The document discusses various mechanisms of enzyme inhibition, including competitive, non-competitive, uncompetitive, and suicide inhibition, along with their clinical significance and effects on enzyme kinetics. It explains how different inhibitors interact with enzymes, affecting reaction velocity, affinity, and enzyme modification through covalent bonds. Additionally, it highlights the role of allosteric regulation and feedback inhibition in metabolic pathways, as well as specific examples and applications of these mechanisms.

1. Ch 3: Mechanisms of Enzyme Inhibition
Saida Almashharawi
2015-2016
Clinical Enzymology
CLS 433
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2. A. Competitive Inhibition
i. Here inhibitor molecules are competing with the normal substrate molecules for
binding to the active site of the enzyme, because the inhibitor is a structural
analog of the substrate
ii. Since E-I (enzyme–inhibitor complex) can react only to reform the enzyme and
inhibitor, the number of enzyme molecules available for E-S formation is reduced.
Suppose 100 molecules of substrate and 100 molecules of inhibitor are competing for
100 molecules of the enzyme. So, half of enzyme molecules are trapped by the
inhibitor and only half the molecules are available for catalysis to form the product.
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iii. Since effective concentration of enzyme is reduced, the reaction velocity is
decreased.

3. 3
A. Competitive Inhibition (cont.)
iv. In competitive inhibition, the inhibitor will be a structural analog of
the substrate. There will be similarity in three dimensional structure
between substrate (S) and inhibitor (I).
For example, the succinate dehydrogenase reaction is inhibited by
malonate (Fig. 5.19).

4. 4
A. Competitive Inhibition (cont.)
v. Competitive inhibition is usually
reversible. Or, excess substrate
abolishes the inhibition.
In the previous example of 100 moles
of E and 100 moles of I, if 900 moles of
S are added, only 1/10th of enzyme
molecules are attached to inhibitor and
90% are working with substrate.
Thus 50% inhibition in the first example
is now decreased to 10% inhibition
(Fig. 5.20).

5. vi. From the graphs, it is obvious that in
the case of competitive inhibition,
the Km is increased in presence of
competitive inhibitor.
Thus competitive inhibitor apparently
increases the Km. In other words,
the affinity of the enzyme towards
substrate is apparently decreased in
presence of the inhibitor.
vii. But Vmaxis not changed. Clinical
significance of such inhibition is
shown in Box 5.10
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A. Competitive Inhibition (cont.)

6. 6

7. 7

8. B. Non-competitive Inhibition
(Irreversible)
i. A variety of poisons, such as
iodoacetate, heavy metal ions (lead,
mercury) and oxidising agents act as
irreversible non-competitive inhibitors.
There is no competition between substrate
and inhibitor. See Fig. 5.22.
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9. ii. The inhibitor usually binds to a different
domain on the enzyme, other than the
substrate binding site. Since these
inhibitors have no structural
resemblance to the substrate, an
increase in the substrate concentration
generally does not relieve this
inhibition.
The saturation curve of this type of
inhibition is shown in Fig. 5.23. A
comparison of the two
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B. Non-competitive Inhibition
(Irreversible) (cont.)

10. • types of inhibitions is shown in Table 5.6. Examples are:
iia. Cyanide inhibits cytochrome oxidase.
iib. Fluoride will remove magnesium and manganese ions and so will inhibit
the enzyme, enolase, and consequently the glycolysis.
iic. Iodoacetate would inhibit enzymes having-SH group in their active
centers.
iid. BAL (British Anti Lewisite; dimercaprol) is used as an antidote for heavy
metal poisoning.
The heavy metals act as enzyme poisons by reacting with the SH group.
BAL has several SH groups with which the heavy metal ions can react and
thereby their poisonous effects are reduced
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B. Non-competitive Inhibition
(Irreversible) (cont.)

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iii. The inhibitor combines with the enzymes by forming a covalent bond and
then the reaction becomes irreversible
The velocity (Vmax) is reduced. But Km value is not changed, because the
remaining enzyme molecules have the same affinity for the substrate.
iv. Increasing the substrate concentration will abolish the competitive
inhibition, but will not abolish non-competitive inhibition.
B. Non-competitive Inhibition
(Irreversible) (cont.)

12. 12

13. C. Uncompetitive Inhibition
Here inhibitor does not have any affinity for free enzyme.
Inhibitor binds to enzyme–substrate complex; but not to the free enzyme.
In such cases both Vmax and Km are decreased (Fig. 5.24).
Inhibition of placental alkaline phosphatase (Reganiso-enzyme) by
phenylalanine is an example of uncompetitiveinhibition.
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14. D. Suicide Inhibition
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i. It is a special type of irreversible inhibition of enzyme activity.
It is also known as mechanism based inactivation. The
inhibitor makes use of the enzyme's own reaction mechanism
to inactivate it (mechanism based inactivation).
ii. In suicide inhibition, the structural analog is converted to a
more effective inhibitor with the help of the enzyme to be
inhibited. The substrate-like compound initially binds with the
enzyme and the first few steps of the pathway are catalyzed.
iii. This new product irreversibly binds to the enzyme and
inhibits further reactions.

