Topic 3 - Enzyme Notes

Enzymes are protein molecules which can be defined as biological catalysts. They speed up chemical reactions but
remain unchanged at the end of the reaction.
Intracellular and Extracellular enzymes
● Enzymes that operate within cells are called intracellular
● Enzymes that are secreted by cells and catalyse reactions outside cells are extracellular. An example of
this is the digestive system. Some organisms secrete enzymes outside their bodies - fungi.
Lock and Key and Induced Fit
Enzymes are globular proteins
Like all GP, they are coiled into a precise
three-dimensional shape with hydrophilic R
groups on the outside of the molecule,
ensuring they are soluble.
LOCK AND KEY HYPOTHESIS
● Enzymes have active sites which
another molecule (the substrate) can bind to.
Shape of the site is complementary to the
substrate. Specific.
● Substrate is the key whose shape fits the
lock of the enzyme
● Substrate is held in place by
temporary bonds with form between the substrate and some R groups of the enzyme’s amino acids.
● Combined to form an enzyme-substrate complex
● When enzyme catalyzes the reaction, substrate molecules can split into 2 or more molecules or may
catalyse the joining of two or more substrates (formation of dipeptide)
Interaction between the R groups of enzyme and atoms of substrate can break or form bonds which means
one or two or more products can be made
● This forms the enzyme-product complex (formed before release of product)
● When reaction is complete,
product/products leave the active
site.

Picture to the right shows an induced fit diagram.

Now, how does induced fit differ from the lock and
key hypothesis?
Induced fit is the modern hypothesis for enzyme
action. It's the same as the lock and key but adds the
idea that the enzyme and sometimes substrate
can change shape slightly to ensure a perfect fit.
In the diagram, the active site undergoes a change in
its conformation to ensure a better fit between the substrate and active site.

Enzymes reduce ACTIVATION ENERGY

As catalysts, enzymes increase the rate of chemical reaction which is essential in living cells.
In chemical reactions, substrate will not be converted to a product unless it is temporarily given some extra energy
and this energy is called activation energy.
- One way to increase the rate of reaction is to increase the energy of reactants by heating them
BUT,
 In our body, our temp is maintained at 37C so even if we raise the
temperature to 37C, it's not enough to give substrates the activation
energy they need to change to products.
SO,
Enzymes avoid this problem by decreasing the activation energy of the
reaction which they catalyse. HOW?
- By holding the substrates in such a way that their
molecules can reaction more easily.

FACTORS THAT AFFECT ENZYME ACTIONS:

The effect of ENZYME CONCENTRATION
The graph to the left shows an investigation in which different concentrations of enzymes were added to the same
vol of hydrogen peroxide solution.
TREND OF THE GRAPH:
● All five curves are similar. Reactions begin very quickly and then gradually slow.
Because the quantity of hydrogen peroxide is the same, they will all
produce the same amount of O2, and eventually meet.
EXPLANATION FOR GRAPH AND RATE:
● In order to compare results, the initial rate is needed to be found first. (1/t)
● In these conditions, reaction rate is directly proportional with the enzyme
concentration. When there's an increase in enzyme concentration, there's an
increase in rate of reaction
● This is because more enzyme present means more active sites available for
the substrate to bind, the initial rate of reaction increases.

The effect of SUBSTRATE CONCENTRATION

As substrate concentration increases, rate of reaction also increases.
This is because when there's more substrate molecules around,
the more often an enzyme’s active site can bind with one.
However, if we go on increasing the substrate concentration
and keeping the enzyme constant, there comes a point where
every enzyme active site is working continuously.
If more substrate is added, enzymes cannot work faster and
substrate are lining up for active site to be vacant.
The enzyme is then working at its maximum rate, known as Vmax.

The effect of TEMPERATURE

WHEN TEMPERATURES ARE LOW:
At low temperatures, the reaction takes place very slowly. This is because substrate molecules are moving
relatively slowly. Substrate molecules will not often collide with the active site, and so binding between
substrate and enzyme is slower/rare.
BUT, WHEN TEMPERATURE INCREASES:
- The enzyme and substrate molecules move faster as theres
more kinetic energy. Collisions more frequently means that substrate
molecules enter the active site more often. Moreover, when they do
collide, they do so with more energy = easier for bonds to be formed or
broken.
 BUT, WHAT HAPPENS WHEN TEMPERATURE CONTINUES TO INCREASE TOO MUCH:
The speed of movement of the substrate and enzyme molecules continues to increase. HOWEVER,
Above a certain temp, the structure of the enzyme vibrates so energetically that some of the bonds holding
the enzyme molecule begins to lose its shape and activity, this is called DENATURATION.
IRREVERSIBLE.
Eventually, the substrate molecule no longer fits at all, so the rate of reaction slows down.
The temperature at which an enzyme catalyses a reaction at the maximum rate is called the optimum temperature. Humans have
an op temp of around 40C. Different enzymes have different op temp.

