Lect.
5
Enzyme Inhibition
Enzyme Inhibition
• In order to function effectively, biological systems
must be able to regulate the activity of enzymes.
Special agents called inhibitors can bind on to
enzymes and inhibit or block their activity.
• Types of Inhibition
1. Irreversible Inhibition
Some inhibitors will bind to enzymes very tightly,
either by covalent or non-covalent means.
Once bound, they will not dissociate very easily
from the enzyme .
• These inhibitors are called irreversible
inhibitors; they have a high affinity for the
enzyme.
2. Reversible Inhibition.
• The defining property of reversible inhibition
is the ease with which the inhibitors can
dissociate from the enzyme under certain
conditions.
2.1. Competitive Inhibition
• The inhibitor molecule typically resembles the
substrate and can therefore bind directly to the
active site of the enzyme.
• Once bound, the inhibitor prevents the
substrate from occupying the active site.
• Competitive inhibitors typically have a much
higher affinity for the active site than the
natural substrate.
• However, if we increase the concentration of the
additional substrate can outcompete the
inhibitor for the active site. Thus increasing
substrate concentration can remove the effect of
the competitive inhibitor.
• The mechanism of competitive inhibition be
expressed as follows:
The kinetics for competitive inhibition
𝑘1
• 𝐸+𝑆 𝐸𝑆 1
𝑘2
𝑘3
• 𝐸+𝐼 𝐸𝐼 2
𝑘4
𝑘5
• 𝐸𝑆 𝐸+𝑃 3
• At Equiliprium
• The Equilibrium constant for eq. 1
𝐶𝐸 𝐶𝑆
• 𝑘𝑚 = 4
𝐶𝐸𝑆
• The Equilibrium constant for eq. 2
𝐶𝐸 𝐶𝐼
• 𝑘𝐼 = 5
𝐶𝐸𝐼
• 𝐶𝐸 𝑡𝑜𝑡𝑎𝑙 = 𝐶𝐸 + 𝐶𝐸𝑆 + 𝐶𝐸𝐼
𝐶𝐸 𝐶𝑆 𝐶𝐸 𝐶𝐼
• 𝐶𝐸 𝑡𝑜𝑡𝑎𝑙 = 𝐶𝐸 + +
𝑘𝑚 𝑘𝐼
𝐶𝐸 𝑡𝑜𝑡𝑎𝑙
• 𝐶𝐸 = 𝐶𝑆 𝐶𝐼
(1+ + )
𝑘𝑚 𝑘𝐼
𝐶 𝐸 𝐶𝑆 𝐶𝐸 𝑡𝑜𝑡𝑎𝑙 ∗ 𝐶𝑆
• 𝐶𝐸𝑆 = = 𝐶𝑆 𝐶𝐼
𝑘𝑚 𝑘𝑚 (1+ + )
𝑘𝑚 𝑘𝐼
𝑘5 ∗ 𝐶𝐸 𝑡𝑜𝑡𝑎𝑙 ∗ 𝐶𝑆 𝑉𝑚𝑎𝑥 𝐶𝑆
• 𝑟𝑝 = 𝑘5 𝐶𝐸𝑆 = 𝐶 𝐶 = 𝐶𝐼
𝑘𝑚 (1+ 𝑆 + 𝐼 ) 𝑘𝑚 1+ +𝐶𝑆
𝑘𝑚 𝑘𝐼 𝑘𝐼
• If we compare it by Michaeli’s Menten eq.
• They look similar and what we have done with the inhibition ,
𝐶𝐼
we’ve added an addition term 1 + this increase the
𝑘𝐼
denominator so the rate will slow with the inhibition.
• The reaction rate decreases due to the presence
of inhibitor.
• The maximum reaction rate is not affected by
the presence of a competitive inhibitor.
• A larger a mount of substrate is required to
reach the maximum rate.
