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•Second level
•Third level
•Fourth level
•Fifth level
LECTURE 4 : ENZYME INHIBITION

&
REGULATION OF ENZYMES ACTIVITY

2. (Indented Learning Outcomes)
ILOs
1.Explain the different types of enzyme inhibition.
2.Compare reversible competitive to noncompetitive enzyme
inhibitors.
3.Identify other types of enzyme inhibitors.
4.Identify examples of drugs acting as enzyme inhibitors.

3. Outlines

Enzymes as
Biological
Catalysts
The Properties
of Enzymes
Enzyme
classification
Active site of
enzymes, its
criteria
Enzyme
specificity
Mechanism of
enzyme
catalysis
Enzyme kinetics
Factors
affecting
enzyme activity
Enzyme
Inhibition
Regulation of
enzyme activity.
Applications of
Enzyme Action

4. Enzymes inhibitors
Enzyme inhibitors
Reversible
competitive Non competitive
Irreversible enzyme
inhibition(enzyme
poison)

5. Compare between different inhibitors as regards the
following points of comparison:
competitive inhibitor Non competitive inhibitor

Inhibitor structure

Site of binding
Effect on Km , Vmax
Reversible or not
Vmax
Examples

7. 3-IRREVERSIBLE INHIBITOR (Enzyme poison)
The inhibitor cannot be easily removed
from enzyme
Inhibitor combines to the enzyme
through covalent bonds
reducing the number of working enzyme
molecules activity
The kinetics of irreversible inhibitors are similar
to reversible non competitive inhibitors(Decrease
Vmax)

8. 3-IRREVERSIBLE INHIBITOR
Km is Same & Vmax are Lowered
It resembles enzyme Kinetics of
non competitive inhibitors
For example, Lead inhibits
irreversibly ferrochelatase enzyme
(an enzyme involved in heme
synthesis

9. Examples of irreversible inhibitors
1- Penicillin and
Amoxacillin (β-Lactam
inhibitors) is an antibiotic ,
inhibits bacterial
transpeptidase.
2-Aspirin as anti-platelet
aggregator on cyclo-
oxygenase
Inhibit prostaglandins and
thromboxane synthesis.
Allopurinol (suicide
inhibitor): for treatment of
gout

10. Penicillin

•Transpeptidase is needed for bacterial cell wall synthesis
transpeptidase transpeptidase
Penicillin is an antibiotic , inhibits
bacterial transpeptidase
.

11. Aspirin

Fatty acid
Prostaglandin
Inflammation
(fever and
headache)
Thromboxanes
Platelet
aggregation
(thrombus)
Cyclooxygenase
Aspirin
(antiplatelet aggregator
and anti-inflammatory)

12. A special class of irreversible inhibitor
(Suicide inhibitors)

Inhibitor is structural analogue to
the substrate on which the
enzyme act giving product
inhibitors that are converted by
the enzyme itself to a form that is
covalently linked to the enzyme
The product inactivate the
enzyme

13. Excess meat , coffee intake

Purine nucleotide catabolism

Hypoxanthine

Xanthine

Uric acid

-
Suicide inhibitors in gout treatment
xathine oxidase
xathine oxidase

14. Allopurinol(suicide inhibition):


used in treatment of GOUT that result from increased uric acid level in blood
N
N

15. Purine nucleotide catabolism

Hypoxanthine

AlloXanthine

Decreased Uric acid

-
Suicide inhibitors in gout treatment
xathine oxidase
xathine oxidase
Competitive
inhibition
Irreversible
inhibition

16. Allopurinol as suicide enzyme inhibitor
Uric acid production is diminished and xanthine and hypoxanthine levels in the
blood rise.
Xanthine oxidase enzyme is now unable to act on normal substrate.
Allopurinol is a substrate for xanthine oxidase, but the product binds so tightly
to enzyme
Allopurinol is similar to hypoxanthine in structure
Allopurinol is xanthine oxidase inhibitor

17. REGULATION OF ENZYMES
ACTIVITY

18. Intended Learning Outcomes (ILOs)

By the end of this lecture, the student should be able to:
1.Identify different mechanisms for the regulation of enzyme activity.
2.Describe long-term mechanism for the regulation of enzyme activity.
3.Describe short-term mechanisms for the regulation of enzyme activity.
4.Define isozymes and describe their clinical importance
5.Define the functional and nonfunctional plasma enzymes
6.Identify the role of enzymes in the diagnosis and follow up of diseases
7.Identify the role of enzymes as lab tools and in therapy of some diseases

19. A B C D

E1

E2

E3

20. Enzyme regulation
Long term regulation=Quantity
(takes hours to days)
Hormonally regulated according to
physiological needs
Short term regulation=Activity
(takes second to minutes)

21. protein aa aa aa aa aa aa aa aa aa aa aa
transcription
cytoplasm
nucleus
translation
Long term regulation

22. Long term regulation
Enzyme
quantity
Enzyme
synthesis at
gene level
Induction Repression
Enzyme
degradation

23. Increase protein (Enzyme) biosynthesis
A- Induction
B- Repression
Decrease protein (Enzyme) biosynthesis

Enzymes subjected to regulation of
synthesis are often those that are needed
under selected physiologic conditions

24. Pyruvate kinase (a regulatory enzyme)Gene Regulation :
Well fed Fasting
Insulin
Glucagon
Gene Induction of
pyruvate kinase

Gene Repression of
pyruvate kinase

Stimulate glycolysis Inhibit glycolysis

25. Mechanisms of Regulation of catalytic
activities
1. Substrate
Availability
2.ALLOSTERIC
REGULATION
3. Reversible
COVALENT
MODIFICATION
4-cAMP/PKA and
Ca2+/Calmodulin
5-Compartmention
6-Multienzyme
complex
7- Secretion of
enzyme as
zymogen(irreversible
covalent modification)