15. -. The anti-inflammatory action of Aspirin is also based on the
suicide inhibition.
Arachidonic acid is converted to prostaglandin by the enzyme
Cyclo-oxygenase.
Aspirin acetylates a serine residue in the active center of cyclo-
oxygenase, thus prostaglandin synthesis is inhibited, and so
inflammation subsides.
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D. Suicide Inhibition (cont.)

16. E. Allosteric Regulation
i. Allosteric enzyme has one catalytic site where the substrate binds and
another separate allosteric site where the modifier binds (allo = other)
(Fig. 5.25).
ii. Allosteric and substrate binding sites may or may not be physically
adjacent.
iii. The binding of the regulatory molecule can either enhance the activity of
the enzyme (allosteric activation), or inhibit the activity of the enzyme
(allosteric inhibition).
iv. In the former case, the regulatory molecule is known as the positive
modifier and in the latter case as the negative modifier
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17. 17

18. v. The binding of substrate to one of the
subunits of the enzyme may enhance
substrate binding by other subunits.
This effect is said to be positive co-
operativity.
If the binding of substrate to one of the
subunits decreases the avidity of
substrate binding by other sites, the
effect is called negative co-operativity.
vi. In most cases, a combination is
observed, resulting in a sigmoid shaped
curve (Fig. 5.26).
.
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E. Allosteric Regulation (cont.)

19. 19

20. 20
• Salient features of allosteric regulation are enumerated in Box 5.11
E. Allosteric Regulation (cont.)

21. F. Key enzymes
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i. Body uses allosteric enzymes for regulating metabolic pathways.
Such a regulatory enzyme in a particular pathway is called the key enzyme
or rate limiting enzyme.
ii. The flow of the whole pathway is constrained as if there is a bottle neck at
the level of the key enzyme.

22. iii. The allosteric inhibitor is most effective when substrate concentration is
low. This is metabolically very significant. When more substrate molecules
are available, there is less necessity for regulation.
Two best examples are given in detail below:
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F. Key enzymes

23. G. Feedback Inhibition
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The term feedback inhibition or end-product inhibition means
that the activity of the enzyme is inhibited by the final
product of the biosynthetic pathway
In this pathway, if D inhibits E1, it is called feedback inhibition.
Usually such end product inhibition is effected allosterically.

24. 24

25. 25
Part 2
Metabolic Inhibitors

26. Covalent Modification
The activity of enzymes may be increased or decreased by covalent
modification. It means, either addition of a group to the enzyme protein
by a covalent bond; or removal of a group by cleaving a covalent bond.
Examples of a covalent modefication:
- Zymogen activation by partial proteolysis,
- Addition or removal of a particular group brings about covalent
modification of enzyme protein. This is a reversible reaction.
1- Commonest type is the reversible protein phosphorylation.
The phosphate group may be attached to serine, threonine or tyrosine
residues
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27. 27

28. When hormone binds to the membrane bound receptor, hormone receptor
complex (HR) is formed.
The enzyme adenylate cyclase is activated. This activation is mediated through
a G protein.
The active adenylate cyclase converts ATP to cyclic AMP (cAMP) which acts as a
second messenger.
It activates protein kinase, by binding to the regulatory subunit of the enzyme.
The active catalytic subunit will phosphorylate the enzyme.
In some cases, the receptor itself has tyrosine kinase activity which is switched
on when hormone binds to receptor, e.g. insulin.
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Examples of a covalent modification

29. 29

30. 30

31. 2- ADP ribosylation is another covalent modification,
where an ADP-ribose from NAD+ is added to enzyme /protein.
For example, ADP ribosylation of alpha subunit of G-protein
leads to inhibition of GTPase activity; hence G protein remains
active.
- Cholera toxin and pertussis toxin act through ADP ribosylation.
-ADP ribosylation of glyceraldehyde-3-phosphate dehydrogenase
results in inhibition of glycolysis.
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32. 32

33. 33

34. 34

35. Stabilization
Enzyme molecules undergo usual wear and tear and finally get degraded.
Such degradation if prevented can lead to increased overall enzyme
activity. This is called stabilization of enzyme.
Degradation of Tryptophan pyrrolase is retarded by tryptophan.
Phospho fructo kinase is stabilized by growth hormone.
Enzymes having SH groups (Papain, Urease, Succinate dehydrogenase) are
stabilized by glutathione (G-SH).
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36. Compartmentalization
The activity of enzymes catalysing the different steps in a metabolic pathway
may be regulated by compartmentalization of enzymes.
Certain enzymes of the pathway may be located in mitochondria whereas
certain other enzymes of the same pathway are cytoplasmic.
For example heme synthesis, urea cycle gluconeogenesis.
The intermediates have to be shuttled across the mitochondrial membrane
for this purpose which provides a point where controls can be exerted.
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37. Assignment
• Q1: make a comparison between the following types
of inhibition, and mention the effects on Vmax, Km,
Substrate saturation curve, Lineweaver Burk plot
(by plotting the curves)
- Competitive inhibitor
- non – Competitive inhibitor
- Un- Competitive inhibitor
Note: your Assignment to be received before Thursday
11/Feb./2016
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