chemistry of enzyme

2. Objective of clinical enzymology:
• To diagnosis and treatment of the disease.
• Measurement of the Various diagestive enzymes in
the body fluids.
• Measurement of cellular enzyme released into the
plasma as a consiquences of tissue damaged.
• Measurement of serum ALP in the bone and liver
disease.
• Estimation of serum ACP to diagnose the prostatic
cancer.

3. Introduction of enzyme:
 It act’s as catalyst i.e That accelerate the process of
metabolism activity in the body.
 All enzyme are protein in nature.
 The enzyme act’s on specific substrate. Substrate is a
substance on which the enzyme act’s the specific and
product will formed.
Eg: Amylase + starch--- Maltose and dextrose
(enzyme) (Substrate) (product)

4. Enzyme act as a protein in nature:-
 All the enzyme are protein in nature with large molecular weight .
 Few enzyme are simple protein while some are conjugated protein.
 In such enzyme the non protein part is called prosthetic group or
coenzyme and the protein part is called as apoenzyme.
 Holoenzyme = apo-enzyme + coenzyme
(protein part) ( prosthetic group)

5. HOLO-ENZYME
APO -
ENZYME
CO-
ENZYME
HOLO
ENZYME

6. Co-enzyme:-
 Certain enzyme require a specific, thermostable, low mol.
Weight, non-protein organic substance is called as Co-
enzyme.
 Enzyme may be simple protein or complex enzyme
containing a nonprotein part called the prosthetic group.
 The coenzyme may bind covalently or non covalently to the
apoenzyme.

7. Classification of Coenzymes:
 Coenzyme can be classified according to the group whose
transefer they facilitate. Based on this concept we may
classified coenzymes as follows:
1) For transfer of group other than Hydrogen:
for eg: Pyridoxal phosphate,Folate Coenzyme ,Biotin
Cobamide coenzyme , Lipoic acid.
2) For transfer of Hydrogen:
for eg: NAD+, NADP+ ,FMN, FAD

8. Silent features of Co-enzymes:
• Co-enzymes are heat stable.
• They are low molecular weight substances.
• Generally the coenzyme combines loosely with the enzyme
molecule.
• The enzyme and coenzyme can be easily by dialysis.
• Inside the body when the reaction is completed coenzyme is
released from the apoenzyme and goes to other reaction
sites.

9. Defination of enzyme:
Enzyme may be defined as biocatalysts which
accelerate the metabolic rate of reaction and
synthesized by living cells.
They are protein in nature, colloidal and
thermolabile in character and specific in their action.

10. Enzyme are broadly classify into two
main categories:-
1) Intracellular enzyme:-
 They are function within cells where they are
synthesized.
2) Extracellular enzyme:-
 They are function outside the cells.

11. According to IUB enzyme is classified into 6 classes which can be
listed as follows:-
a) Each enzyme is characterized by a code number ( enzyme code no. or
EC no.)
b) The second figure indicate the type of group involved in the reaction.
c) Third figure denotes the reaction more precisely indicating substrate
on which the group acts.
d) The fourth figure is the serial number of the enzyme. Briefly, the four
digits characterized by:-
i) class, ii) sub class iii) sub-sub class,and iv) serial no. of a perticular
enzyme.

12. Classification :
1) Oxidoreductase
2) Transferase
3) Hydrolases
4) Lyases
5) Isomerases
6) Ligases

13. 1) Oxidoreductase:-
• This group of enzyme will catalyse Oxidation of
one substrate with simultaneous reduction of
another substrate. This may be represented as:
Eg: Alcohol dehydrogenase, lactate dehydrogenase,
glucose-6-phosphate dehydrogenase.
Alcohol + NAD+ Alcohol Dehydrogenase Aldehyde + NADH + H+
EC.1.1.1.1

