
Bacteria ferment glucose and galactose more quickly than
lactose so the production of cheese and yoghurt is faster.
Lactase is obtained from a type of yeast that grows
naturally in milk.
o Kluveromyces lactis.
o Biotechnology companies culture the yeast and extract
lactase, pufify it and sell to food manufacturing companies.
 Lactose intolerance
Lactose is a disaccharide that is produced in
lactating mammals as an energy source for
newborns.
Lactose is digested into glucose and galactose
by the enzyme lactase this occurs within the
small intestine.
As mammals typically only drink milk as part of
the initial growth process, the production of
lactase typically decreases after infancy.
Without lactase, lactose will pass intact into the
large intestine, where it is broken down by
probiotic bacteria.
As part of the bacterial fermentation process,
large amounts of gas are produced.
This leads to the various ailments associated
with lactose intolerance including abdominal
bloating, cramps and flatulence.
A proportion of the human population possess
a mutation that maintains lactase production in
adulthood.
 Immobilized enzymes used in industry
Immobilised enzymes are utilised in a wide
variety of industrial practices:
Biofuels Enzymes are used to breakdown
carbohydrates to produce ethanol-based fuels
Medicine Enzymes are used to identify a
range of conditions, including certain diseases
and pregnancy
Biotechnology Enzymes are involved in a
number of processes, including gene splicing
Food production Enzymes are used in the
production and refinement of beers and dairy
products
Textiles Enzymes are used in the processing
of fibres (e.g. polishing cloth)
Paper Enzymes assist in the pulping of wood
for paper production