Download Enzymes: Basic Concepts and Kinetics

Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Enzymes: Basic Concepts & Kinetics
Enzymes are highly specific, powerful catalysts of biological
systems that help to accelerate reactions taking place in
organisms by factors of millions or more.
Learning Objectives:
After interacting with this Learning Object, the learner will
be able to:
Describe Enzymes and their components.
Recall Energetics of enzymatic reactions.
List out models for enzyme-substrate binding.
Define kinetics of enzymatic reactions.
Define enzyme inhibition.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Enzymes & their componentss
Most enzymes are made up of a
protein part known as the apoenzyme
as well as a cofactor which can either
be an organic molecule known as a
coenzyme or
a metal ion. These
cofactors are essential for the enzyme
to be catalytically functional and the
complete
functional
enzyme
is
referred to as the holoenzyme.
Pyruvate dehydrogenase is a complex
enzyme
which
uses
Thiamine
pyrophosphate as its coenzyme while
carbonic anhydrase uses zinc ion as its
cofactor.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Enzymes & their componentss
Enzymes are classified on the basis of the
reactions that they catalyze. Most
enzymes are named by adding the suffix
‘ase’ either to their substrate or the type
of activity they carry out. However as
more enzymes came to be known, it
became increasingly difficult to name
them in this manner. Classification by
international organizations has therefore
led to six enzyme classes with many
subgroups within each class, depending
upon the type of reaction being
catalyzed. Every enzyme has a unique,
four-part classification number known as
the Enzyme Commission number (E.C.
number), in which subclass number gives
finer details about that particular enzyme
reaction.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Energetics of enzymatic reactions
Conversion of substrate to product
proceeds through formation of a
transition state. The free energy of
activation of an uncatalyzed reaction is
very high. Enzymes form favorable
interactions with the substrate and
facilitate formation of the transition
state by lowering the free energy of
activation. The transition state then
dissociates to give the product and
regenerates free enzyme. For a reaction
to be spontaneous, the ∆G must be
negative. It must be emphasized that
enzymes do not alter the equilibrium of
a reaction.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Models for enzyme-substrate binding
Fischer’s hypothesis is aptly defined as
the ‘lock-and-key’ hypothesis. Any
lock, which is analogous to an
enzyme, can have only one suitable
key of appropriate shape and size to
open it. The various available keys,
which are analogous to the thousands
of substrates available, can attempt
to open the lock but only one will be
the perfect fit that is capable of
opening the lock. Similarly only one
particular substrate will fit into the
active site of the enzyme and the
enzymatic reaction can occur
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Models for enzyme-substrate binding
According to the Fischer’s hypothesis,
enzymes and their substrates possess
specific complementary geometric
shapes that fit exactly into each other.
This model accounts for the specificity
of enzymes but fails to account for
stabilization of the transition state.
Koshland modified this hypothesis and
suggested that the active site of an
enzyme gets continually reformed
based on the interactions that it
establishes
with
the
substrate
molecule. This accounts for both the
enzyme
specificity
and
the
stabilization of the transition state
since the enzyme is not considered to
be a rigid molecule.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Kinetics of enzymatic reactions
Enzymes catalyze the formation of
product from
its substrate via an
enzyme-substrate intermediate complex.