𝑘𝑀 /𝑟𝑚𝑎𝑥
• 2. non- competitive Inhibitor
• Some enzymes have a permanent allosteric site
that can bind inhibitors. These inhibitors, called
non-competitive inhibitors because they do not
compete for the active site, can bind to enzyme
regardless of whether the substrate is bound or
not. These inhibitors block enzymatic activity
• The mechanism of noncompetitive inhibition
can be expressed as follows:
Derivation of enzyme kinetics for
noncompetitive inhibitors
𝑘1 𝐶𝐸 𝐶𝑆
• 𝐸+𝑆 𝐸𝑆 1 𝑘𝑀 =
𝑘2 𝐶𝐸𝑆
𝑘3 𝐶𝐸 𝐶𝐼
• 𝐸+𝐼 𝐸𝐼 2 𝑘𝐼 =
𝑘4 𝐶𝐸𝐼
𝑘5 𝐶𝐸𝑠 𝐶𝐼
• 𝐸𝑆 + 𝐼 𝐸𝑆𝐼 3 𝑘𝐼 =
𝑘6 𝐶𝐸𝑠𝐼
𝑘7 𝐶𝐸𝐼 𝐶𝑆
• 𝐸𝐼 + 𝑆 𝐸𝐼𝑆 4 𝑘𝑀 =
𝑘8 𝐶𝐸𝐼𝑆
𝑘9
• 𝐸𝑆 𝐸+𝑃 5
• 𝐶𝐸𝑇 = 𝐶𝐸 + 𝐶𝐸𝑆 + 𝐶𝐸𝐼 +
𝐶𝐸 𝐶𝑆
• 𝐶𝐸𝑇 = 𝐶𝐸 + + 𝐶𝐸𝐼 + 𝐶𝐸𝐼𝑆 + 𝐶𝐸𝑆𝐼
𝑘𝑀
• Uncompetitive Inhibitor
• In some instance the binding of the substrate to
the active site changes the conformation of the
enzyme and creates an allosteric site that was
not previously there. Now, a certain inhibitor
can bind to the enzyme substrate complex and
block its activity.
• Uncompetitive Inhibitor can't be overcome by
increase the substrate concentration.
Derivation of enzyme kinetics for
Uncompetitive Inhibitor
Example (1)
Other influence in enzyme activity
• 1. Substrate Effects
• Substrates may affect enzyme kinetics either by
activation or by inhibition.
• Substrate activation may be observed if the enzyme
has two (or more) binding sites, and substrate
binding at one site enhances the affinity of the
substrate for the other site(s).
• The result is a highly active ternary complex,
consisting of the enzyme and two substrate
molecules, which subsequently dissociates to
generate the product.
• Substrate inhibition may occur in a similar way,
except that the ternary complex is nonreactive.
2. Effect of pH
• The rate of an enzyme reaction is strongly
influenced by the pH of the reaction solution
both in vivo and in vitro
• The optimum pH is different for each enzyme.
• For example, pepsin from the stomach has an
optimum pH between 2 and 3.3,
• while the optimum pH of amylase, from saliva
(səˈlīvə), is 6.8.
• Chymotrypsin “kīmōˈtripsən”, from the
pancreas, has an optimum pH in the mildly
alkaline region between 7 and 8.
• The reason that the rate of enzyme reaction is
influenced by pH can be explained as follows:
1. Enzyme is a protein which consists of ammo acid
residues (that is, amino acids minus water).
2. The amino acid residues possess basic, neutral, or acid
side groups which can be positively or negatively
charged at any given pH.
3. let's consider one acidic amino acid, glutamic acid,
which is acidic in the lower pH range. As the pH is
3. An enzyme is catalytically active when the amino
acid residues at the active site each possess a
particular charge. Therefore, the fraction of the
catalytically active enzyme depends on the pH.
3. Effect of temperature
The rate of a chemical reaction depends on the temperature according to
Arrhenius equation as
• An increase in the temperature increases the rate of reaction, since the
atoms in the enzyme molecule have' greater energies and a greater
tendency to move.
• However, the temperature is limited to the usual biological range. As the
temperature rises, denaturation processes progressively destroy the
activity of enzyme molecules.
• This is due to the unfolding of the protein chain after' the breakage of
weak (for example, hydrogen) bonds, so that the overall reaction velocity
drops.
• For many proteins, denaturation begins to occur at 45 to 50°C.. Some
enzymes are very resistant to denaturation by high
• temperature, especially the enzymes isolated from thermophilic
organisms found in certain hot environments.
4. effect of shear
• Enzymes had been believed to be susceptible to mechanical
force, which disturbs the elaborate shape of an enzyme
molecule to such a degree that denaturation occurs.
• The mechanical force that an enzyme solution normally
encounters is fluid shear, generated either by flowing fluid,
the shaking of a vessel, or stirring with an agitator.
• The effect of shear on the stability of an enzyme is important
for the consideration of enzyme reactor design, because the
contents of the reactor need to be agitated or shook in order
to minimize mass transfer resistance.
Home Work