14. 2) Transferase:
• This class of Enzyme transfer one group (other
than hydrogen) from one substrate to another
substrate.This may be represented as:
Eg: Aspartate and alanine transaminase, hexokinase,
aminotransferase.
Hexose + ATP Hexokinase Hexose-6-phosphate + ADP
EC 2.7.1.1

15. 3) Hydrolases:
• This group of enzyme hydrolyses bond by adding
water and then breaking bond.
Eg: Glucose-6-phosphatase, pepsin, trypsin, esterase.
Acetylcholine + H20 Acetylcholine Hydrolase Choline + acetate
EC.3.1.1.7

16. 4) Lyases:
• This Enzyme can removal of one group from
substrate or breakdown of bond by mechanism other
than hydrolysis.
Eg: Fumarase, aldolase, HMG CoA lyase, histidine
decarboxylase.
Fructose- 1-6-bisphosphate Aldolase Glyceraldehyde 3- phosphate
+ Dihydroxy acetone phosphate
EC.4.2.1.2

17. 5) Isomerases:
• These enzymes can produce optical, geometric or
positional isomers of substrate.
Eg: isomerase, UDP glucose, triose phosphate
isomerase, racemases, retinal isomerase.
Glyceraldehyde-3-phosphate triose phosphate isomerase Dihydroxy
acetone phosphate
EC.5.3.1.1

18. 6) Ligases:
• These enzymes links two substrate together,
usually with the simultaneous hydrolysis of ATP .
Eg: DNA ligases, Acetyl CoA carboxylase, glutamine
synthetase, PRPP synthetase.
Acetyl CoA + Co2 + ATP Acetyl CoA Carboxylase Malonyl CoA
+ ADP + Pi
EC.6.4.1.2

19. ACTIVE SITE
 The active site ( or active centre) of an enzyme
represents as the small region at which the
substrate(s) binds and participates in the catalysis
Salient features
• The active site is made up of amino acids
which are far from each other in the linear
sequence of amino acids.
• The active site is not rigid in structure and
shape. It is rather flexible to promote the
specific substrate binding.

20. Mechanism of action
 Michaelis and Menton (in 1993), have proposed a hypothesis
for enzyme action.
 the enzyme molecule (E) first combines with a substrate
molecule (S) to form an enzyme-substrate (ES) complex
which further dissociates to form product (P) and enzyme
(E) back

21. MODELS OF ENZYME-SUBSTRATE COMPLEX FORMATION
Template or Lock and key Model
o proposed by Emil Fischer
o the active site already exists in proper conformation even in
absence of substrate
o the active site by itself provides a rigid, pre-shaped template
fitting with the size and shape of the substrate molecule
o it cannot explain change in enzymatic activity in presence of
allosteric modulators

23. Induced- Fit or Koshland Model
o Proposed by Koshland in 1958
o The important features of this model is the flexibility of the
region of active site
o the substrate during its binding induces conformational changes
in the active site to attain the final catalytic shape and form
o It explains:
– Enzymes become inactive on denaturation
– Saturation kinetics
– Competitive inhibition
– Allosteric modulation

25. Factors affecting enzyme activities:-
1) substrate concerntration:-
 By increasing the conc of substrate there is an increasing rate of
formation of end product.
 A point is finally reached when enzyme saturated with substrate conc.
Have no effect on the rate of production of end product.
 The substrate conc. That produces half the maximum velocity or
speed is turn as Km value.
Where , K = Constant.
M =Michalis constant.

26. Michalis constant:-
E + S K1 ES K3 E + P
K2
Km = K2 + K3
K1
Thus, Km is substrate conc. at half the Vmax. It
denotes 50% of enzyme molecule are bound with
substrate molecule at
that particular substrate
concerntration.

