Enzymes (AS Biology)

Enzymes are essential for life to exist. The mode of action of enzymes and the factors that affect their activity are
explored in this topic. Prior knowledge for this topic is an understanding that an enzyme is a biological catalyst that
increases the rate of a reaction and remains unchanged when the reaction is complete.

There are many opportunities in this topic for candidates to gain experience of carrying out practical investigations and
analysing, interpreting and evaluating their results.

3.1 Mode of action of enzymes

1. state that enzymes are globular proteins that catalyse reactions inside cells (intracellular enzymes) or are secreted
to catalyse reactions outside cells (extracellular enzymes)
2. explain the mode of action of enzymes in terms of an active site, enzyme–substrate complex, lowering of activation
energy and enzyme specificity, including the lock-and-key hypothesis and the induced-fit hypothesis
3. investigate the progress of enzyme-catalysed reactions by measuring rates of formation of products using catalase
and rates of disappearance of substrate using amylase
4. outline the use of a colorimeter for measuring the progress of enzyme-catalysed reactions that involve colour changes

Enzymes as proteins
Enzymes are biological catalysts
ʻBiologicalʼ because they function in living systems
ʻCatalystsʼ because they speed up the rate of chemical reactions without being used up or changed

The following points are also important:

Enzymes are globular proteins. They fold up into precise shapes.

Almost all metabolic reactions which take place in living organisms are catalysed by enzymes; enzymes are therefore
essential to life.
Metabolic pathways are controlled by enzymes in a biochemical cascade of reactions. Virtually every metabolic
reaction within living organisms is catalysed by an enzyme – enzymes are therefore essential for life to exist
Metabolism: all the chemical reactions that take place inside cells, including anabolism and catabolism
Catabolic (also, catabolism): pathways in which complex molecules break down into simpler ones
Anabolic (also, anabolism): pathways that require an energy input to synthesise complex molecules from simpler
ones
Many enzyme names end in -ase; for example, amylase and ATPase.

What is an enzyme?
An enzyme is a globular protein produced by a living organism that acts as a biological catalyst in a chemical
reaction by reducing activation energy.

An enzyme is a biological catalyst that accelerates metabolic reactions. Enzymes are globular proteins as they have a
roughly spherical shape and are water soluble. Enzymes functioning inside a cell are intracellular, but those that are
secreted by cells and catalyse reactions outside cells are described as extracellular.

Substrate : the substance on which an enzyme can act.

Types of enzymes
Synthetases join two molecules together by forming a new chemical bond between them. They are classified as ligases,
and they use ATP as an energy source for the reaction. Synthases catalyse synthesis without the use of ATP. They belong
to the lyase group of enzymes.

Intracellular and extracellular enzymes

An enzyme can be either intracellular or extracellular. Sometimes, they can be both.

Enzymes can be intracellular or extracellular referring to whether they are active inside or outside the cell
respectively
Intracellular enzymes are produced and function inside the cell
Extracellular enzymes are secreted by cells and catalyse reactions outside cells (eg. digestive enzymes in the gut)
Digestive enzymes in the gut are extracellular enzymes. Some organisms secrete enzymes outside their bodies.
Fungi, for example, often do this in order to digest the food on which they are growing.

Type of Intracellular Extracellular

enzyme

Example Catalase Amylase

Function of - Hydrogen peroxide is produced as a byproduct of - Digestion is usually carried out by extracellular enzymes.
enzyme many metabolic reactions. - This is because the macromolecules being digested are too
- It is harmful to cells. large to enter the cell
- Catalase converts hydrogen peroxide into water - Amylase is involved in carbohydrate digestion, it hydrolyses
and oxygen, preventing any damage to cells or starch into simple sugars.
tissues. - It is secreted by the salivary glands and the pancreas, for
digestion of starch in the mouth and small intestine
respectively.

Intracellular enzymes:

Some enzymes are intracellular. Some enzymes stay and function inside cells. Examples of reactions catalysed by
intracellular enzymes include photosynthesis and respiration.
Catalase helps break down hydrogen peroxide. Catalase is an intracellular enzyme. Certain reactions in the cell
release a byproduct called hydrogen peroxide which is lethal to the cell in large amounts. Catalase is involved in
breaking down this hydrogen peroxide into water and oxygen, which are safe for the cell.

Extracellular enzymes:

Some enzymes are extracellular. Many enzymes also function outside of cells to participate in reactions for the
organism as a whole. Digestive enzymes are good examples of extracellular enzymes.
Amylase is an enzyme present in the saliva. Amylase is one of the digestive enzymes present in the saliva. It
catalyses the breakdown of starch into maltose in the mouth.
Trypsin is an enzyme present in the small intestine. Trypsin is another digestive enzyme that is found in the small
intestine. It continues the breakdown of peptide bonds in large protein molecules which starts in the stomach.

Mode of enzyme action

Enzymes: Biological catalysts that speed up the rate of a biochemical reaction by lowering the activation energy of the
reaction

Key words:
active site: an area on an enzyme molecule where the substrate can bind
 lock-and-key hypothesis: a hypothesis for enzyme action; the substrate is a complementary shape to the active
site of the enzyme, and fits exactly into the site; the enzyme shows specificity for the substrate
induced-fit hypothesis: a hypothesis for enzyme action; the substrate is a complementary shape to the active
site of the enzyme, but not an exact fit – the enzyme, or sometimes the substrate, can change shape slightly to
ensure a perfect fit, but it is still described as showing specificity
activation energy: the energy that must be provided to make a reaction take place; enzymes reduce the
activation energy required for a substrate to change into a product
colorimeter: an instrument that measures the colour of a solution by measuring the absorption of different
wavelengths of light

Mode of enzyme action

Enzymes have an active site where specific substrates bind forming an enzyme- substrate complex
The active site of an enzyme has a specific shape to fit a specific substrate
Extremes of heat or pH can change the shape of the active site, preventing substrate binding – this is called
denaturation
Substrates collide with the enzymes active site and this must happen at the correct orientation and speed in order for
a reaction to occur

The specificity of an enzyme is a result of the complementary nature between the shape of the active site on the
enzyme and its substrate(s)
The shape of the active site (and therefore the specificity of the enzyme) is determined by the complex tertiary
structure of the protein that makes up the enzyme:
Proteins are formed from chains of amino acids held together by peptide bonds
The order of amino acids determines the shape of an enzyme
If the order is altered, the resulting three-dimensional shape changes
Note: Enzymes are functional proteins which are used to catalyse reactions. They all exhibit primary, secondary
and tertiary structure, and some which have more than one polypeptide chain have quaternary structure (such as
pyruvate dehydrogenase, an enzyme in the link reaction of respiration).

An enzyme-substrate complex forms when an enzyme and its substrate join together
The enzyme-substrate complex is only formed temporarily, before the enzyme catalyses the reaction and the
product(s) are released
Formation of Enzyme-Substrate Complex

1. Enzyme and substrate collide effectively in the correct orientation

2. Substrate binds to active site by non-covalent bonds (e.g. ionic bonds, hydrogen bonds) with R groups of binding
amino acids, forming enzyme-substrate complex
3. R groups of catalytic amino acids strain critical bonds in substrate, catalysing conversion of substrate to product
4. Active site provides a microenvironment that favours formation or breakage of particular bonds
5. Product released from active site and enzyme can be reused for binding of another substrate

Catabolism and Anabolism

Enzyme reactions can either be catabolic or anabolic

Catabolic reactions involve the breakdown of complex molecules into simpler products, which happens when a single
substrate is drawn into the active site and broken apart into two or more distinct molecules
Examples of catabolic reactions include cellular respiration and hydrolysis reactions

Anabolic reactions involve the building of more complex molecules from simpler ones by drawing two or more
substrates into the active site, forming bonds between them and releasing a single product
Examples of anabolic reactions include protein synthesis, photosynthesis and condensation reactions.
Anabolic and catabolic:

What is activation energy?

Activation energy: the energy that must be provided to make a reaction take place; enzymes reduce the activation energy
required for a substrate to change into a product.