The effect of pH
Most enzymes work fastest at pH of around 7. Some like protease pepsin prefers acidic conditions. Every enzyme
has a different optimum pH.
What is pH? It is the measure of the concentration of hydrogen ions in a
solution.
HOW DOES THIS EFFECT RATE?
● Hydrogen ions can interact with R groups of amino acids
- example, by affecting ionisation of groups
● This affects ionic bonding between groups which affects
the 3-dimensional arrangement of enzymes. The shape of the
active site may change and therefore reduce the chances of
substrate fitting.
A pH that is very different from the OPTIMUM PH CAN CAUSE
DENATURATION:

INHIBITORS:

Competitive, reversible inhibition

It is possible for some other molecules to bind to an enzyme’s active
site if it is very similar in shape to the enzyme’s substrate. This would
then inhibit the enzyme’s function.
- If an inhibitor molecule binds to the site, there is competition
between the it and the substrate for the site
- BUT, if there are larger conc of substrates than the inhibitor,
substrate can easily bind so the enzyme's function is unaffected and there's not much competition.
- HOWEVER, if the conc of the inhibitor is large or the conc of the substrate falls, chances of substrate
colliding with site is decreased. The enzyme’s function is then inhibited and therefore it is known as a
competitive inhibitor.

Competitive inhibitor is reversible and can be done by increasing the concentration of substrate
 Non-competitive Inhibitor
Also reversible. Non-competitive inhibitors take place when a molecule binds to another part of the enzyme rather
than the active site.
HOW DOES IT EFFECT ACTIVITY?
● While the inhibitor is bound to the enzyme, it disrupts the normal arrangement of hydrogen bonds and
hydrophobic interactions holding the enzyme molecule in the 3-dimensional shape.
● The distortion ripples across the molecule to the active site, making enzyme unsuitable for substrate.
● When an inhibitor is attached to an enzyme, the enzyme's function is blocked no matter how substrate is
present, this is a non-competitive inhibitor. The non-competitive inhibitor binds to the allosteric site.

HOW TO CONTROL IT:

To use the end-product of a chain of reactions
as a non-competitive, reversible inhibitor.
As the enzyme converts substrate to product, it is slowed down because the end-product binds to another part of
the enzymes and prevents more substrate binding.
HOWEVER, end-product can lose its attachment to enzyme and go used elsewhere, allowing enzyme to reform to
its active state.
As product levels fall, enzymes are able to top them up again. This is end-product inhibition.
Enzyme Affinities (Vmax and Km)
The rate of reaction when the enzyme is saturated with substrate
is the maximum rate of reaction is called the Vmax.
The relationship between rate of reaction and concentration of
substrate depends on the affinity of the enzyme for its substrate,
this is expressed in km (michaelis constant) of the enzyme, an
inverse measure of affinity.
- Km is the concentration
of substrate (from the graph)
which permits the enzyme to
achieve half Vmax.
Michaelis-Menten is Km.
At this point, half the active
sites for enzymes are occupied
by the substrate.
- The higher the affinity of
the enzyme for the substrate, the
lower the Michaelis-Menten
constant and the quicker the
reaction will proceed to its maximum rate (steep graph)
FINDING KM AND VMAX FROM A DOUBLE-RECIPROCAL
PLOT?
The point where the line of the graph intersects the x-axis is -1/Km.
From the value of -1/km, we calculate the Km by multiplying it.
 Whereas,Vmax is found where it intercepts on the y-axis. However, this gives 1/vmax, and you’ll need to find Vmax.

COMPARING NON-COMPETITIVE AND COMPETITIVE

The difference between Vmax:
Competitive has unchanged Xmax whereas non-competitive
has a decreased value of Vmax. The Km for competitive is
increased but the Km for the non-competitive is unchanged.

WHAT ARE THE SIGNIFICANCE OF VMAX AND KM

VALUES?
- An enzyme’s preference for different substrates can be compared quantitatively
- By understanding what affects the enzyme efficiency, scientists may be able to design better catalysts
- For a commercial enzyme, the performance of the same enzyme from organisms can be compared
- Calculations involved can be applied to other fields of biochemistry, - antibody - antigen binding.
IMMOBILISING ENZYMES
Enzymes are expensive. No company wants to buy them over again, so we can recycle them in some way.
Immolised enzyme is an enzyme attached to an inert, soluble material, such as calcium alginate (made by
reacting sodium alginate and enzyme solution with calcium chloride).
Example:
- Enzyme lactase can be immobilised using alginate beads. (Sodium alginate solution and lactase + calcium chloride), milk is
then run through the column of lactase-containing beads. Lactase hydrolysis the lactase in milk to glucose and galactose,
making it lactose free.
ADVANTAGES OF IMMOBILISING ENZYMES:
- This process means that immobilised enzymes are more tolerant of temperature and pH changes than
enzymes in solution (because their molecules are held firmly in shape by the
alginate so does not denature easily - or that the enzymes are embedded
in the beads so not fully exposed to the pH, temp, etc.
- Using immobilized enzymes means that you can keep and re-use
the enzymes, and the product is enzyme-free. = Reduced enzyme-cost
- Possibility of operating continuously