Enzymes are protein catalysts that accelerate chemical reactions. They lower the activation energy of reactions allowing metabolic processes to occur faster. Enzymes achieve specificity through their tertiary structure and active site. The induced fit model describes how the active site changes shape upon substrate binding. Enzyme kinetics follow the Michaelis-Menten model where the substrate first binds the enzyme before products are released. Reaction rates depend on factors like substrate/enzyme concentration, temperature, pH, and inhibitors. Enzymes can be immobilized in beads to make them reusable.

1. Unit-3
Enzymes
- By Suman Tiwari
(M.Sc. , M. Ed.)

2. Enzymes
• Enzymes are proteins that act as biological catalysts (biocatalysts).
Catalysts accelerate chemical reactions. The molecules upon
which enzymes may act are called substrates, and the enzyme
converts the substrates into different molecules known
as products. Almost all metabolic processes in the cell
need enzyme catalysis in order to occur at rates fast enough to
sustain life.
• The study of enzymes is called enzymology
• In 1877, German physiologist Wilhelm Kühne(1837–1900) first
used the term enzyme.
• An enzyme's name is often derived from its substrate or the
chemical reaction it catalyzes, with the word ending in -
ase.Examples are lactase, alcohol dehydrogenase and DNA
polymerase.

3. • All enzymes are globular proteins with a specific tertiary structure,
which catalyse metabolic reactions in all living organisms. This means
that they speed up chemical reactions, but are not ‘used-up’ as part of
the reaction.
• Enzymes are relatively large molecules, consisting of hundreds of amino
acids which are responsible for maintaining the specific tertiary
structure of the enzyme. Each enzyme has a specific active site shape,
maintained by the specific overall tertiary structure. Therefore the
tertiary structure must not be changed.
Enzymes

4. • Enzymes can function both inside cells (intracellular) or outside cells
(extracellular).
• Extracellular enzyme action occurs outside the cell, which produces
the protein. For example, some enzymes in digestive systems are
extracellular as they are released from the cells that make them,
onto food within the digestive system spaces.
• Spiders and flies are two examples of animals that have taken
extracellular digestion a step further. They secrete an enzyme soup
into or on their food. In spiders, this is injected into the prey's body.
The enzyme soup digests the prey's body contents (specific enzymes
breaking down proteins to amino acids, lipids into fatty acids and
glycerol and polysaccharides into monosaccharides) and the spider
simply sucks up the resulting already digested food. Saprophytic
fungi also secrete enzymes through their hyphal tips in order to
digest their food.
Types of Enzymes

5. •Intracellular enzyme action occurs inside the cell, which
produces the enzyme. For example, some enzymes in
digestive systems are found in the cytoplasm of cells or
attached to cell membranes and the reaction takes place
inside the cell.
•Enzymes that act inside cells are responsible for
catalysing the millions of reactions that occur in
metabolic pathways such as glycolysis in the
mitochondria and in the photosynthetic pathway in the
chloroplast. The lysosome contains many enzymes that
are mainly responsible for destroying old cells.
Types of Enzymes

6. • To explain the observed specificity of enzymes, in 1894 Emil Fischer
proposed that both the enzyme and the substrate possess specific
complementary geometric shapes that fit exactly into one another.
This is often referred to as "the lock and key" model. This early
model explains enzyme specificity, but fails to explain the
stabilization of the transition state that enzymes achieve.
The Lock and Key Model

7. • In 1959, Daniel Koshland suggested a modification to the lock and
key model:
• According to the induced fit model, the enzyme’s active site is not a
completely rigid fit for the substrate
• Instead, the active site will undergo a conformational change when
exposed to a substrate to improve binding
• This theory of enzyme-substrate interactions has two advantages
compared to the lock and key model:
• It explains how enzymes may exhibit broad specificity (e.g. lipase
can bind to a variety of lipids)
• It explains how catalysis may occur (the conformational change
stresses bonds in the substrate, increasing reactivity)
Induced fit hypothesis

8. Enzyme Models

9. • Molecules that have enough energy undergo reaction are converted
to products. The energy required for the molecules to undergo a
change in known as activation energy. In laboratory experiments
energy is provide to the system to increase the activation energy of
the molecules to produce products. But in the living system it is not
possible to increase the temperature to such elevated state of
energy, which could not support life.
Activation energy