Activation energy is the energy required in any chemical reaction to break the bonds in reactant molecules so that new
bonds are formed to make the product. An enzyme lowers the activation energy required for the reaction. However,
overall energy released during reaction is maintained.

Enzymes work by lowering the activation energy of a reaction

All chemical reactions are associated with energy changes

For a reaction to proceed there must be enough activation energy
Activation energy is the amount of energy needed by the substrate to become just unstable enough for a reaction to
occur and for products to be formed
Enzymes speed up chemical reactions because they influence the stability of bonds in the reactants
The destabilisation of bonds in the substrate makes it more reactive
Enzymes work by lowering the activation energy of a reaction and in doing so they provide an alternative energy
pathway

Enzymes perform the critical task of lowering the activation energies of chemical reactions inside the cell. Enzymes do
this by binding to the reactant molecules, and holding them in such a way as to make the chemical bond-breaking and
bond-forming processes take place more readily. It is important to remember that enzymes do not change the reaction's
∆G. In other words, they do not change whether a reaction is exergonic (spontaneous) or endergonic. This is because
they do not change the reactants' or products' free energy. They only reduce the activation energy required to reach the
transition state.

The transitions state is the intermediary state of the reaction, when the molecule is neither a substrate or product.

By definition, the transition state is the transitory of molecular structure in which the molecule is no longer a substrate
but not yet a product. All chemical reactions must go through the transition state to form a product from a substrate
molecule. The transition state is the state corresponding to the highest energy along the reaction coordinate. It has more
free energy in comparison to the substrate or product; thus, it is the least stable state. The specific form of the
transition state depends on the mechanisms of the particular reaction.

In the equation S → X → P, X is the transition state, which is located at the peak of the curve on the Gibbs free energy
graph.
How enzyme lowers activation energy of bond formation

1. Enzyme has an active site which is complementary to the substrate molecules

2. resulting in the formation of an enzyme-substrate complex
3. Orientation substrate molecules accurately for bond formation / Increases proximity of substrate molecules for bond
formation / Active site provides suitable environment for condensation reaction / distortion of bonds within substrate
molecules
4. Reactants require less energy to reach transition state

How enzymes work / Mechanisms of enzyme action

Lock-and-key hypothesis

Enzymes are globular proteins

This means their shape (as well as the shape of the active site of an enzyme) is determined by the complex tertiary
structure of the protein that makes up the enzyme and is therefore highly specific
In the 1890ʼs the first model of enzyme activity was described by Emil Fischer:
He suggested that both enzymes and substrates were rigid structures that locked into each other very precisely,
much like a key going into a lock
This is known as the ʻlock-and-key hypothesisʼ
This was later modified and adapted to our current understanding of enzyme activity, permitted by advances in
techniques in the molecular sciences
The enzyme is the lock, and the substrate is the key. Only the correct size key, which is the substrate, enters the
keyhole, which is the active site of the lock, which is the enzyme.

Lock-and-key Hypothesis

Enzyme is a rigid structure

Only substrates exactly complementary to conformation of active site are able to bind for catalysis
Explains substrate specificity: Enzyme is specific to one type of substrate

Induced-fit hypothesis

The modified model of enzyme activity is known as the ʻinduced-fit hypothesisʼ

Although it is very similar to the lock and key hypothesis, in this model the enzyme and substrate interact with each
other:
The enzyme and its active site (and sometimes the substrate) can change shape slightly as the substrate
molecule enters the enzyme
These changes in shape are known as conformational changes
This ensures an ideal binding arrangement between the enzyme and substrate is achieved
This maximises the ability of the enzyme to catalyse the reaction
Induced Fit Hypothesis

Active site is not in precise complementary conformation to substrate

Changes 3D conformation slightly upon binding of substrate, moulding into a specific conformation
Explains group specificity: Enzyme is able to catalyse reactions for a variety of substrates with similar structural or
chemical properties

In 1959 the lock and key hypothesis was modified in the light of evidence that enzyme molecules are more flexible than is
suggested by a rigid lock and key. The modern hypothesis for enzyme action is known as the induced fit hypothesis. It is
basically the same as the lock and key hypothesis, but adds the idea that the enzyme, and sometimes the substrate, can
change shape slightly as the substrate molecule enters the enzyme, in order to ensure a perfect fit. This makes the
catalysis even more efficient.

When the reaction is complete, the product or products leave the active site. The enzyme is unchanged by this process,
so it is now available to receive another substrate molecule. The rate at which substrate molecules can bind to the
enzyme’s active site, be formed into products and leave can be very rapid. The enzyme catalase, for example, can bind
with hydrogen peroxide molecules, split them into water and oxygen, and release these products at a rate of 10 million
molecules per second.
The example of lysozyme
The interaction between the substrate and the active site, including the slight change in shape of the enzyme (induced
fit) which results from the binding of the substrate, is clearly shown by the enzyme lysozyme. Lysozyme is found in
tears, saliva and other bodily secretions. It acts as a natural defence against bacteria. It does this by breaking the
polysaccharide chains that form the cell walls of the bacteria.

Induced fit mechanism

1. When substrate enters / binds to the active site

2. it causes active site to change conformation thus providing a more precise fit
3. This allows catalysis to occur / allows interaction of catalytic R groups of active site with substrate

Collagenase is an enzyme that breaks down collagen in damaged tissue and helps healthy tissue to grow.

Functions of amino acid residues at collagenase active site

1. Catalytic residues act on bonds in substrate and help to catalyses conversion of substrate to product
2. Amino acids in collagenase have specific R groups which facilitates cleaving of peptide bonds e.g. acid-base catalysis
3. Contact residues help to hold substrate at correct orientation / position via weak interactions such as hydrogen
bonds, ionic bonds and hydrophobic interactions

Mode of action of enzymes

Properties of enzymes

Enzymes speed up the rate of a chemical reaction by lowering the activation energy of the reaction

Mechanisms of enzyme action

In the lock-and-key hypothesis, the enzyme is a rigid structure, where only substrate that are exactly
complementary to the conformation of the active site are able to bind to the active site for catalysis
In the induced fit hypothesis, active site is not in precise complementary conformation to the substrate before binding
to the substrate
 A change in the 3D conformation of the enzyme’s active site is induced, thus moulding into a precise conformation
This hypothesis explains for group specificity of enzymes where an enzyme is able to catalyse reactions for a variety
of structurally or chemically similar substrates

Formation of enzyme-substrate complex

When enzymes and substrates collide in the correct orientation / effectively, substrate binds to the active site
through temporary / non-covalent bonds such as ionic bonds, hydrogen bonds
forming enzyme-substrate complex
Action of the catalytic amino acids causes a strain on the bonds in the substrates
Active site provides a microenvironment to make or break covalent bonds in a controlled way
Product released from the active site and the enzyme can be reused to take up another substrate

Measuring enzyme activity

Measuring Rate of Enzyme-Catalysed Reaction

1. Measuring rate of formation of product

e.g. rate of oxygen production for
catalase
−
2 2
→ 2 2 2 H O 2 H O + O
2. Measuring rate of disappearance of substrate
e.g. rate of disappearance of starch for
amylase
−→ starch maltose

The progress of enzyme-catalysed reactions can be investigated by:

Measuring the rate of formation of a product using catalase

Measuring the rate of disappearance of a substrate using amylase

Although the graphs in Figures 2 and 3 differ, the explanation for their shapes is the same:

At first there is a lot of substrate (hydrogen peroxide / starch) but no product (water and oxygen / maltose).
It is very easy for substrate molecules to come into contact with the empty active sites on the enzyme molecules.
All enzyme active sites are filled and the substrate is rapidly broken down into its products.
The amount of substrate decreases as it is broken down, resulting in an increase in the amount of product.
As the reaction proceeds, there is less and less substrate and more and more product.
The product produced per unit time decreases as there are fewer substrate molecules and so some active sites may
not be filled at any one moment.
 The rate of reaction continues to slow as the substrate concentration decreases.
The graphs flatten out because all the substrate has been used up and so no new product can be produced.

