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1. REGULATION OF ENZYMES ACTIVITY
&

Clinical Importance of Enzymes and Isozymes

2. 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

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

5. Mechanisms of Regulation of catalytic
activities
1.ALLOSTERIC
REGULATION
2. Reversible
COVALENT
MODIFICATION
3.1. Substrate
Availability
3.2.-cAMP/PKA and
3.3.Ca2+/Calmodulin
3.4-Compartmention
3.5-Multienzyme
complex
3.6- Secretion of
enzyme as
zymogen(irreversible
covalent modification)

6. 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

7. 2. Reversible Covalent modification

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

8. 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.

9. 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

10. 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.

11. 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

12. 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

13. 3.4-Compartmentation
Physical separation of
antagonistic pathways in
different subcellular locations
fatty acid
synthesis in
the cytosol
fatty acid
oxidation in the
mitochondria

14. 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).

15. 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)

16. 3.6-Secretion of enzymes as inactive proenzymes or zymogens

17. Secretion of enzymes as inactive proenzymes or
zymogens

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

19. Proenzyme examples
Plasmin
Plasminogen
Cleavage of small peptide










Antithrombin III

20. 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

21. Enzyme Clinical Application
21

22. Enzyme Clinical Application
Diagnosis of diseases
1. Plasma enzymes
Functional Non functional
Isoenzymes
Alkaline
phosphatase
Creatinine kinase
2. Enzymes as lab
tools
Therapy
A. Enzyme itself as
therapy
B. Enzyme inhibitors
in therapy

23. Functional
Plasma Enzymes
Non-Functional
Plasma Enzymes

Synthesis

by the liver Different body organs
(Released by normal cell turnover

Function

Perform specific functions in blood No physiological function in blood
Substrates In
blood

Present Absent

Plasma levels

Higher than those in tissues Very low under normal conditions
They increase only in case of tissue damage
Examples

Enzymes of blood coagulation and
fibrinolysis,

Pseudocholine esterase

Creatine kinase
Aminotransferases (ALT & AST)
Alkaline phosphatase
Amylase & lipase
Enzymes in diagnosis of diseases

24. They are
normally found
intracellularly
released in
plasma in
excess amount
in case of
tissue damage
Indicator of
cellular
damage
Nonfunctional plasma enzymes

25. Enzyme Tissue source Diagnostic use
Creatine kinase Heart, Skeletal muscle, Brain myocardial infarction
Skeletal muscle disorders
ALT Liver Hepatocellular disorders (e.g., viral
hepatitis)
Higher levels than AST and remains elevated
for longer time (longer half-life)
AST Heart, Liver, Skeletal muscle, RBCs

Hepatocellular disorders (e.g., viral
hepatitis)
Historically, a cardiac marker
Alk.phosphatas
e(ALP)
Intestine, Liver, Bone, Placenta, RBCs


Hepatobiliary obstruction
Rickets and osteomalacia
Amylase Pancreas, Salivary glands

Acute pancreatitis
Lipase Pancreas

Acute pancreatitis
The diagnostic value of nonfunctional plasma enzymes

26. Physically distinct
forms of the same
catalytic activity. They
are synthesized by
different tissues
Isoenzymes can be
identified and separated
by electrophoresis
Isoenzymes
•Catalyze the same
reaction
•Act on the same
substrate
•Use the same
coenzymes
Same

•Physical
properties
•Electrophoretic
mobility,
Different

27. Examples of clinically important
isoenzymes:

1-Creatine kinase.
2-Alkaline phosphatase

28. Creatinekinase(CK)
Three isoforms are present
-CK 1 (BB) (Brain)
-CK2 (MB) (Heart)
-CK3 (MM) (Skeletal muscle)
In myocardial infarction there
is Increased total CK and CK-MB > 6%
of total CK

29. Alkaline phosphatase
ALP isoenzymes, present in blood, are derived from bone, liver,
intestine, or placenta of pregnant woman
Fastest Slowest
Liver----bone---- intestinal forms---placenta

The hepatic isoform is increased in hepatobiliary obstruction

30. Enzymes as lab tools
•Enzymes may be used as laboratory tools.
1.The fungal enzyme: glucose oxidase is used for measuring blood
glucose.
2. Specific enzymes are used to measure some blood analytes : such
as urea, uric acid and cholesterol
3.Taq polymerase, from Thermus aquaticus, is used in polymerase
chain reaction (PCR).
4. In enzyme-linked immuno-assay (ELISA), an enzyme is used to
label the immune complex of the assay.

31. Enzyme Clinical Application
Diagnosis of diseases
1. Plasma enzymes
Functional Non functional
Isoenzymes
Alkaline
phosphatase
Creatinine kinase
2. Enzymes as lab
tools
Therapy
A. Enzyme itself as
therapy
B. Enzyme inhibitors
in therapy

32. Some enzymes are used as Therapeutic agents
♥ Streptokinase:
Used in clearing
blood clots in
myocardial
infarction
Act by activating
plasminogen to
form plasmin.
Plasmin cleaves
fibrin into several
soluble
components

33. Plasminogen

Sterptokinase

Plasmin


Fibrin
FDP

34. ♥ Asparaginase: enzyme used in therapy of adult
leukemia ,.

35. Asparginase

Aspargine Aspartic acid

Aspargine is required as nutrition for tumor
cells

Asparginase used in treatment of Leukaemia