1. ENZYMES
-Jayati Shrivastava

2. What are Enzymes?
Enzymes are biological molecules, typically proteins,
that act as catalysts in biochemical reactions.
Enzymes speed up chemical reactions by lowering
the activation energy required for the reaction to
occur.
Enzymes are specific for the reactions they catalyze
and can recognize and bind to specific substrates.

3. • Enzymes can be regulated by various mechanisms,
including feedback inhibition and allosteric
regulation.
• Enzymes are often named based on the substrate
they act on, with the suffix "-ase" added to the
substrate name.
• Enzymes can be found in all living organisms and are
essential for many biological processes, including
digestion, metabolism, and DNA replication.

4. Properties of Enzymes
• Enzymes are proteins that act as biological catalysts,
speeding up chemical reactions without being consumed in
the process.
• Enzymes are highly specific, meaning that each enzyme is
designed to catalyze a particular chemical reaction or set of
reactions.
• Enzymes are sensitive to their environment, with their
activity being influenced by factors such as temperature, pH,
and the presence of certain ions or chemicals.

5. • Enzymes can be regulated by various mechanisms,
including feedback inhibition, allosteric regulation, and
post-translational modifications such as
phosphorylation or glycosylation.
• Enzymes can exist in different forms, including
apoenzymes (inactive enzymes without their cofactors)
and holoenzymes (active enzymes with their cofactors).
• Enzyme kinetics describe the quantitative relationship
between enzyme activity and substrate concentration,
which can be modeled using the Michaelis-Menten
equation.

6. • Enzymes are involved in a wide range of biological
processes, including digestion, metabolism, DNA
replication, and signal transduction.
• Enzymes can be used in various industrial
applications, including food production,
pharmaceuticals, and biotechnology.

7. How does Enzyme work ?
• Enzymes work by lowering the
activation energy required for a
chemical reaction to occur,
which makes the reaction
proceed at a faster rate. This is
accomplished by bringing the
reactants, called substrates,
into close proximity and in the
correct orientation for the
reaction to occur.

8. Basic steps of Enzyme action
• Enzymes have an active site, which is a specific region on the
enzyme's surface that binds to the substrate molecule.
• When the substrate molecule binds to the active site, an enzyme-
substrate complex is formed. The active site is complementary in
shape and chemical properties to the substrate molecule.
• The formation of the enzyme-substrate complex brings the
substrate molecule into close proximity to the enzyme's catalytic
groups, which can then facilitate the chemical reaction.

9. • The catalytic groups on the enzyme interact with the
substrate molecule, altering its chemical bonds and reducing
the activation energy required for the reaction to occur. This
lowers the energy barrier for the reaction, making it easier
for the reaction to proceed.
• After the reaction is complete, the products are released
from the active site of the enzyme, and the enzyme is free
to bind to another substrate molecule and repeat the
process.
• Enzymes are highly specific for their substrates, due to the
complementary shape and chemical properties of the active
site. This specificity allows enzymes to selectively catalyze
specific reactions.

11. Enzymes activators
• Allosteric activators: Allosteric activators are molecules that
bind to a specific site on the enzyme, called the allosteric site,
causing a change in the enzyme's shape that increases its
activity. This can be a positive feedback loop, where the
product of a reaction acts as an activator for an earlier step in
the reaction.
• Coenzymes: Coenzymes are organic molecules that bind to the
enzyme and help it carry out its function. Some coenzymes,
like NAD+ and FAD, act as electron carriers and can transfer
electrons between different enzymes, thereby increasing their
activity.

12. • Metal ions: Some enzymes require metal ions, such as
Mg2+, Zn2+, or Fe2+, as cofactors to function properly.
These ions can help to stabilize the enzyme's structure or
participate in the reaction itself, increasing the enzyme's
activity.
• pH regulators: Some enzymes require specific pH conditions
to function optimally. Substances that help regulate pH,
such as buffers, can therefore act as activators by
maintaining the optimal pH range for the enzyme.
• Hormones: Some hormones can act as enzyme activators by
binding to specific receptors on the cell membrane and
triggering a signaling cascade that activates enzymes
involved in the response.

13. Enzymes Inhibitors
Competitive inhibitors:
Competitive inhibitors bind to
the active site of the enzyme,
preventing the substrate from
binding and reducing the
enzyme's activity. The degree of
inhibition can be overcome by
increasing the substrate
concentration.

14. • Non-competitive inhibitors: Non-
competitive inhibitors bind to a site
on the enzyme that is distinct from
the active site, altering the enzyme's
shape and reducing its activity.
Increasing the substrate
concentration will not overcome
this type of inhibition.

16. • Irreversible inhibitors: Ithe
inhibitor binds to the enzyme and
forms enzyme-inhibitor complex
which dissociates slowly or does
not dissociate at all. Such
inhibitors often form a dead
complex where the enzyme
becomes useless.
• Drugs such as penicillin are
irreversible inhibitors of the
enzyme- transpeptidase in
bacteria. True irreversible
inhibitors include oxidizing agents
or alkylating agents, that cause a
more permanent chemical
modification, usually at or near
the active site

17. • Feedback Inhibitors

18. Factor affecting Enzyme activity
• Temperature: Enzymes have
an optimal temperature at
which they work most
efficiently. Above or below
this temperature, the enzyme
activity can decrease or stop
altogether.