Investigating catalase activity

In this investigation, the rate of product formation is used to measure the rate of an enzyme-controlled reaction:
Hydrogen peroxide is a common but toxic by-product of metabolism
This means it must be broken down quickly
Catalase is an enzyme found in the cells of most organisms that breaks hydrogen peroxide down into water and
oxygen
Hydrogen peroxide and catalase are combined and the volume of oxygen generated is measured in a set time
The rate of reaction can then be calculated

All biological material contains catalase. You could mash up some potato tuber or celery stalks, mix them with water and
filter the mixture to obtain a solution containing catalase. This can then be added to hydrogen peroxide in a test tube.
Use relatively small tubes, so that there is not too much gas in the tube above the liquid.

You could measure the rate of oxygen formation by collecting the gas in a gas syringe and recording the volume every
minute until the reaction stops. OR: You could collect the oxygen in an inverted measuring cylinder over water instead.
When the enzyme and substrate are first mixed, there are a large number of substrate molecules. At any moment,
virtually every enzyme molecule has a substrate molecule in its active site. The rate at which the reaction occurs
depends only on how many enzyme molecules there are and the speed at which the enzyme can convert the substrate into
product, release it, and then bind with another substrate molecule. However, as more and more substrate is converted
into product, there are fewer and fewer substrate molecules to bind with enzymes. Enzyme molecules may be ‘waiting’
for substrate molecules to hit their active sites. As fewer substrate molecules are left, the reaction gets slower and
slower, until it eventually stops.
The curve of a graph such as the one in Figure 3.6 is therefore steepest at the beginning of the reaction: the rate of an
enzyme-controlled reaction is always fastest at the beginning. This rate is called the initial rate of reaction. You can
measure the initial rate of the reaction by calculating the slope of a tangent to the curve, as close to time 0 as possible.
An easier way of doing this is simply to read off the graph the amount of oxygen given off in the first 30 seconds. In this
case, the rate of oxygen production in the first 30 seconds is 2.7 cm of oxygen per 30 seconds, or 5.4 cm per minute.
3 3

Course of a reaction: Initially, there’s a large number of substrates and every enzyme has a substrate in its active site.
The rate at which the reaction occurs depends only on how many enzymes there are and the speed at which the enzyme
can convert the substrate into product, release it, and then bind with another substrate. However, overtime, there are
fewer substrates to bind with enzymes; the reaction gets slower, until it eventually stops. The rate of an enzyme-
controlled reaction is always fastest at the beginning.

Investigating amylase activity

In this investigation, the rate of substrate disappearance is used to compare rates of reaction under different
conditions:
Amylase is a digestive enzyme that hydrolyses starch into maltose and glucose
Amylase functions best at pH 7 and 37 °C (all enzymes operate best under specific conditions)
Amylase and starch are combined and this reaction mixture is then tested for starch at regular time intervals
This can be done by taking samples from the reaction mixture at each time interval and adding each sample to
some iodine in potassium iodide solution (starch forms a blue-black colour with this solution)
In this way, the time taken for starch to be broken down can be measured
The investigation can be repeated under a variety of conditions (eg. by altering pH or temperature) and the
reaction rates can then be compared
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 potassium iodide solution. If starch is still present, you will obtain a
blue-black colour. If there is no starch present, the iodine solution will remain orange-brown.

To obtain quantitative results, you could 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, then measure the absorbance (Figure 3.2). The darker the blue-black colour, the greater
the absorbance, and the greater the concentration of starch.

Colorimetry to measure enzyme activity

A colorimeter is able to measure light absorbance (how much light is absorbed) or light transmission (how much light
passes through) a substance
Colorimetry can be used in any enzyme-catalysed reaction that involves colour change
As the colour breaks down the transmission increases or light absorption decreases and this can be used to measure
the rate of the reaction
For example, a colorimeter can be used to follow the progress of a starch-amylase catalysed reaction as the amylase
breaks the starch down into maltose
This can be carried out as follows:
Colorimeter calibration: this is an important step in a colorimetric investigation and in this case a weak iodine
solution can be used to calibrate the colorimeter as the end point (or 100% transmission)
Preparation of a starch solution of known concentration (stock solution), from which a range of concentrations
are made using serial dilutions (method outlined in diagram below)
 Following calibration and switching on the red filter (to maximise the percentage transmission or absorbance),
the colorimeter is used to measure the percentage absorbance or percentage transmission values
A calibration graph is then plotted of starch concentration (X-axis) vs percentage absorbance or percentage
transmission (Y-axis)

Serial dilution of starch to make a range of concentrations:

3.2 Factors that affect enzyme action

1. investigate and explain the effects of the following factors on the rate of enzyme-catalysed reactions:
temperature
pH (using buffer solutions)
enzyme concentration
substrate concentration
inhibitor concentration
2. explain that the maximum rate of reaction (V max ) is used to derive the Michaelis–Menten constant (K ), which is
m

used to compare the affinity of different enzymes for their substrates

3. explain the effects of reversible inhibitors, both competitive and non-competitive, on enzyme activity
4. investigate the difference in activity between an enzyme immobilised in alginate and the same enzyme free in
solution, and state the advantages of using immobilised enzymes

Factors affecting rate of enzyme-catalysed reaction

Key words:

Vmax
: the theoretical maximum rate of an enzyme-controlled reaction, obtained when all the active sites of the
enzyme are occupied
Michaelis–Menten constant (K ): the substrate concentration at which an enzyme works at half its maximum rate (½
m

V ), used as a measure of the efficiency of an enzyme; the lower the value of K , the more efficient the enzyme.
max m

competitive inhibition: when a substance reduces the rate of activity of an enzyme by competing with the substrate
molecules for the enzyme’s active site; increasing substrate concentration reduces the degree of inhibition;
increasing inhibitor concentration increases the degree of inhibition
non-competitive inhibition: when a substance reduces the rate of activity of an enzyme, but increasing the
concentration of the substrate does not reduce the degree of inhibition; many non-competitive inhibitors bind to areas
of the enzyme molecule other than the active site itself
immobilised enzymes: enzymes that have been fixed to a surface or trapped inside beads of agar gel

Factors Affecting Rate of Enzyme-Catalysed Reaction

1. Substrate Concentration

At low substrate concentration, rate of reaction increases as substrate concentration increases

Substrate concentration is limiting

Not all enzyme active sites are occupied
Increase in substrate concentration increases frequency of effective collisions between enzyme active sites and
substrate molecules
More enzyme-substrate complexes formed per unit time
At high substrate concentration, rate of reaction becomes constant as substrate concentration increases

Substrate concentration in no longer limiting, enzyme concentration is limiting

All enzyme active sites are occupied at any one time
Any added substrate molecules will be unable to bind to any active site
Rate of enzyme-substrate complexes formed is maximum

2. Enzyme Concentration

At low enzyme concentration, rate of reaction increases as enzyme concentration increases

Enzyme concentration is limiting

Increase in enzyme concentration provides more active sites, thus increases frequency of effective collisions between
active sites and substrate molecules
More enzyme-substrate complexes formed per unit time

At high enzyme concentration, rate of reaction becomes constant as enzyme concentration increases

Enzyme concentration is no longer limiting, substrate concentration is limiting

Not enough substrate molecules competing for available active sites

3. Temperature

As temperature increases from 5°C to 40°C, rate of reaction increases (~doubles for every 10°C)

Increase in temperature increases kinetic energy, which increases frequency of effective collisions between substrate
and enzyme
More enzyme-substrate complexes formed per unit time

At optimum temperature of 40°C, rate of reaction is maximum

Rate of enzyme-substrate complexes formed is maximum

As temperature increases beyond 40°C, rate of reaction decreases drastically

Thermal agitation disrupts hydrogen bonds, ionic bonds and hydrophobic interactions
Rapid denaturation and loss of specific 3D conformation of active site

Note: Optimum temperature decreases as reaction time increases

Enzymes exposed to higher temperature for longer time

Increasing denaturation with more enzymes denatured

4. pH

At optimum pH, rate of reaction is maximum

Rate of enzyme-substrate complexes formed is maximum

At pH slightly above and below optimum, rate of reaction decreases drastically

Change in ionisation of amino acid R groups disrupts hydrogen bonds and ionic bonds
Rapid denaturation and loss of specific 3D conformation of active site

5. Inhibitor concentration

There are two types of inhibitors: competitive inhibitors and non–competitive inhibitors.