10. •The only possibility to produce products without
increasing the energy of the molecule is to decrease the
activation energy of the system. This is possible only
because of the mediation of enzyme. It is through
enzymes that life carries out such reactions at relatively
low temperature suitable for life. The enzymes being
proteins do this job by lowering the energy of activation
so that a much greater fraction of the substrate
molecules has sufficient energy to react without a
temperature increase, thus substrates are converted into
products. A careful study of graph can further help in the
understanding of this enzyme action mechanism.
Activation energy

11. • Enzyme kinetics is the investigation of how enzymes bind
substrates and turn them into products.
• Leonor Michaelis and Maud Leonora Menten proposed a
quantitative theory of enzyme kinetics, which is referred to
as Michaelis–Menten kinetics .
• The major contribution of Michaelis and Menten was to think of
enzyme reactions in two stages. In the first, the substrate binds
reversibly to the enzyme, forming the enzyme-substrate complex.
The enzyme then catalyzes the chemical step in the reaction and
releases the product.
The course of a reaction:
Enzyme Kinetics

12. •G. E. Briggs and J. B. S. Haldane, who derived kinetic
equations that are still widely used today.
•Enzyme rates depend on solution conditions and
substrate concentration. To find the maximum speed of
an enzymatic reaction, the substrate concentration is
increased until a constant rate of product formation is
seen. This is shown in the saturation curve on the right.
Saturation happens because, as substrate concentration
increases, more and more of the free enzyme is
converted into the substrate-bound ES complex. At the
maximum reaction rate (Vmax) of the enzyme, all the
enzyme active sites are bound to substrate, and the
amount of ES complex is the same as the total amount of
enzyme.
The course of a reaction

13. The course of a reaction

14. •For ex.
•If Km = 0.05 milli Molar ,Glucose ( Hexokinase)
•Km = 5 mM, Glucose (Glucokinase)
•Km indicates when the enzyme is going to be active for
the given substrate.
•In the above case as hexokinase have low Km= 0.05, it
means it has higher affinity to start the reaction for the
glucose substrate even at such a low concentration.
•On the other hand glucokinase has a value of ,5mM , so
it means it has lower affinity to start the reaction.

15. •Vmax is the saturated point.( Where all enzymes are
involved in making enzyme substrate complex).
•The capacity of any enzyme to use the substrate is
calculated by using Vmax.
•In the above case hexokinase will have lower Vmax.
•Whereas glucokinase will have higher Vmax.
Word Problem
Formula to be used: V0 = Vmax X [S]
Km + [S]

18. Competitive Inhibitor-
Km ,Vmax same
Non Competitive Inhibitor-
Km same , Vmax

19. • Competitive Inhibitor is analogous to substrate so, it competes with
substrate. In the previous graph inhibitor is meeting at the same
point (1/Vmax) and 1/Km value is closer to zero so it indicates an
increase in the value of Km, hence a decrease in the affinity of
enzyme. So it could be concluded that competitive inhibitor
increases the Km value of any reaction. More substrate is required
for an enzyme reaction involving competitive inhibitor to reach the
same value of Vmax.
• Non competitive Inhibitors do not have structural analogue as it
binds to enzymes on other site than the active sites. But they do
change the confirmation of active site, thereby the substrate will
not be able to fit well. This will decrease the reaction velocity. In this
type of inhibition Km value remains same but the Vmax value will be
changed. Vmax value will decrease in this situation.

20. •Vmax is only one of several important kinetic parameters.
The amount of substrate needed to achieve a given rate
of reaction is also important. This is given by
the Michaelis-Menten constan (Km), which is the
substrate concentration required for an enzyme to reach
one-half its maximum reaction rate; generally, each
enzyme has a characteristic KM for a given substrate.
Another useful constant is kcat, also called the turnover
number, which is the number of substrate molecules
handled by one active site per second.
•The efficiency of an enzyme can be expressed in terms
of kcat/Km. This is also called the specificity constant.