26. 1. Allosteric regulation
A B C D

E1

E2

E3
KEY ENZYME
Rate limiting enzyme
REGULATORY ENZYME
Usually irreversible
The rate-limiting enzyme of metabolic
pathway catalyzes the first committed
(unique) step of the pathway

27. 1-Allosteric Regulation
(Allos= another site)
Allosteric
Enzyme
Active site
bind to
substrate Allosteric site bind
to allosteric modifier

29. Allosteric Regulation
Allosteric enzymes have 2 binding sites
1-Active site binds
substrate
2-Allosteric site binds
regulator (Effectors or
modifiers)
Allosteric enzymes are almost always
composed of multiple subunits.

30. 1. Allosteric Regulation
Binding of Allosteric effectors to
allosteric site.
Increase the enzyme
activity(positive
allosteric modifier )
Inhibit the enzyme
activity(negative
allosteric modifier
Through:
– Altering K
0.5
– Modifying
Vmax
– Or both

31. Allosteric effectors

Homotropic
effectors
Substrate itself,
Mostly positive
Cooperative binding
Multi-subunit enzymes
Heterotropic
effectors
The effector may be
different from the substrate
May be negative (feed back
inhibition) or positive(feed
forward stimulation )

32. Binding of the substrate
at one active site
Facilitates binding of
other substrate
molecules at other
active sites on other
subunits
Cooperativity of binding
binding of oxygen to
hemoglobin
Homotropic effectors
Cooperative binding
=
Cooperative binding of the substrate to the protein subunits of the enzyme

33. Feed- Back Inhibition=Heterotropic effectors

A B C D

E1

E2

E3










Product which binds
to an allosteric site on
a regulatory enzyme
Allosteric inhibition of
first enzyme

35. 2. Reversible Covalent modification
Result
in
marked
change
in
enzyme
activity
of a phosphate Removal
group by cleaving the
.covalent bond
of a phosphate Addition
group to the enzyme
covalent bond protein by

36. 2. Reversible Covalent modification

Reversible binding of phosphate group to the enzyme (phosphorylation-
dephosphorylation).
It is hormonally regulated

37. Phosphorylation/Dephosphorylation

Addition of P done by PROTEIN
KINASES, which transfer P from ATP
to an enzyme
Activates or inactivates the enzyme via
P transfer to (OH) Serine, Threonine
or Tyrosine residues
PHOSPHATASES ...dephosphorylate
the enzyme **thus activate or
inactivate the enzyme.

38. Glycogen synthase
(an enzyme that synthesizes glycogen)

Activated by
Dephosphorylation
Glycogen
synthase
Activated by
phosphorylation
Glycogen
phosphorylase
P
Glycogen phosphorylase
(an enzyme that degrades glycogen)

2. Reversible covalent modification

•Two forms of the enzyme (active and inactive): Depending on the specific
enzyme
•The phosphorylated form may be more or less active than the
unphosphorylated enzyme

39. 3.1. Substrate availability

If the substrate concentration is much greater
than Km, the enzyme’s active site is saturated
with substrate and the enzyme is maximally
active
If the actual concentration of a substrate in a
cell is much less than the Km, the activity of
the enzyme is very low.

40. 3.2.cAMP/protein kinase A (PKA)
receptor
hormone
γ
β
α
GDP GTP
Adenylate
Cyclase
ATP cAMP
Protein
kinase A
(Active)
Protein
kinase A
(Inactive)
Phosphorylation
of cellulr proteins
Phosphorylation of target enzyme
Activation of its kinase activity
cAMP causes separation of the catalytic subunits
of PKA from the regulatory subunits
cAMP activates protein kinase A
(PKA is composed of 2 regulatory and 2 catalytic
subunits).
Hormone binding to the appropriate receptor
leads to increase in second messenger cAMP.
C C
C C
R R

41. 3.3. Ca2+/Calmodulin complex
Activates target enzymes
Binding of Ca2+ induces
conformational changes in
calmodulin and its activation
Ca2+/calmodulin complex is a part or a
subunit of some enzymes

42. 3.4-Compartmentation
Physical separation of
antagonistic pathways in
different subcellular locations
fatty acid
synthesis in
the cytosol
fatty acid
oxidation in the
mitochondria

43. 3.5-Multienzyme complex These are multiple enzymes that
are encountered in a certain
pathway and are bound together
for better flow of substrates and
products between the sequential
reactions,
Fatty acid synthase
multienzyme complex
(the main enzyme system in the
cytoplasmic pathway for fatty
acid synthesis).

44. 3.6-Secretion of enzymes as inactive proenzymes or zymogens
Irreversible covalent modification (proteolytic cleavage=Proenzyme
Activation)
Cleavage of small peptide
Reveal the catalytic site
(active enzyme)
(inactive zymogen)

45. 3.6-Secretion of enzymes as inactive proenzymes or zymogens

46. Secretion of enzymes as inactive proenzymes or
zymogens

47. Proenzyme examples
pepsin
Pepsinogen
Cleavage of small peptide in the intestine

48. Proenzyme examples
Plasmin
Plasminogen
Cleavage of small peptide










Antithrombin III

49. Enzyme Regulation
Long term
Synthesis
Induction
Repression
Short term
Allosteric
Reversible covalent
modification
Substrate
availability
cAMP/PKA and
Ca2+/Calmodulin
Compartmentaion
Multienzyme
complex
zymogen
Activity