19. • pH: Enzymes have an optimal pH at which they
work most efficiently. Changes in pH can cause
changes in the enzyme's structure and alter its
activity.

20. • Substrate concentration: Enzyme activity is
directly proportional to the concentration of
substrate molecules available for the enzyme to
bind with. However, once the enzyme is saturated
with substrate, further increases in substrate
concentration will not increase the rate of the
reaction.

21. • Enzyme concentration:
Increasing the amount of enzyme
present will increase the rate of
the reaction, as long as there is
enough substrate present for the
additional enzyme to bind with.

22. Enzyme Kinetics
• Enzyme kinetics is the study of the rates and
mechanisms of enzyme-catalyzed reactions.
Enzyme kinetics involves measuring the rate of an
enzyme-catalyzed reaction under different
conditions and analyzing the data to determine the
parameters that describe the reaction.

23. Here are some key concepts and terms related to
enzyme kinetics:
• Enzyme-substrate complex: The complex formed
when an enzyme binds to its substrate.
• Michaelis-Menten equation: A mathematical
equation that describes the relationship between
substrate concentration and reaction rate for an
enzyme-catalyzed reaction.

24. • Enzyme kinetics parameters: Parameters used to
describe enzyme-catalyzed reactions, including
the Michaelis constant (Km), the maximum
velocity (Vmax), and the catalytic efficiency
(kcat/Km).
• Km: The Michaelis constant, which is the
substrate concentration at which the reaction rate
is half of the maximum velocity.
• Vmax: The maximum velocity, which is the
maximum rate of reaction when the enzyme is
saturated with substrate.

25. • kcat: The turnover number, which is the number of
substrate molecules converted to product per unit
time when the enzyme is fully saturated with
substrate.
• Catalytic efficiency: The ratio of kcat to Km, which
provides a measure of how efficiently an enzyme
converts substrate to product.
• Lineweaver-Burk plot: A graphical representation of
the Michaelis-Menten equation that allows for the
determination of Km and Vmax.

26. Michaelis-Menten derivation for simple steady-state
kinetics
• It is a mathematical model that is used to analyze
simple kinetic data. The model has certain
assumptions, and as long as these assumptions are
correct, it will accurately model your experimental
data. The derivation of the model will highlight
these assumptions

27. • In an enzyme catalyzed reaction the substrate
initially forms a reversible complex with the enzyme
(i.e. the enzyme and substrate have to interact for
the enzyme to be able to perform its catalytic
function). The standard expression to show this is
the following:

28. • ASSUMPTION 1:
• There is no product present at the start of the
kinetic analysis
• Therefore, as long as we monitor initial reaction
rates we can ignore the reverse reaction of E+P
going to ES

29. • ASSUMPTION 2:
• During the reaction an equilibrium condition is
established for the binding and dissociation of the
Enzyme and Substrate (Briggs-Haldane
assumption)
• Thus, the rate of formation of the ES complex is
equal to the rate of dissociation plus breakdown

30. • ASSUMPTION 3:
• [E] << [S]
• The enzyme is a catalyst, it is not destroyed and can
be recycled, thus, only small amounts are required
• The amount of S bound to E at any given moment is
small compared to the amount of free S
• It follows that [ES] << [S] and therefore [S] is constant
during the course of the analysis (NOTE: this
assumption requires that the reaction is monitored
for a short period, so that not much S is consumed
and [S] does not effectively change - see next
assumption)

31. • ASSUMPTION 4:
• Only the initial velocity of the reaction is measured
• [P] = 0 (reverse E + P reaction can be ignored)
• [S] » [S]initial
• ASSUMPTION 5:
• The enzyme is either present as free enzyme or as
the ES complex
• [E]total = [E] + [ES]

32. Michaelis-Menten derivation using these assumptions:
• Rate of ES formation = k1[E][S] + k-2[E][P]
• Assumption #1 says we can ignore the k-2 reaction, therefore:
• Rate of ES formation = k1[E][S]
• Assumption #5 says [E] = [E]total - [ES], therefore:
• Rate of ES formation = k1([E]total - [ES])[S]
• The rate of ES breakdown is a combination of the dissociation and
the conversion to product:
• Rate of ES breakdown = k-1[ES] + k2[ES]
• Rate of ES breakdown = (k-1 + k2)[ES]
• Assumption #2 says the rate of ES formation equals the rate of
breakdown:

33. • k1([E]total - [ES])[S] = (k-1 + k2)[ES]
• Rearrange to define in terms of rate constants:
• ([E]total - [ES])[S] / [ES] = (k-1 + k2) / k1
• ([E]total [S] / [ES]) - [S] = (k-1 + k2) / k1
• Define a new constant, Km = (k-1 + k2) / k1
• ([E]total [S] / [ES]) - [S] = Km
• Solve for the [ES] term (for reasons that will be given in the
next step):
• [ES] = [E]total [S] / (Km + [S])
• The actual reaction velocity measured at any given moment
is given by:
• V = k2[ES]
• Multiple both sides of the above equation by k2:
• k2[ES] = k2[E]total [S] / (Km + [S])

34. • thus
• V = k2[E]total [S] / (Km + [S])
• The maximum possible velocity (Vmax) occurs
when all the enzyme molecules are bound with
substrate [ES] = [E]total, thus:
• Vmax = k2[E]total
• Substituting this into the prior expression gives:
• V = Vmax [S] / (Km + [S])
• This is the mathematical expression that is used to
model your experimental kinetic data
• It is known as the Michaelis-Menten equation

35. Experimental approach
• The general approach is to add a known concentration of
substrate to the enzyme and to determine the initial
reaction rate for that concentration of substrate
• Reaction rates are typically given as moles (or micromole) of
product produced per unit of time (sec or min) per mole (or
micromole) of enzyme
• The experiment is repeated for a wide range of substrate
concentrations
• A table of [S] versus V datapoints are collected
• These datapoints are plotted (V versus S) and should fit a
curve that agrees with the Michaelis-Menten equation

36. • The Vmax and Km terms are intrinsic properties
of the particular enzyme/substrate combination
that you are studying
• They will be determined from the features of the
V versus S plot

37. • Vmax
• There are a limited number of enzyme molecules
and they can only perform a single reaction at a
time. Thus, at high [S] the enzymes can be saturated
• Under saturating conditions the reaction is going as
fast as it can, and additional increases in [S] do not
increase the reaction rate.
• The maximum observable rate is Vmax and the data
will asymptote to this value at high [S]
• At low [S] the reaction rate is generally linearly
proportional to the [S] (i.e. at low [S] if you double
[S] the V will double)

38. • Km
• Km = (k-1 + k2) / k1 = (rate of breakdown of
ES)/(rate of formation of ES)
• Km is similar, but not exactly equal to, a
dissociation constant (Kd) for the ES complex
• If k-1 >> k2, then Km » Kd
• Due to this similarity to the expression for Kd, a
low value of Km is often interpreted as a high
affinity of the enzyme for the substrate, and a large
value for Km is often interpreted as a weak affinity
of the enzyme for the substrate
• Km has units of molar concentration (just like the
units for [S])

39. • There is a mathematical treatment that allows for the
determination of Km from the experimental V versus [S] data
• Consider the situation when the [S] being evaluated results in
a value of V that is exactly 1/2 of the maximum reaction
velocity:
• V = Vmax [S] / (Km + [S])
• 1/2Vmax = Vmax [S] / (Km + [S])
• 1/2 = [S] / (Km + [S])
• Km + [S] = 2[S]
• Km = [S]
• Thus, Km equals the substrate concentration that results in
exactly one half the maximum possible reaction velocity

41. Lineweaver-Burke (the "double reciprocal" plot)
The Michaelis-Menten equation can be rearranged by
taking the reciprocal, to yield:
• The Michaelis-Menten equation can be rearranged
by taking the reciprocal, to yield:
If X = 1/[S] and Y=1/V then this is a linear equation with
a slope of Km/Vmax and a Y intercept of 1/Vmax

42. Since the plot of 1/[S] versus 1/v data should
be a straight line, it is easier to fit a linear
function to the data in this form, and Vmax
and Km can be readily determined from the
plot

43. The effects of the reversible competitive inhibitor on
the kinetics are as follows:
• If no inhibitor is present (i.e. if [I] = 0) then the
equations are the same
• As inhibitor is added, the effect is to modify the
apparent value of Km. In particular, the apparent Km
will be increased by a value equal to (1 + [I]/KI). If Km is
increased, the reaction velocity v will decrease.
• Note that as [S] gets very large the value of the
denominator is essentially equal to [S] and v @ vmax.
Thus, the reaction velocity can be driven to vmax with a
high enough substrate concentration

44. • The diagnostic criteria for reversible competitive
inhibition is that while the apparent Km is affected
by addition of the inhibitor, the value of vmax does
not change
Figure: Effect of reversible competitive inhibitor

45. • How is the Lineweaver-Burke double reciprocal plot
affected by the presence of a reversible
competitive inhibitor?
Figure : Double reciprocal plot with reversible
competitive inhibitor

46. • Noncompetitive Inhibitors
• Noncompetitive inhibitors react with both E and ES
(this is because the noncompetitive inhibitor does
not bind at the same site in the enzyme as the
substrate)
• Inhibition cannot be overcome by increasing the
concentration of S
• The effect on kinetics is as if the enzyme were less
active (vmax is reduced), but that the affinity for
substrate is unaffected (Km remains the same)
since the substrate binding site is not occupied by
the noncompetitive inhibitor.

47. Figure: Effect of reversible noncompetitive inhibitor

48. Figure: Double reciprocal plot with noncompetitive inhibitor