Biochemistry Second Year

1. INTRODUCTION
1. Enzymes are biological catalysts.
2. Almost all enzymes are proteins.
3. They use their dynamic structure to bind substarates and bring them into optimal orientation
to make or break chemical bonds.
4. Almost all briochemical reactions are enzyme-catalysed.
5. Most important properties:
a) High catalytic power
b) High specificity
CO2 + H2O
Carbonic
H2CO3
anhydrase
Enzyme + Substrate E-S E + Prduct
Enzyme substrate complex
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2. 6. Enzymes work by stabilization of transition states.
Biomedical Importance:
1. Many diseases (Errors of Met.) are due to synthesis of abnormal enyzmes.
2. Cell injuries result in leakage of certain enzymes into plasma, the
measurement of which is useful in diagnosis.
3. Enzyme therapy
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3. BASIC CONCEPTS
1. Enzymes are highly SPECIFIC for:
a) Type of reaction
b) Type of substrate
* No wasteful side reactions
Examples: Proteolytic enzymes:
H O H O H O H O
----N—C—C—N—C—C--- + H2O ------N—C---C + +
H3N—C---C---
H R1 H R2 H R1 O- R2
Peptide Caroxyl Component Amino Component
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4. Subtilisin: Not specific for side chain
Trypsin: Hydrolyse the peptide bond on the
Side of Lys and Arg only.
Thrombin: More specific
Arginine Glycine
DNA PolymeraseI:
Highly specific
Directed by template, chance of
Mismatch 1 in 106 replications.
H O H O
---N—C—C--------N—C—C---
H H R2
Lysine or arginine
Hydrolysis site
H O H O
---N—C—C--------N—C—C---
H H
Arginine Glycine
Hydrolysis site
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5. Optical Specificity
Most enzyme are optically specific e.g.: Glycolytic enzymes work on D-isomers.
Enzymes that work on amino acid, work mostly on L-isomer.
Exception: Isomerases (e.g. epimerase) no optical specificity.
2. Regulation of the Catalytic Activity:
a) Feed-back Inhibition:
A B C D Z
End of product
Enzyme inhibited by Z
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7. 3.Enzymes in diagnosis:
* Non-functional plasma enzymes:
* Cellular necrosis non-functional
enzymes useful in diagnosis
Examples:
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8. Serum Enzyme Major Diagnostic Use
Aminotransferases
Aspartate aminotransferase (AST,
or SGOT)
Alanine aminotransferase (ALT, or
SGPT)
Myocardial infarction
Viral hepatitis
Amylase Acute Pancreatitis
Ceruloplasmin Hepatoenticular degeneration (Wilson’s disease)
Creatinine phosphokinase Muscle disorders and myocardial infarction.
γ-Glutamyl transpeptidase Various liver diseases
Lactate dehydrogenase (isozymes) Myocardial infarction
Lipase Acute pancreatitis
Phosphatase, acid Metastatic carcinoma of the prostate
Phosphatase, alkaline (isozymes) Various bone disorders, obstructive liver diseases.
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9. 4. ENZYME KINETICS
The active site:
* The site for substrate attachment
* It contains amino acid involved directly in making or breaking of
chemical bonds.
Properties of active site:
* Small
* It is 3-dimensional
* Substrate bind to E by multiple weak bonds (electrostatic, H-
bonding, Van der Waals
and hydrophobic bonds).
* Clefts, H2O is excluded.
* Specificity depends on arrangement of atoms in active stie.
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12. Transformation of Energy by Enzymes
Light Energy Chemical bond energy
Food Small molecules.
ATP
Chemical Energy Mechanical Energy
Photo-
Synthesis
digestion
Metabolism
Muscle
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13. Free Energy:
Ist
Law of Thermodynamics:
Total energy of a system and its surrounding is constant.
∆E = EB—EA = Q ---- W
∆E depends on initial and final states.
2nd
Law (Prediction of spontaneity)
Condition for a spontaneous process:
∆s System + ∆s surrounding > 0
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14. 600
C 200
C
400
C 400
C
1 M NaCL H2O
0.5 M NaCL 0.5 M Nacl
Figure 8-6
Processes that are driven by an increase in the entropy of a system : (A) diffusion
of heat; and (B) diffusion of a solute.
A
B
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15. Gibbs Free Energy
Gibbs combined the 2 laws:
∆G = ∆H - T ∆S
∆G= ∆E - T ∆ S
For a reaction if ∆G is negative, the reaction is spontaneous.
If ∆G = O, Equilibrium
If ∆G is positive, no spontaneity
Glucose CO2 + H2O
Glucose CO2 + H2O
Combustion
Enzymes
Same ∆G
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18. K1[E][S] = (k1 + k3) [ES]
[ES] = Km =
[ES] =
[E][S}
K2 + k3 + k1
K2 + k3
k1
[E] [S]
KM
[E] = [ET] – [ES]
[ES] = ([ET] - [ES]) [S] / KM
[ES] = [ET]
[S] / Km
1 + [S] / Km
[ES] = [Et]
[S]
[S] + Km
Substitute for [ES] in equation (1)
V = k3 [Et]
[S]
[S] + Km
V = Vmax
S
S + Km
Michaelis-Menten equation
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20. Significance of Km
Range of Km 10-1
- 10-7
M
Km has 2 meanings:
i) [S] which fill ½ active sites (1/2 Vmax)
ii) Under certain conditions, the Km is a measure of
strength of ES complex (affinity).
Km = , if K2 >>k3 then
Km = = KES =
K2 + k3
k1
k2
k1
[E][S]
[ES]
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21. KES = Dissociation constant of ES
If Km is high weak binding of E + S
If Km is low strong binding E + S
Significance of Vmax:
Vmax reveals the turn-over number for an enzyme
which is the number of subst.
Molecules converted into product by one enzyme
molecule in unit time.
Vmax = k3[Et], k3 = turnover number
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22. Enzyme
Inhibition of Enzymes:
• Used by cells for regulation
• Many drugs and toxins act by inhibition of enzymes.
1. Irreversible Inhibition
H3C—C CH3
O
----CH2OH + F---P=O
O
H3C---C
H
isopropylphosphofluoridate
(DIPF)
H
H3C---C---CH3
O
CH2—O—P—O + HF
O
H3C---C---CH3
H
Enzyme
Action of a nerve agent on
Acetylcholinesterase
Inactivation of acetylcholineserase by
diisopropylphosphofluoridate (DIPF)
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24. B. Non-Competitive Inhibition:
• Act by decreasing the turnover number
• Vmax decreased, Km, same.
E ES E + P
E1 ESI
+S
+
I
+S
+ I
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