Both types of inhibitors slow down or stop enzyme activity

Increasing the concentration of an inhibitor, therefore, reduces the rate of reaction and eventually, if inhibitor
concentration continues to be increased, the reaction will stop completely
When there are competitive inhibitors present, increase in substrate concentration reduces effect of inhibition
When there are non–competitive inhibitors present, increase in substrate concentration has no effect on inhibition
Effect of changing substrate concentration on rate of reaction of enzyme catalysed reaction (with and without inhibitors):

Competitive and noncompetitive inhibition affect the reaction's rate differently. Competitive inhibitors affect the initial
rate but do not affect the maximal rate; whereas, noncompetitive inhibitors affect the maximal rate.

Rate: Temperature

Enzymes have a specific optimum temperature – the temperature at which they catalyse a reaction at the maximum
rate
Lower temperatures either prevent reactions from proceeding or slow them down:
Molecules move relatively slow
Lower frequency of successful collisions between substrate molecules and active site of enzyme
Less frequent enzyme-substrate complex formation
Substrate and enzyme collide with less energy, making it less likely for bonds to be formed or broken (stopping
the reaction from occurring)
Higher temperatures speed up reactions:
Molecules move more quickly
Higher frequency of successful collisions between substrate molecules and active site of enzyme
More frequent enzyme-substrate complex formation
Substrate and enzyme collide with more energy, making it more likely for bonds to be formed or broken (allowing
the reaction to occur)
However, as temperatures continue to increase, the rate at which an enzyme catalyses a reaction drops sharply, as
the enzyme begins to denature:
Bonds (eg. hydrogen bonds) holding the enzyme molecule in its precise shape start to break
This causes the tertiary structure of the protein (ie. the enzyme) to change
This permanently damages the active site, preventing the substrate from binding
Denaturation has occurred if the substrate can no longer bind
Very few human enzymes can function at temperatures above 50°C
 This is because humans maintain a body temperature of about 37°C, therefore even temperatures exceeding
40°C will cause the denaturation of enzymes
High temperatures causes the hydrogen bonds between amino acids to break, changing the conformation of
the enzyme

Effect of temperature on enzyme activity

1. As temperature increases from 5 - 45 °C, the quantity of product formed increases

2. This is due to increased kinetic energy, which increases the rate of effective collisions between substrate and
enzyme
3. Thus, forming more enzyme-substrate complexes per unit time / higher rate of enzyme-substrate complexes formed
4. At optimum temperature 45 °C, maximum rate of ES complex formation, resulting in maximum amount of product
formed
5. As temperature increases further from 45 - 55 °C, the quantity of products formed decreases due to greater thermal
agitation
6. Thus, hydrogen bonds, ionic bonds and hydrophobic interactions are disrupted, leading to rapid denaturation and loss
of specific 3D conformation of active site

Why optimum temperature is lower for longer duration

1. Enzymes exposed to higher temperature for longer time

2. Increasing denaturation with more enzymes denatured
3. Due to breaking of hydrogen bonds, ionic bonds and hydrophobic interactions
4. Resulting in loss of specific 3D conformation of active site

Why a particular enzyme has higher optimum temperature

1. More cysteine amino acid residues, thus forming more disulfide bonds that are covalent in nature and are stronger
2. Ref. to more ionic / hydrogen / hydrophobic bonds between the R groups at tertiary level
 3. Ref. to more thermal energy required to break these bonds / overcome R group interactions
4. Resulting in (enzyme) being able to withstand / more stable at / does not denature at higher temperatures
A: thermostable

The optimum working temperature differs from enzyme to enzyme. Some work best at around 10°C, while others continue
to work well at 80°C. Each enzyme in the human body has a different optimum working temperature.

Our body temperatures have, however, evolved to be 37°C because:

Although higher body temperatures would increase the metabolic rate slightly, the advantages are offset by the
additional energy (food) that would be needed to maintain the higher temperature.
Proteins, other than enzymes, may be denatured at higher temperatures.
At higher temperatures, any further rise in temperature, e.g. during illness, might denature the enzymes.

Denaturing enzymes at high temperatures is used to prevent the spoilage (breakdown) by various enzymes found in food
materials. This is the basis for heating food before canning or bottling it and for blanching vegetables before freezing.

It would be dangerous to maintain a body temperature of 40 °C, as even a slight rise above this would begin to denature
enzymes.

Enzymes from other organisms may have different optimum temperatures. Some enzymes, such as those found in bacteria
which live in hot springs, have much higher optimum temperatures. Some plant enzymes have lower optimum
temperatures, depending on their habitat.

Rate: pH

All enzymes have an optimum pH or a pH at which they operate best

Enzymes are denatured at extremes of pH
Hydrogen and ionic bonds hold the tertiary structure of the protein (ie. the enzyme) together
Below and above the optimum pH of an enzyme, solutions with an excess of H ions (acidic solutions) and OH ions
(alkaline solutions) can cause these bonds to break
This alters the shape of the active site, which means enzyme-substrate complexes form less easily
Eventually, enzyme-substrate complexes can no longer form at all
At this point, complete denaturation of the enzyme has occurred
Where an enzyme functions can be an indicator of its optimal environment:
Eg. pepsin is found in the stomach, an acidic environment at pH 2 (due to the presence of hydrochloric acid in
the stomachʼs gastric juice)
Pepsinʼs optimum pH, not surprisingly, is pH 2
 When investigating the effect of pH on the rate of an enzyme-catalysed reaction, you can use buffer solutions to
measure the rate of reaction at different pH values:
Buffer solutions each have a specific pH
Buffer solutions maintain this specific pH, even if the reaction taking place would otherwise cause the pH of the
reaction mixture to change
A measured volume of the buffer solution is added to the reaction mixture
This same volume (of each buffer solution being used) should be added for each pH value that is being
investigated

Note: Temperature can both affect the speed at which molecules are moving (and therefore the number of collisions
between enzyme and substrate in a given time) and can denature enzymes (at high temperatures). pH, however, does
not affect collision rate but only disrupts the ability of the substrate to bind with the enzyme, reducing the number of
successful collisions until eventually, the active site changes shape so much that no more successful collisions can
occur.

pH is a measure of the concentration of hydrogen ions in a solution. The lower the pH, the higher the hydrogen ion
concentration. Hydrogen ions can interact with the R groups of amino acids – for example, by affecting ionisation (the
negative or positive charges) of the groups. This affects the ionic bonding between the groups, which in turn affects the
three-dimensional arrangement of the enzyme molecule. The shape of the active site may change and therefore reduce the
chances of the substrate molecule fitting into it. A pH which is very different from the optimum pH can cause denaturation
of an enzyme.
Rate: Enzyme concentration

Enzyme concentration affects the rate of reaction

The higher the enzyme concentration in a reaction mixture, the greater the number of active sites available and the
greater the likelihood of enzyme-substrate complex formation
As long as there is sufficient substrate available, the initial rate of reaction increases linearly with enzyme
concentration
If the amount of substrate is limited, at a certain point any further increase in enzyme concentration will not increase
the reaction rate as the amount of substrate becomes a limiting factor

Rate: Substrate concentration

The greater the substrate concentration, the higher the rate of reaction:
As the number of substrate molecules increases, the likelihood of enzyme-substrate complex formation increases
If the enzyme concentration remains fixed but the amount of substrate is increased past a certain point,
however, all available active sites eventually become saturated and any further increase in substrate
concentration will not increase the reaction rate
When the active sites of the enzymes are all full, any substrate molecules that are added have nowhere to bind
in order to form an enzyme-substrate complex
For this reason, in the graph below there is a linear increase in reaction rate as substrate is added, which then
plateaus when all active sites become occupied
If the concentration of enzyme is fixed at a constant level and substrate concentration is increased, the rate of reaction
increases in proportion to the increase in substrate concentration. If a higher concentration of substrate is used the
active sites become fully occupied. They are said to be fully saturated at the point where they are all working as fast as
they can. The rate of reaction is at its maximum (V max
). After that, the addition of more substrate will have no effect on
the rate of reaction. In other words, when the substrate is in excess the rate of reaction levels off.