During the initial stages of the reaction,
the equilibrium favors product formation
rather than dissociation of the [ES]
complex to give back the substrate. The
number of moles of product formed per
second during these stages determines
the reaction velocity for that particular
enzyme. Vo has an almost linear relation
with substrate concentration when the
substrate concentration is low but
becomes
independent
at
higher
concentrations
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Kinetics of enzymatic reactions
The Michaelis-Menten model for enzyme
kinetics assumes that the breakdown of
[ES] complex to give back free substrate is
negligible and also assumes steady-state
conditions whereby the rates of formation
and breakdown of the [ES] complex are
equal. The reaction velocity increases
linearly with substrate concentration
when [S] is low but becomes independent
at higher concentrations. The maximal
velocity that can be achieved by an
enzyme refers to the state in which all its
catalytic sites are occupied. The substrate
concentration at which the reaction
velocity is equal to half its maximal value
is known as the Michaelis constant, Km.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Kinetics of enzymatic reactions
The Lineweaver-Burk equation or the
double reciprocal plot is a useful tool
that can be plotted using simple
experimental data from kinetics
experiments. This equation is derived
from the Michaelis-Menten equation
by taking reciprocals on both sides and
then plotting a graph of 1/Vo Vs 1/
[S]. The Y-intercept on this graph can
be used to deduce the value of Vmax
while the X-intercept gives the value
of Km.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Enzyme inhibition
Enzyme inhibition can either be
reversible, where the inhibitor can
dissociate quickly from the enzyme, or
irreversible, where the inhibitor
dissociates very slowly from the
enzyme and can covalently modify the
enzyme thereby rendering it unsuitable
for
further
catalysis
reactions.
Reversible inhibition can be further
classified
as
competitive,
uncompetitive and mixed inhibition.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Enzyme inhibition
In competitive inhibition, the inhibitor
molecule is structurally similar to the
substrate and therefore binds to the
enzyme at the active site. Binding of
inhibitor
prevents
substrate
from
binding, thereby decreasing the reaction
rate. The Vmax in this type of inhibition
remains the same and only the Km is
altered. Competitive inhibition can be
overcome by suitably increasing the
substrate concentration, which allows
the substrate to out-compete the
inhibitor for the enzyme’s active site.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Enzyme inhibition
In the case of uncompetitive inhibition,
the substrate and inhibitor both have
different binding sites on the enzyme .
However, the inhibitor binds only to the
enzyme-substrate complex and not to
the enzyme alone. Binding of inhibitor to
the ES complex prevents any further
reaction and no product formation is
observed. Both the Km and Vmax are
found to decrease with this type of
inhibition.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Enzyme inhibition
A mixed inhibitor also binds to the enzyme
at a site distinct from the substrate
binding site with the difference being that
it can bind either to enzyme or ES
complex. Binding of either one brings
about conformational changes in the
enzyme structure thereby affecting
binding of the other. This inhibition can be
reduced but not overcome by increasing
substrate concentration. Both Km and
Vmax are altered in this type of inhibition.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Enzymes & their components
1. Enzymes: Majority of enzymes are proteins
(some are RNAs) that increase the rates of
biochemical reactions in living organisms.
They are highly specific molecules that
catalyze only a particular group of
reactions and like other catalysts, they are
regenerated at the end of a reaction. The
rates of enzyme catalyzed reactions are
often more than million times faster than
those of un-catalyzed ones. Molecular
weights of enzymes range from 12,000 to
more than 1 million daltons. Enzymes offer
the advantages of high reaction rates,
substrate
specificity,
capacity
for
regulation and mild reaction conditions
when compared to any other chemical
catalysts.
2. Apoenzyme: The protein part of anenzyme
that requires other additional components in
order to make it catalytically functional is
known as the apoenzyme or apoprotein.
3. Cofactor: Many enzymes require the presence
of an additional component known as a cofactor
for their catalytic activity. These cofactors can
either be metal ions such as Fe2+, Mg2+, Mn2+
etc. or organic molecules known as coenzymes.
A coenzyme or metal ion that is very tightly or
covalently bound to the apoenzyme is known as
the prosthetic group.
4. Holoenzyme: The complete, catalytically
functional enzyme containing both the protein
part as well as its cofactor is known as the
holoenzyme.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Enzymes & their components
5. Enzyme classification: Enzymes are
classified on the basis of the reactions that
they catalyze. Most enzymes are named by
adding the suffix ‘ase’ either to their substrate
or the type of activity they carry out.
Classification by international organizations
has led to six enzyme classes with many
subgroups within each class, depending upon
the type of reaction being catalyzed. Every
enzyme has a unique, four-part classification
number known as the Enzyme Commission
number (E.C. number)
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Energetics of enzymatic reactions
1. Enzyme: The biocatalyst responsible for
bringing about an increase in the rate of
reaction for conversion of substrate to
product.