21. •Enzyme concentration:
•As the concentration of enzymes is increased, there
are more available active sites for substrates to fit into.
More enzyme-substrate complexes are formed, more
products are formed and the rate of reaction is
increased. The limiting factor is the enzyme
concentration. Once all substrates have formed
enzyme substrate complexes, a further increase in
concentration will have no effect on the rate of
reaction. At this point, the limiting factor is the
substrate concentration. During comparison, look at
initial rate to ensure differences in reaction rate are
caused only by differences in enzyme concentration.
Factors that affect
enzyme action

22. •Substrate concentration:
•As the concentration of the substrates increases, there
are greater chances of collision with enzyme. More
enzyme-substrate complexes are formed, more products
are formed and the rate of reaction is increased. The
limiting factor is the substrate concentration. Once all
enzymes are occupied and working at maximum rate
(Vmax), a further increase in substrate concentration will
have no effect on the rate of reaction. At this point, the
limiting factor is the enzyme concentration.
Factors that affect
enzyme action

23. Factors that affect
enzyme action

24. •Temperature:
As the temperature increases, the kinetic energy and the
enzyme activity increases as there’s more collisions until
optimal temperature is reached (usually 40C). At optimal
temperature, maximum rate of reaction is achieved. If
the temperature continues to increase beyond optimal
temperature, the rate of the reaction begins to decrease
as more kinetic energy breaks the hydrogen bonds in the
secondary and tertiary structure of enzyme. This
changes the shape of the enzyme and its active site and
causes the substrate to no longer fit. The enzyme is
denatured.
Factors that affect
enzyme action

25. Factors that affect
enzyme action

26. •pH:
Any change in the pH value of the medium around the
enzyme will cause ionic and hydrogen bonds to be
damaged, this will change the 3-D shape of the enzyme
and deform the active site. The substrate will therefore
not be able to fit into active site so the reaction slows
down or stops. The effects of pH is reversible within
certain limits but if the pH is far from optimal value, the
enzyme gets denatured.
Factors that affect
enzyme action

27. •Inhibitors interfere with enzyme activity and reduce the
rate of an enzyme catalysed reaction. Therefore, as the
concentration of inhibitors increases, the rate of reaction
decreases.
•Reversible competitive inhibitor: has a similar shape
to the substrate and fits into the active site. This reduces
the number of enzyme-substrate complexes formed and
the rate of reaction decreases. It is said to be reversible
because it can be reversed by increasing the concentration
of the substrate.
Enzyme inhibitors

28. Enzyme inhibitors

29. •The reversible non-competitive inhibitor: has a
different shape to the substrate and fits into a site
other than the active site. While the non-competitive
inhibitor is bound, the tertiary structure of the entire
enzyme is distorted, preventing the formation of
enzyme-substrate complexes and decreasing the rate
of reaction regardless of substrate concentration.
Enzyme inhibitors

30. Enzyme inhibitors

31. •End-product inhibition: used to control metabolic
reactions via non-competitive reversible inhibitors. As
the enzyme converts substrate to product, it is slowed
down because the end product binds to another part
of the enzyme and prevents more substrate binding.
However, the end-product can lose its attachment to
the enzyme and go on to be used elsewhere, allowing
the enzyme to reform into its active state.
Enzyme inhibitors

32. •Theoretical maximum rate velocity (Vmax):
The reaction rate is measured at different substrate
concentrations while keeping the enzyme concentration
constant. As substrate concentration is increased,
reaction rate rises until the reaction reaches its
maximum rate.
Enzyme Kinetics

33. •Michaelis–Menten constant (Km):
•The substrate concentration that corresponds to half of
Vmax is Km.
Km measures the affinity of the enzyme for the
substrate. The higher the affinity, the more likely the
product will be formed when a substrate molecule
enters the active site. The higher the affinity of the
enzyme for the substrate, the lower the substrate
concentration needed for this to happen. The higher the
affinity, the lower the Km and the quicker the reaction
will proceed to Vmax.

34. 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.
Advantages:
• 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 molecule
embedded in the beads are not fully exposed to temperature or pH
changes.
Enzyme immobilization

35. Enzyme immobilization

36. • Disadvantages:
• Active site may be distorted by immobilizing. Substrate passes
through matrix when immobilized. Some product is retained within
matrix
• Video Link:
• https://www.youtube.com/watch?v=oiTVJ91-YxQ
Enzyme immobilization

37. • https://www.youtube.com/watch?v=R8MVcoufUfs
• https://www.youtube.com/watch?v=1Fa2sSIt4_I
• https://www.youtube.com/watch?v=3DfKVr_IHyI
• https://www.youtube.com/watch?v=WsqVFDhqBaY

38. Thank You