If substrate concentration is continually increased but enzyme concentration is kept constant, there eventually comes a
point where every enzyme active site is working continuously. At this point, the substrate molecules are effectively
ʻqueuing upʼ for an active site to become available.At this stage, the enzyme is working at its maximum possible rate,
known as V max
(V stands for velocity).

Enzyme affinity is the tendency of the enzyme to bind to the substrate. A high affinity means it binds readily, at low
concentration. A low affinity means it binds reluctantly, only at higher concentration.

Effect of substrate concentration at low vs high substrate concentrations

At low substrate concentrations, the rate of reaction increases as substrate concentration increases

Substrate concentration is limiting

Not all enzyme active sites are occupied / more active sites available / free / empty
 An increase in substrate concentration increases the frequency of effective collisions between the enzyme active
sites and substrate molecules
which increases the number of enzyme-substrate complexes formed per unit time / rate of enzyme-substrate
complexes formation

At high substrate concentrations, the rate of reaction becomes constant as substrate concentration increases

Substrate concentration is no longer limiting / enzyme concentration is limiting

All enzyme active sites are occupied at any one time
Any added substrate molecules will be unable to bind to any active site
as the number of enzyme-substrate complexes formed per unit time / rate of enzyme-substrate complexes formation
is at its maximum

Rate: Inhibitor concentration

V max = the maximum rate of reaction at a specific enzyme concentration

K m = the affinity of an enzyme for a particular substrate = the substrate concentration at ½ V max

There are two types of inhibitors:

Competitive inhibitors have a similar shape to that of the substrate molecules and therefore compete with the
substrate for the active site
Non-competitive inhibitors bind to the enzyme at an alternative site, which alters the shape of the active site and
therefore prevents the substrate from binding to it
Both types of inhibitors slow down or stop enzyme activity
Increasing the concentration of an inhibitor, therefore, reduces the rate of reaction and eventually, if inhibitor
concentration continues to be increased, the reaction will stop completely
For competitive inhibitors, countering the increase in inhibitor concentration by increasing the substrate concentration
can increase the rate of reaction once more (more substrate molecules mean they are more likely to collide with
enzymes and form enzyme-substrate complexes)
For non-competitive inhibitors, increasing the substrate concentration cannot increase the rate of reaction once
more, as the shape of the active site of the enzyme remains changed and enzyme-substrate complexes are still
unable to form

Effect of changing substrate concentration on rate of reaction of enzyme catalysed reaction (with and without inhibitors):
While a competitive inhibitor will lower the initial rate of reaction (by occupying some of the available active sites),
eventually the same amount of product will be produced as would have been produced without the competitive inhibitor
(the maximal rate is not affected).

Non-competitive inhibitors lower the initial rate of reaction and the maximal rate of reaction (a lower amount of product is
produced than would normally be produced).

Competitive Inhibitor: Mode of Action

Structurally similar to substrate

Competes with substrate for binding to active site
Binds to active site non-permanently or permanently
Prevents substrate from binding to active site
Graph of Competitive Inhibition

Initial rate of reaction is reduced

Increase in substrate concentration reduces effect of inhibition
Same V max ∵ substrate outcompetes inhibitor
Higher K m
∵ higher substrate concentration required to reach V max

Final amount of product formed is the same

Non-Competitive Inhibitor: Mode of Action

Bears no structural resemblance to substrate

Binds to site other than active site
Changes 3D conformation of active site such that it is no longer complementary to substrate
Prevents substrate from binding to active site
Graph of Non-Competitive Inhibition

Initial rate of reaction is reduced

Increase in substrate of concentration has no effect on inhibition
Lower V max ∵ a certain proportion of enzyme molecules rendered inactive
Same K m
∵ affinity of enzyme for substrate unaffected
Final amount of product formed is the same (or sometimes a lower amount of product is produced than would
normally be produced for a given time)

Action of competitive inhibitor

1. The competitive inhibitor is structurally similar to the substrate molecule

2. and competes with the substrate molecule for binding to the enzyme active site
3. The inhibitor binds to the enzyme active site via covalent bonds / permanently
4. and prevents substrate from binding to the active site

Graph of competitive inhibitor

Shape of the curve

1. The curve is lower than the curve when no inhibitor is present

2. The curve eventually reaches a plateau at the same level (same V max ) as the curve when no inhibitor is present

Explanation

1. The competitive inhibitor binds to the active site of the enzyme non-permanently
2. At low substrate concentration, inhibitor competes with the substrate for active site binding, and therefore it is
lower than the curve when no inhibitor is present
3. At high substrate concentration, the effect of inhibitor would be negligible / substrate outcompetes inhibitor, and
therefore the curve eventually reaches a plateau at the same level as the curve when no inhibitor is present

Graph of non-competitive inhibitor

Shape of the curve

1. The curve is lower than the curve when no inhibitor is present

2. The curve eventually reaches a plateau at half the level (lower V max ) as the curve when no inhibitor is present

Explanation

1. The non-competitive inhibitor binds to a site other than the active site of the enzyme
2. which causes a change in specific 3D conformation of the enzyme’s active site, thus preventing substrate molecules
from binding / making active site no longer complementary to substrate
3. A certain proportion of the enzyme molecules are rendered inactive resulting in a lower V max

V max & the Michaelis-Menten Constant

1. V max (maximum rate of reaction): the maximum velocity (V) or rate of a reaction which occurs at a certain
concentration of substrate for any enzyme under fixed conditions of pH and temperature.
 2. Michaelis-Menten Constant (K ): the substrate concentration at which an enzyme functions at half its maximum rate
m

(½ V ). This is a measure of the affinity of an enzyme for its substrate. K is inversely related to the affinity.
max m

Some enzymes catalyse several different reactions and act on different substrates. K m
is a way to compare the affinity
of an enzyme for its different substrates. K m
is also used to determine the effect of inhibitors on an enzyme.

Substrate concentration affects the rate of catalysis in an enzyme-substrate reaction

When the substrate concentration is fixed (and enzyme concentration is kept constant) the initial rate of reaction is
fastest and as active sites become engaged, the reaction rate falls
The Michaelis-Menten model describes the kinetics of such enzyme catalysed reactions
In this model, two values are used to describe an enzyme catalysed reaction, the maximal rate or maximal velocity
(V max ) and the Michaelis-Menten constant (K ) m

These values are derived from the reaction rate at different substrate concentrations
The maximum rate of reaction (V max
) is used to derive the Michaelis–Menten constant (K ), which is used to
m

compare the affinity of different enzymes for their substrates

Michaelis-Menten enzyme kinetics

The Michaelis-Menten model is used to investigate the kinetics of enzyme catalysed reactions (enzyme kinetics is an
area in biochemistry that studies how different variables affect reaction rates)
The rate of reaction is measured at different substrate concentrations, producing a graph like the one below
The two important values deduced are the V (maximum rate of reaction at saturating substrate concentrations) and
max

the K , which is the substrate concentration at ½ V

m (also known as the Michaelis-Menten constant)
max

The Michaelis-Menten constant is the substrate concentration at which the enzyme works at half its maximum
rate
At this point, half of the active sites of the enzyme are occupied by substrate molecules
The higher the affinity of the enzyme for the substrate, the lower the substrate concentration needed for this to
occur
This is why the Michaelis-Menten constant is a measure of the affinity of an enzyme for its substrate
Enzyme affinity is the tendency of the enzyme to bind to the substrate. A high affinity means it binds
readily, at low concentration. A low affinity means it binds reluctantly, only at higher concentration.
There is an inverse relationship between the K m and the affinity of an enzyme for its substrate
An enzyme with a high K m
has a low affinity for its substrate and an enzyme with a low K m
has a high affinity for its
substrate
Turnover numbers, which are related to V max and K m values, for four enzymes:

The table above shows the great variation in efficiency that is possible between enzymes, and the fact that V max
and K m

are independent of each other.