2. Substrate(s): The molecules present at the
beginning of a reaction that are modified by
means of the enzyme are known as substrates.
An enzymatic reaction may have one or more
substrates depending upon the reaction.
3. Product(s): The molecules produced as a
result of an enzymatic reaction are known as
the products. A reaction may yield one or
more products with the enzyme being
regenerated at the end of the reaction.
4.Transition state: Enzymatic reactions proceed
through formation of a transition state i.e. an
intermediate state between substrate and
product having higher free energy than that of
either the substrate or product. The transition
state is the least stable species of the reaction
pathway due to its high free energy and is
therefore the most seldom occupied.
5. Free energy: The Gibbs free energy is a
useful thermodynamic property that can be used
to understand enzymatic reaction mechanisms.
The free energy difference between the
reactants and products as well as the
energy.required to activate conversion of
reactants to products can provide information
about the spontaneity and rate of a reaction.
The free energy change (∆G) of a reaction is
only dependent on the free energy of the
reactants and products and not on the path of
the reaction.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Energetics of enzymatic reactions
A reaction can occur spontaneously only if the
∆G is negative.
6. Free energy of activation (∆G‡): The rate of
any reaction is dependent on the Gibbs free
energy of activation (∆G‡) i.e. the energy
difference between the substrate and the
transition state. Enzymes function to lower this
energy gap by forming favourable interactions
with the substrate, thereby facilitating
formation of the transition state and allowing a
larger number of molecules to overcome this
energy barrier. Enzymes do not function by
modifying the reaction equilibrium but only
alter the reaction rate.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Models for enzyme-substrate binding
1. Fischer’s Lock-and-Key hypothesis: Emil
Fischer in 1894 postulated that enzymes and
their substrates possess specific complementary
geometric shapes that fit exactly into each
other like a key fits in a lock. This model
accounts for the specificity of enzymes but fails
to account for stabilization of the transition
state.
2. Koshland’s induced fit model: Daniel
Koshland in 1958 suggested that the active site
of an enzyme gets continually reformed and
reshaped based on the interactions that it
forms with the substrate molecule. Therefore,
rather than assuming a rigid shape, the
enzyme’s active site gets molded.
into the exact shape and position required to
carry out catalysis of the substrate. This
accounts for both the enzyme specificity and the
stabilization of the transition state.
3. Active site: All enzymes possess an active site
that is lined with around 4-5 suitable amino acid
residues that are responsible for catalysis of the
reaction. Cofactors that are also involved in the
reaction are usually bound at or near the active
site.
4. Enzyme-substrate [ES] complex: Enzymatic
reactions proceed through formation of a
transition state i.e. an intermediate state
between substrate and product having higher
free energy than that of either the substrate or
product. This state results from binding of the
substrate to the active site of the enzyme
resulting in an [ES] complex.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Kinetics of enzymatic reactions
1. Michaelis-Menten enzyme kinetics: A simple
model to explain the kinetic characteristics of
enzymatic reactions was proposed by Leonor
Michaelis and Maud Menten in 1913. According
to this model, formation of the [ES] complex
intermediate is essential for the enzymatic
catalysis reaction to take place. A group of
enzymes that does not obey the MichaelisMenten enzyme kinetics is comprised of the
allosteric enzymes. These enzymes have
multiple active sites and binding of substrate to
one site affects, positively or negatively, the
binding of substrate to the remaining sites.
2. Rate constants: An enzyme [E] combines
with its substrate [S] to form the enzymesubstrate complex [ES] with a rate constant of
k1. This complex can either form the product
[P] with rate constant k2 or can dissociate to
undergo a reverse reaction with rate constant
k-1 which results in release of the substrate.1 In
the Michaelis-Menten model for enzyme kinetics,
it has been assumed that dissociation of the [ES]
complex to give back the substrate is negligible.
This condition applies for the initial stages of a
reaction when the equilibrium favours product
formation
3. Reaction velocity (Vo): The rate of increase
in product concentration [P] with time when the
concentration is low is known as the reaction
velocity (Vo). It is also often defined as the
number of moles of product formed per second.