27. Enzymes and [S]
[S]
Initial
reaction
rate
/
arbitrary
units

28. Enzymes and [S]
[S]
Initial
reaction
rate
/
arbitrary
units

29. Enzymes and [S]
[S]
Initial
reaction
rate
/
arbitrary
units
Increasing [S]
increases collision
rate and increases
reaction rate

30. Enzymes and [S]
[S]
Initial
reaction
rate
/
arbitrary
units All active sites are
occupied. Enzymes
are working at
maximum rate.
All active sites
are not occupied

31. Enzymes and [S]
[S]
Initial
reaction
rate
/
arbitrary
units Maximum
turnover number
or Vmax has been
reached

32. 2) Enzyme concerntration:-
 The enzyme concerntration also effects the rate of formation
of end product.
 If the excess substrate is present and doubling the enzyme
conc. Usually the double rate of formation of end product.
 And a point is reached where the substrate is saturated with
the enzyme there is no further increasing production of end
product.

34. Enzymes and temperature
Temperature /
o
C
Increasing kinetic
energy increases
successful
collision rate
Reaction
rate
/
arbitrary
units

35. Enzymes and temperature
Temperature /
o
C
Permanent disruption
of tertiary structure
leads to loss of active
site shape, loss of
binding efficiency and
activity
Reaction
rate
/
arbitrary
units

37. 5) Radiation:-
 Enzyme are highly sensitive to short wave therapy ( high
energy) radiation such as a,b or g rays.
 High energy radiation forms peroxides which causes
oxidation of enzymes resulting in loss of enzyme activity.
 The enzyme activity is lost by irradition which may support
indirect effects on the DNA of genes.

38. 6) oxidation:
 The sulfhydryl(SH) group of many enzyme are essential for
enzyme activity.
 Oxidation of these SH group by many oxidising agents
including the oxygen of air forms the disulfide(S-S) linkage
and results in loss of enzyme acitivity.
SH S
E + ½ O2 --- E + H2O
SH S

39. Enzymes and inhibitors
• Inhibitors are molecules that prevent
enzymes reaching their maximum turnover
numbers
• Some inhibitors compete with the substrate
for the active site
Active site directed inhibition
Non-active site directed inhibition

40. Active site directed inhibition
• Inhibitor resembles the substrate enough to
bind to active site and so prevent the
binding of the substrate:
Substrate
Inhibitor
Enzyme

41. Active site directed inhibition
• Inhibitor resembles the substrate enough to
bind to active site and so prevent the
binding of the substrate:
Substrate
Enzyme/Inhibitor
complex
Enzyme
activity is lost

42. Enzymes and active site directed
inhibition
[S]
Initial
reaction
rate
/
arbitrary
units At low [S], the enzyme is more
likely to bind to the inhibitor and
so activity is markedly reduced
Uninhibited
Inhibited

43. Enzymes and active site directed
inhibition
[S]
Initial
reaction
rate
/
arbitrary
units As [S] rises, the enzyme is
increasingly likely to bind to the
substrate and so activity increases
Uninhibited
Inhibited

44. Enzymes and active site directed
inhibition
[S]
Initial
reaction
rate
/
arbitrary
units At high [S], the enzyme is very
unlikely to bind to the inhibitor and so
maximum turnover is achieved
Uninhibited
Inhibited

45. Competitive Inhibition
 When the active site or catalytic site of an enzyme is
occupied by a substance other than the substrate of
that enzyme, its activity is inhibited. The type of
inhibition is known as competitive inhibition
 a type of reversible reaction
 both ES and EI complexes are formed during the
reaction
 Km value increases where as Vmax remains
unchanged

47. Clinical significance of compitative inhibitors:
1) Sulfonamide:
It is a antibacterial drugs, bacteria synthesized folic acid with glutamic acid.
Bacterial wall is impermeable to folic acid, to transport folic acid inside the
bacterial cells requires PABA but sulphonamide structurally similar to
PABA.
Sulphonamide inhibit’s the actions of PABA and bacterial cell die due to
lackness of folic acid.
2) Methotrexate:
 It is a anti-cancer drugs.
 It is a 4-amino-N10 methyl folic acid.
 it is a structural analogs of folic acid and it inhibits folate reductase enzyme
and stops the multiplication of DNA and cells.