Knowing the values of V max

and K m
has a number of applications. Significance of V max
and K m
values:

It enables scientists to make computerised models of biochemical pathways or even the behaviour of whole cells
because it helps to predict how each reaction in a proposed pathway will proceed and therefore how the enzymes will
interact. The consequences of changing conditions such as temperature, pH or the presence of inhibitors can be built
into the models.
An enzyme’s preference for different substrates can be compared quantitatively.
By understanding what affects enzyme efficiency, scientists may in future be able to design better catalysts, linking
this to genetic engineering.
For a commercially important enzyme, the performance of the same enzyme from different organisms can be
compared.
The calculations involved can be applied to other fields of biochemistry, such as antibody–antigen binding.
Knowing K means the proportion of active sites occupied by substrate molecules can be calculated for any substrate
m

concentration.

Enzyme inhibitors
An enzyme's activity can be reduced or stopped, temporarily, by a reversible inhibitor
There are two types of reversible inhibitors:
Competitive inhibitors have a similar shape to that of the substrate molecules and therefore compete with the
substrate for the active site
Non-competitive inhibitors bind to the enzyme at an alternative site, which alters the shape of the active site and
therefore prevents the substrate from binding to it
Non-competitive inhibitors attach themselves to the enzyme at a binding site which is not the active site. This is known
as the allosteric site.

Upon attaching to the enzyme, the inhibitor alters the shape of the enzyme's active site in such a way that substrate
molecules can no longer occupy it, and so the enzyme cannot function. As the substrate and the inhibitor are not
competing for the same site, an increase in substrate concentration does not decrease the effect of the inhibitor.

Comparison of competitive and non-competitive inhibition of enzymes:

 Competitive inhibition Non-competitive inhibition

Inhibitor chemically resembles the substrate molecule and Inhibitor chemically unlike the substrate molecule but reacts with the
occupies (blocks) the active site bulk of the enzyme, reducing access to the active site

With a low concentration of inhibitor, increasing concentration With a low concentration of inhibitor, increasing concentration of
of substrate eventually overcomes inhibition as substrate substrate cannot prevent binding of inhibitor – some inhibition remains
molecules displace inhibitor at high substrate concentration

Example: O competes with CO for active site of rubisco

2 2 Example: alanine non-competitively inhibits pyruvate kinase

Certain irreversible inhibitors bind tightly and permanently to an enzyme and destroy its catalytic properties entirely.
These drastic effects occur at low concentrations of inhibitor and we may describe these substances as poisons.
Examples include:

cyanide ions which block cytochrome oxidase in terminal oxidation in cell aerobic respiration
the nerve gas sarin blocks a neurotransmitter (acetyl cholinesterase) in synapse transmission.

Reversible Inhibition

Inhibitor binds via weak non-covalent bonds

Effect is temporary

Irreversible Inhibition

Inhibitor binds via covalent bonds

Causes permanent damage

Feedback inhibition in metabolic pathways

Metabolic Pathways

1. No accumulation of products as they become substrates of subsequent reactions

2. Reactants are modified in a series of small steps enabling greater control
3. Each step is catalysed by a specific enzyme which acts as a point of control
4. Reactions proceed rapidly due to build-up of high local concentrations of substrate
5. Multienzyme complex orders sequence of reactions which increases efficiency

Reversible inhibitors can act as regulators in metabolic pathways

Metabolic reactions must be very tightly controlled and balanced, so that no single enzyme can ʻrun wildʼ and
continuously and uncontrollably generate more and more of a particular product
Metabolic reactions can be controlled by using the end-product of a particular sequence of metabolic reactions as a
non-competitive, reversible inhibitor:
As the enzyme converts substrate to product, the process is itself slowed down as the end-product of the
reaction chain binds to an alternative site on the original enzyme, changing the shape of the active site and
preventing the formation of further enzyme-substrate complexes
The end-product can then detach from the enzyme and be used elsewhere, allowing the active site to reform and
the enzyme to return to an active state
This means that as product levels fall, the enzyme begins catalysing the reaction once again, in a continuous
feedback loop
This process is known as end-product inhibition
Producing both amino acids and nucleotides is controlled through feedback inhibition. Additionally, ATP is an allosteric
regulator of some of the enzymes involved in sugar's catabolic breakdown, the process that produces ATP. In this way,
when ATP is abundant, the cell can prevent its further production. Remember that ATP is an unstable molecule that can
spontaneously dissociate into ADP. If too much ATP were present in a cell, much of it would go to waste. Alternatively,
ADP serves as a positive allosteric regulator (an allosteric activator) for some of the same enzymes that ATP inhibits.
Thus, when relative ADP levels are high compared to ATP, the cell is triggered to produce more ATP through sugar
catabolism.

Other than regulation by cellular metabolic reaction products, regulation can also be done by cofactors and coenzymes,
ions, and organic molecules. These molecules in the cell provide enzymatic regulation, such as allosteric modulation, and
competitive and noncompetitive inhibition.

Allosteric regulation

Some inhibitor molecules bind to enzymes in a location where their binding induces a conformational change that reduces
the enzyme's affinity for its substrate. This type of inhibition is an allosteric inhibition. More than one polypeptide
comprise most allosterically regulated enzymes, meaning that they have more than one protein subunit. When an allosteric
inhibitor binds to an enzyme, all active sites on the protein subunits change slightly such that they bind their substrates
with less efficiency. There are allosteric activators as well as inhibitors. Allosteric activators bind to locations on an
enzyme away from the active site, inducing a conformational change that increases the affinity of the enzyme’s active
site(s) for its substrate(s).

Allosteric Activation

Allosteric activator binds and stabilises active form of enzyme

Increases affinity of enzyme for substrate
Allosteric Inhibition

Allosteric inhibitor binds and stabilises inactive form of enzyme

Decreases affinity of enzyme for substrate

Allosteric enzyme

1. Enzymes that can change their conformation and hence activity

2. upon binding of effector to regulatory site / a site other than the active site
3. Binding of positive effector / allosteric activator results in increased affinity for substrate binding at the active site
OR Binding of positive effector / allosteric activator maintains the active form of the enzyme allowing substrates to
bind to the active site → increase in rate of reaction
4. Binding of negative effector / allosteric inhibitor results in decreased affinity for substrate binding at the active site
OR Binding of negative effector / allosteric inhibitor maintains the inactive form of the enzyme and substrates are not
able to bind to the active site → decrease in rate of reaction
5. Allosteric enzyme exhibits subunit cooperativity / binding of a substrate molecule to the active site of one subunit
facilitates the binding of (other) substrate molecules to the active sites of the other subunits

Cooperativity in enzymology, is a phenomenon in which the shape of one subunit of an enzyme consisting of several
subunits is altered by the substrate (the substance upon which an enzyme acts to form a product) or some
other molecule so as to change the shape of a neighbouring subunit. The result is that the binding of a second substrate
molecule to the second subunit of the enzyme differs in strength or velocity from that of the first, the third from the
second, and so on. If the change in shape of the first subunit makes easier the binding of substrate to the second
subunit, the effect is called positive cooperativity. In negative cooperativity, the binding of a molecule to the first
subunit makes more difficult the binding of substrate to the second.

Immobilising enzymes
Enzymes have an enormous range of commercial applications – for example, in medicine, food technology and industrial
processing. Enzymes are expensive. No company wants to have to keep buying them over and over again if it can recycle
them in some way. One of the best ways of keeping costs down is to use immobilised enzymes.

Immobilising lactase enzyme

The enzyme lactase can be immobilised using alginate beads. Milk is then allowed to run through the column of lactase-
containing beads. The lactase hydrolyses the lactose in the milk to glucose and galactose. The milk is therefore lactose-
free, and can be used to make lactose-free dairy products for people who cannot digest lactose.

Enzyme immobilisation has several advantages compared with just mixing up the enzyme with its substrate. If you just
mixed lactase with milk, you would have a very difficult task to get the lactase back again. Not only would you lose the
lactase, but also you would have milk contaminated with the enzyme. Using immobilised enzymes means that you can
keep and re-use the enzymes, and that the product is enzyme-free. Another advantage of this process is that the
immobilised enzymes are more tolerant of temperature changes and pH changes than enzymes in solution. This may be
partly because their molecules are held firmly in shape by the alginate in which they are embedded, and so do not
denature as easily. It may also be because the parts of the molecules that are embedded in the beads are not fully
exposed to the temperature or pH changes.