Vo
is linearly proportional to substrate
concentration [S] when [S] is low but becomes
independent of [S] when [S] is high.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Kinetics of enzymatic reactions
4. Vmax: The maximal rate that can be
achieved by an enzyme when all its catalytic
sites are occupied by the substrate is referred
to as Vmax or maximum velocity.
5. Km: The substrate concentration at which
the reaction rate is half its maximal value is
known as Km or the Michaelis constant. Km
value of an enzyme is an indicator of the
affinity that the enzyme has for its substrate
or in other words, it is a measure of the
strength of the [ES] complex. A high Km
indicates weak affinity between enzyme and
substrate while a low Km is indicative of strong
affinity. Michaelis constant. Km value of an
enzyme is an indicator of the affinity that the
enzyme has for its substrate or in other words,
it is a measure of the strength of the [ES]
complex. A high Km indicates weak affinity
between enzyme and substrate while a low Km
is indicative of strong affinity.
6. Enzyme activity: The activity of an enzyme
can be defined either in International Units (U)
or Katal. 1 U of enzyme activity is defined as the
amount of enzyme that catalyzes the conversion
of 1 mmole of substrate into product at 25oC
under the specified assay conditions. 1 Katal is
the amount of enzyme that catalyzes the
conversion of 1 mole of substrate into product
per second. (1 U = 16.67 nanokatal)
7. Specific activity: This can be defined as the
enzyme activity per milligram of protein. It is a
measure of enzyme purity and increases
continuously during a purification process until it
achieves a maximum, constant value indicating
the pure enzyme state.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Kinetics of enzymatic reactions
8. Turnover number (kcat): The turnover
number of an enzyme can be defined as the
maximum number of substrate molecules that
can be converted into product per active site of
the enzyme per unit time. Unit of kcat is s-1 or
min-1 and can obtained from parameters of the
Michaelis-Menten equation (Vmax/[E]).
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Enzyme inhibition
1. Enzyme inhibition: Several molecules are
capable of binding at or near the active site of
enzymes thereby decreasing or inhibiting their
activity. It provides an important control
mechanism in biological systems. Enzyme
inhibition is also an important mechanism that
is exploited during the manufacture of various
drug molecules.
2. Reversible inhibition: Inhibition of an
enzyme can be reversed if there is rapid
dissociation of the enzyme-inhibitor complex.
The inhibitor is associated to the enzyme
molecule by relatively weaker interactions.
3. Irreversible inhibition: In case of
irreversible inhibition, the inhibitor covalently
modifies the enzyme and dissociates very
slowly from the target enzyme because it is
tightly bound. The action of penicillin,
an important antibiotic, is through the
irreversible
inhibition
of
the
enzyme
transpeptidase that is essential for bacterial cell
wall synthesis. Aspirin, the commonly used
analgesic and anti-pyretic also functions by
means of irreversible inhibition of the enzyme
cyclooxygenase.
4. Competitive inhibition: This is a type of
reversible inhibition wherein the inhibitor binds
to the active site of the enzyme thereby
preventing the substrate from binding to it. The
inhibitor and substrate, in this case, are
structural analogues and the reaction rate is
decreased due to fewer enzyme molecules being
bound to the substrate. This type of inhibition
can be overcome by increasing the substrate
concentration.
Molecular & cell Biology
Enzymes: Basic Concepts and Kinetics
Enzyme inhibition
5. Uncompetitive inhibition: In this case, the
substrate and the inhibitor have distinct
binding sites on the enzyme and the inhibitor
binds only to the ES complex and not to the
enzyme alone. Both Km and Vmax are found to
decrease in this type of inhibition.
6. Mixed inhibition: A mixed inhibitor also
binds to the enzyme at a site distinct from the
substrate binding site with the difference being
that it can bind either to E or ES. Binding of
either one brings about conformational changes
in the enzyme structure thereby affecting
binding of the other.This inhibition can be
reduced but not overcome by increasing
substrate
concentration.
Non-competitive
inhibition is a special case of mixed inhibition
where inhibitor binding reduces catalytic
activity but does not affect substrate binding.