48. c) Methanol:
• It is a very toxic ingestion may lead to blindness and neural disease.
• Methanol is oxidized by alcohol dehydrogenase to formaldehyde is
poisonous and ethanol inhibits the action of alcohol dehydrogenase
enzyme.
Methanol alcoholdehydrogenase Formaldehyde
(-) Inhibits
Ethanol

49. Non-competitive Inhibition
 two types
a) Reversible and b) Irreversible
 the site of attachment of the substrate and inhibitor are different
Reversible non-competitive Inhibition:-
 If the inhibitor can be removed from its site of binding without
affecting the activity of the enzyme, it is called as reversible-
non-competitive Inhibition.
For eg:-
Trypsin inhibitor + trypsin inhibits the action of trypsin enzyme

50. Irreversible Non-competitive Inhibition:-
 If the inhibitor can be removed only at the loss of
enzymatic activity, it is known as Irreversible
Non-competitive Inhibition
 kinetic properties are same
 Km value is unchanged while Vmax is lowered

51. Uncompetitive Inhibitor
• Inhibitor does not have any affinity for free
enzyme
• Inhibitor binds to enzyme-substrate complex; but
not to the free enzyme
• both Vmax and Km are decreased

54. CLINICAL
ENZYMOLOGY

55. OBJECTIVES
• List the clinically important enzymes and isoenzymes.
• State which of the enzymes and isoenzymes are found
in which tissues
• Describe plasma enzyme changes in myocardial
infarction and liver disease
• Outline different ways of measuring plasma enzymes

56. Isoenzymes
 They are physically different form of a same enzyme having
same catalytic activity so they catalyze the same reaction.
 So they are identical or very similar to the enzyme activity
but the enzyme are produced from the different source of
protein.
 They differ in physical, biochemical and immunological
properties also differ in their molecular weight and rate of
migration in the electrophoresis.

57. ENZYME UNITS
One international unit:
 amounts of enzyme that will convert one micromole of
substrate per minute per litre of sample=U/L
One KATAL:
 number of mole of substrate transfer per second per liter
of sample.
KAT or K 60U =1 uKAT
1nKAT=0.06U
1IU=1 micromole/min=16.67nKAT

58. 1) Liver/ Hepatic enzyme:
A) Serum ALP:
 ALP Present in Liver tissue.
 ALP level is high in Liver also in kidney, Intestine,and
placenta.
 The function of ALP in the body are the transport of
phosphate across the cell membrane.
• occurrence: bone , small intestine,liver , Kidney & placenta.

59. Increased level ALP:
> Ricketes
> Osteomalacia
> Osteoblastoma
> Hyperparathyrodism.
> Bone disease.
Decreased level ALP:
> Hypophosphatasia
> Cretinism
> Kwasworker.
Normal Value: 40 – 125 IU/L

60. ISO ENZYME OF ALP:
Iso enzyme are separated at alkaline PH by electrophoresis:
1) Liver ALP:
 Fast moving iso enzyme appear close to anode.
2) Bone Phospatase:
 Appear In diffuse form some times it may overlapping liver Iso-enzyme.
3) Intestinal phospatase:
 Which also migrate diffusely.
4) Kidney phospatase:
 Rarely appear in the serum.
5) Placental Iso enzyme:
 It has got mobility of the same order as in case of liver.

61. B) ALT:
 Also called serum glutamate Pyruvate Transaminase
 High value seen in Acute Hepatitis and viral disease.
Normal value: 5 to 35 KU.
C) Gamma Gutamyl transferase(GGT):
 Old name = Gamma Glutamyl transpeptidase.
 Slightly higher in men due to presence of prostrate gland.
 Moderate increased in hepatitis and prostrate cancer.
 Normal Range = 10 to 30 IU/L

62. GGT increased in:
• obstructive jaundice.
• Hepatitis.
• Alcoholic Cirrosis.
• Plasma GGT level is a very sensitive indicator
to diagnose alcoholic cirrosis.