Enzyme Activity: Immobilised v Free

Enzymes can be added to solutions and are thereby considered ‘free’ or they can be immobilised
Immobilised enzymes are enzymes that have been bound to an inert, stationary and insoluble material such as alginate
The substrate is then passed over the immobilised enzyme and the product is collected
Advantages to this method:
There is no enzyme in the product (the product is uncontaminated) and therefore there is no need to further
process or filter the end product
The immobilised enzyme can be reused multiple times which is both efficient and cost-effective (enzymes are
expensive)
Immobilised enzymes have a greater tolerance of temperature and pH changes (immobilisation often makes
enzymes more stable)
A practical application of immobilised enzymes used in the food industry is in the production of lactose-free milk:
Milk is a valuable source of nutrients containing protein, fat and the carbohydrate Lactose
5-10% of the UK population are lactose intolerant
Lactose is a disaccharide that is broken down into glucose and galactose

Using lactase as shown above is an efficient way to remove lactose from milk and to provide lactose intolerant
individuals with a way of consuming milk without suffering intolerance symptoms:
The enzyme lactase can be immobilised using alginate beads
Milk is run through the column of lactase-containing beads
The lactase hydrolyses the lactose in the milk to glucose and galactose
This ensures the milk is lactose-free
It can also then be used to make other lactose-free dairy products

Advantages and disadvantages of immobilisation:

Advantages Disadvantages

The enzyme is held in a form that can be manipulated easily. It is An immobilisation mechanism that does not alter the shape or the
absent from the product, so no purification steps are required. catalytic ability of the enzyme must be selected.

The enzyme is available for multiple re-use, since it functions as an The creation of stable, hardened pellets is an added expense that
effective catalyst in pellet form. is inevitably reflected in the cost of the industrial product.
 Advantages Disadvantages

An immobilised enzyme is stable at the temperatures and pH at If the enzyme becomes detached it will appear in the product as a
which it is held and used. contaminant, possibly unnoticed.

Enzyme immobilisation (using sodium alginate):

The enzyme is mixed with a solution of sodium alginate

Little droplets of this mixture are then added to a solution of calcium chloride.
The sodium alginate and calcium chloride instantly react to form jelly, which turns each droplet into a little bead. The
jelly bead contains the enzyme.

Can reuse the enzyme as it is not mixed with the solution, and can keep the product enzyme free, thus preventing
contamination.
More tolerant to PH changes as the enzyme molecules are held firmly in shape by the alginate beads, thus don’t
denature easily.
More tolerant to temperature changes as parts of the molecules embedded in the beads are not fully exposed to
temperature or pH changes.
 Active site may be distorted by immobilising
Substrate passes through matrix when immobilised
Some product is retained within matrix

Question regarding immobilised enzymes:

Ans:
two from:
(alginate / immobilisation), is protective / has stabilising effect ;
A enzyme, less / not, exposed (to solution)
hydroxide ions do not penetrate the alginate beads ;
shape of active site (of immobilised enzyme) is, less / not, disrupted / AW ;
A active site is (more) complementary to substrate
A few(er) active sites are, altered / changed
few(er) bond(s) within (immobilised) enzymes break ;
A hydrogen / ionic
R peptide / disulfide
at pH 8 immobilised enzyme is not fully denatured ;
A pH 7 / 7. 5

Coenzymes, cofactors & prosthetic groups

Many enzymes don’t work optimally, or even at all, unless bound to other specific non-protein helper molecules, either
temporarily through ionic or hydrogen bonds or permanently through stronger covalent bonds. Two types of helper
molecules are cofactors and coenzymes. Binding to these molecules promotes optimal conformation and function for their
respective enzymes. Cofactors are inorganic ions such as iron (Fe++) and magnesium (Mg++). One example of an enzyme
that requires a metal ion as a cofactor is the enzyme that builds DNA molecules, DNA polymerase, which requires a bound
zinc ion (Zn++) to function. Coenzymes are organic helper molecules, with a basic atomic structure comprised of carbon
and hydrogen, which are required for enzyme action. The most common sources of coenzymes are dietary vitamins. Some
vitamins are precursors to coenzymes and others act directly as coenzymes. Vitamin C is a coenzyme for multiple
enzymes that take part in building the important connective tissue component, collagen. An important step in breaking
down glucose to yield energy is catalysis by a multi-enzyme complex scientists call pyruvate dehydrogenase. Pyruvate
dehydrogenase is a complex of several enzymes that actually requires one cofactor (a magnesium ion) and five different
organic coenzymes to catalyse its specific chemical reaction. Therefore, enzyme function is, in part, regulated by an
abundance of various cofactors and coenzymes, which the diets of most organisms supply.

There are substances other than substrates and inhibitors that interact with enzymes
Some enzymes can only function properly if another non-protein substance is present
For example, some enzymes are inactive until they combine with a non-protein substance
that changes their tertiary structure (allowing the active site to bind correctly with the substrate)
These substances are broadly known as cofactors

Cofactors

Inorganic ions: Inorganic ions are formed when an element or compound, that does not contain carbon, gains or loses
electrons to become negatively or positively charged, for example: hydrogen ions, phosphate ions, iron ions and sodium
ions.

Some enzymes require inorganic ions to function properly

Particular inorganic ions may help to stabilise the structure of the enzyme or may actually take part in the
reaction at the active site
For example, chloride ions act as a cofactor for amylase
This means that in order for amylase to be able to digest starch into maltose, chloride ions must be present
The inorganic ions that an enzyme requires in order to function are known as inorganic cofactors

Coenzymes

Coenzyme: A coenzyme is a non-protein molecule that helps an enzyme carry out its function but is not used up in the
reaction itself

Larger organic (carbon-containing) cofactors are known as coenzymes

Some coenzymes are permanently bound to the enzyme they assist, often in or near the active site
Some coenzymes only bind temporarily during the reaction
Coenzymes link different enzyme-catalysed reactions into a sequence during metabolic processes, such
as photosynthesis and respiration
Vitamins are an important source of coenzymes. For example, many vitamins in the B vitamin group are used in the
production of important coenzymes, including:
Pantothenic acid (vitamin B5), a key component of coenzyme A (a coenzyme required for the oxidation of
pyruvate during the link reaction that occurs between the glycolysis and Krebs cycle stages of respiration)
Nicotinic acid(vitamin B3), used to produce the coenzymes NAD and NADP (coenzymes required in many different
metabolic reactions, including many of the reactions that take place during photosynthesis and respiration)
Vitamin B2 (riboflavin), used to produce the coenzyme FAD (a coenzyme required in the Krebs cycle during
respiration)

Examples of coenzyme functions

 During many of the reactions in respiration, the coenzymes NAD and FAD are
alternately reduced and oxidised, transferring energy in the form of hydrogen ions
The coenzyme NADP fulfils this same role in chloroplasts during photosynthesis
The coenzymes ATP and coenzyme A act in a different way, by transferring chemical groups. For example:
ATP is responsible for the transfer of phosphate groups between respiration and energy-consuming processes in
cells
Coenzyme A is responsible for the transfer of an acetyl group (-CH₃CO) from fatty acids and glucose during
respiration

Prosthetic groups

Some cofactors are actually a permanent part of the structure of the enzyme they assist
These cofactors are known as prosthetic groups
Prosthetic groups are essential to the enzyme functioning properly, as they help to form the final 3D shape of the
enzyme
For example, by forming part of the active site of the enzyme, a zinc ion acts as the prosthetic group
for carbonic anhydrase (an enzyme found in red blood cells that converts CO₂ and H₂O into carbonic acid, H₂CO₃)

In summary:

Cofactors are non-protein substances (i.e. not made from amino acids) that enzymes require in order to function
properly. Cofactors can be a temporary part of the enzyme or a permanent part (known as a prosthetic group)
Cofactor: inorganic ion, such as iron and magnesium ions, required for optimal enzyme activity regulation
Coenzymes are organic non-protein cofactors. Coenzymes contribute to enzyme-catalysed reactions
by accepting or donating hydrogen ions or chemical groups (e.g. phosphate groups)
coenzyme: small organic molecule, such as a vitamin or its derivative, which is required to enhance an enzyme's
activity

Miscellaneous enzyme information

How human and bacterial amylases break down same substrate

1. (Ref. to different levels of protein structure)

2. Ref. to similar specific 3D conformation / globular structure
3. active sites are complementary to the same substrate
4. Ref. to either induced fit / lock-and-key hypothesis for binding of the substrate to active site
5. resulting in the formation of enzyme-substrate complex
6. activation energy is lowered
7. Similar catalytic amino acid residues present at the active sites of human and bacterial amylase catalyse breaking
down of the same substrate
8. Ref. to similar binding amino acid residues, allowing to bind to the same substrate
9. AVP e.g. Ref. to lack of quaternary structure as amylase is made of one polypeptide
Enzyme compartmentalisation
In eukaryotic cells, molecules such as enzymes are usually compartmentalised into different organelles. This allows for
yet another level of regulation of enzyme activity. Enzymes required only for certain cellular processes are sometimes
housed separately along with their substrates, allowing for more efficient chemical reactions. Examples of this sort of
enzyme regulation based on location and proximity include the enzymes involved in the latter stages of cellular
respiration, which take place exclusively in the mitochondria, and the enzymes involved in digesting cellular debris and
foreign materials, located within lysosomes.

Use of enzymes as industrial and laboratory catalysts

Enzymes as biological catalysts are important components of many industrial processes. Their use is widespread because
they are:

highly specific, catalysing changes in one particular compound or one type of bond
efficient, in that a tiny quantity of enzyme catalyses the production of a large quantity of product
effective at normal temperatures and pressures, and so a limited input of energy (as heat and high pressure) may be
required.

The enzymes selected by industry are e frequently produced from microorganisms – typically from species of fungi or
bacteria. Some examples:

Non-protein biological catalysts

It used to be thought that all biological catalysts were enzymes and were therefore made of protein. We now know that
some reactions in cells are catalysed by RNA molecules, also known as ribozymes. Some ribozymes work on other RNA
molecules, such as those that cut out unwanted sections from messenger RNA. This could answer the 'chicken or egg'
question - which came first, the enzyme (protein) needed to make nucleic acids, or the nucleic acids, needed to make
enzymes? The answer could be RNA which, in this sense at least, is both nucleic acid and 'enzyme'.

Enzymes summary
Enzymes are chemical catalysts that accelerate chemical reactions at physiological temperatures by lowering their
activation energy. Enzymes are usually proteins consisting of one or more polypeptide chains. Enzymes have an active
site that provides a unique chemical environment, comprised of certain amino acid R groups (residues). This unique
environment is perfectly suited to convert particular chemical reactants for that enzyme, scientists call substrates, into
unstable intermediates that they call transition states. Enzymes and substrates bind with an induced fit, which means
that enzymes undergo slight conformational adjustments upon substrate contact, leading to full, optimal binding. Enzymes
bind to substrates and catalyze reactions in four different ways: bringing substrates together in an optimal orientation,
compromising the bond structures of substrates so that bonds can break down more easily, providing optimal
environmental conditions for a reaction to occur, or participating directly in their chemical reaction by forming transient
covalent bonds with the substrates.

Enzyme action must be regulated so that in a given cell at a given time, the desired reactions catalyze and the undesired
reactions are not. Enzymes are regulated by cellular conditions, such as temperature and pH. They are also regulated
through their location within a cell, sometimes compartmentalized so that they can only catalyze reactions under certain
circumstances. Enzyme inhibition and activation via other molecules are other important ways that enzymes are
regulated. Inhibitors can act competitively, noncompetitively, or allosterically. Noncompetitive inhibitors are usually
allosteric. Activators can also enhance enzyme function allosterically. The most common method by which cells regulate
the enzymes in metabolic pathways is through feedback inhibition. During feedback inhibition, metabolic pathway products
serve as inhibitors (usually allosteric) of one or more of the enzymes (usually the first committed enzyme of the
pathway) involved in the pathway that produces them.

Metabolism, all the chemical reactions of life, consists of anabolic reactions, the build up of complex molecules from
smaller ones, e.g. protein synthesis, and catabolic reactions, the breakdown of complex molecules, e.g. oxidation
of sugar in respiration.
All reactions of metabolism are made possible by enzymes. Enzymes are biological catalysts and most are made of
globular protein. An enzyme is highly specific to the type(s) of substrate molecule and type of reaction that they
catalyse.
Enzymes work by forming a temporary complex with a substrate molecule at a special part of the enzyme surface,
called the active site (the lock-and-key hypothesis). Enzymes work by lowering the activation energy needed for a
reaction to occur.
A slight change in shape of the substrate molecule when it binds to the active site helps raise the molecule to a
transition state (the induced fit hypothesis), from which the products may form. The enzyme is released for reuse.
The rate of an enzyme-catalysed reaction is found by measuring the disappearance of the substrate or the
accumulation of the product in a given period of time. The initial rate of reaction is taken since the reaction rate falls
with time under experimental conditions.
A colorimeter can be used to measure the rate of enzyme-catalysed reactions that involve a colour change.
The factors that affect the rate of an enzyme-catalysed reaction include pH and temperature – through their effects
on protein structure. When molecules of substances recognised as inhibitors are in contact with enzyme molecules,
the rate of reaction may be lowered in characteristic ways.
» The Michaelis–Menten constant (K ) is the substrate concentration that sustains half maximum velocity ((½ V )
m max

of an enzyme-catalysed reaction. It is a measure of the degree of affinity of an enzyme for its substrate. K can be m

measured experimentally, and is expressed in units of molarity. Values of K m have been measured for a great many
enzymes, and it has been shown that some enzymes are able to work at maximum velocity at very low
concentrations of substrate.
Industries use enzymes as biological catalysts, often immobilised, with the reactants passed over them. In these
cases, the advantages are the recovery of the enzyme and its availability for reuse. Being immobilised may affect the
enzyme’s efficiency compared to use of the same enzyme when free in the substrate solution.

Enzymes are globular proteins which catalyse metabolic reactions. Each enzyme has an active site with a flexible
structure which can change shape slightly to fit precisely the substrate molecule. This is called the induced fit
hypothesis. When the substrate enters the active site, an enzyme–substrate complex is temporarily formed in which
the R groups of the amino acids in the enzyme hold the substrate in place.
Enzymes may be involved in reactions which break down molecules or join molecules together. They work by lowering
the activation energy of the reactions they catalyse.
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.
Temperature, pH, enzyme concentration and substrate concentration all affect the rate of activity of enzymes.
The greater the concentration of the enzyme, the faster the rate of reaction, provided there are enough substrate
molecules present. 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.
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 an enzyme is completely
denatured, it ceases to function, but denaturation is sometimes reversible.
Each enzyme has an optimum pH. Some enzymes operate within a narrow pH range; some have a broad pH range.
Enzymes are also affected by the presence of inhibitors, which slow down their rate of reaction or stop it
completely. Competitive inhibitors are molecules which are similar in shape to the normal substrate molecules. They
compete with the substrate for the active site of the enzyme. Competitive inhibition is reversible because the
inhibitor can enter and leave the active site.
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. Binding of non-competitive inhibitors may or may not be reversible.
Reversible non-competitive inhibitors bind at a site elsewhere on the enzyme, causing a change in shape of the active
site.
The efficiency of an enzyme can be measured by finding the value known as the Michaelis–Menten constant, K . To m

do this the maximum rate of reaction, V max , must first be determined. Determination of V max involves finding the
initial rates of reactions at different substrate concentrations while ensuring that enzyme concentration remains
constant.
Enzymes can be immobilised – for example by trapping them in jelly (alginate) beads. This is commercially useful
because the enzyme can be re-used and the product is separate from (uncontaminated by) the enzyme.
Immobilisation oft en makes enzymes more stable.