63. 2)Cardiac enzyme:
A) SGOT/ AST:
 Significantly increased in Myocardial infraction.
 Moderately increase in Liver disease.
Normal value: 8 to 40 KU.
B) Lactate Dehydrogenase(LDH):
 LDH activity present in cell cytoplasm.
 It is present high concerntration in heart also in kidney, RBC’S and
skeletal muscle.

64. Isoenzyme of LDH:
1) LDH1:
• Sub unit H4.
• Fastest electrophorotic movility.
• At 600c not destroyed.
• Responseble for heart muscle and less amount for
RBC.
2) LDH2:
• Sub unit H3M1.
• Fastest electrophorotic movility.
• At 600c not destroyed.
• Found in tissue more RBC and less heart muscle.

65. 3) LDH3:
• Sub unit H2M2.
• Fastest electrophorotic movility.
• At 600c Partially destroyed.
• Tissue of Brain.
4) LDH4:
• Sub unit H1M3.
• Slow electrophorotic movility.
• At 600c destroyed.
• Tissue of liver.
5) LDH5:
• M4,
• Slow electrophorotic movility.
• At 600c destroyed.
• Tissue of Skeletal.

68. Creatine kinase increases in:
Muscular Dystrophy.
 Injuries of the skeletal muscle. it is CKMM from which
increases due to destruction of the muscle cell.
Myocardial infarction and other heart diseases the
isoenzyme CKMB will increased markedly.
Creatine kinase insreases also in head injury and
diseases of the brain. It is the CKBB form of CK that is
increased.

69. 69
Iso enzymes of CK
 dimer
 mw=40,000
 subunit M(muscle) and B(brain)==dimer
 product of loci in chromosome 14 and 19
respectively so seen in circulation.
 estimation is important in myocardial
infarction
CK-1 (BB)
maximum electiophoresis mobility
tissue of brain

70. 70
CK-2 (MB)
Intermediate electrophoretic mobility
Tissue of heart
CK-3 (MM)
Least electrophoretic mobility
Tissue of skeletal muscles

71. 71
AST/SGOT:
Also called glutamate oxaloacetate transaminase
(SGOT).
Serum level is 8-40 IU/L
Significantly elevated in myocardial infarction
Moderately increased in liver diseases
Iso enzymes present in Cytoplasm and mitochondria

72. Cardiac troponine
 Protein found in skeletal and contractural fibers of the
heart (cardiac muscle)
 Not enzyme
 Marker of myocardial infarction
 Troponine is a protein complex consisting of 2 subunit
with different structure and function
i) Troponine I
ii) Troponine T
Release into the blood within hours
 peak level at 12 to 16 hours
72

73.  Normal: 0-0.3 ng/ml
 Timing
– Earliest rise: 3-4 hrs
– Peak: 10-24 hrs
– Return to Normal: 1-3 wks

74. LEVELS OF ENZYMES IN MYOCARDIAL INFARCTION
CK
CK-MB
AST
LDH
HBDH
AST and CK rise in 6
hours following acute
myocardial infarction
HBDH and LDH are
elevated much later and
remains high for a
longer period of days

75. 3) Prostrate Cancer Enzyme:
A) Prostrate Specific Antigen (PSA)
Normal range = 1 to 5 mg/l
Above 10 mg/ l indicate Prostrate Cancer.
 PAS is found in the Prostrate tissue as a normal part.
 It is present in Benign Hyperplasia of the prostrate and Malignent tumor of
the prostrate.
 PAS is simple chain of Glycoprotein Which contain 7% carbohydrate and
about 240 amino acid.

76. Diagnosis:
 It is used for early detection of Tumor and also monitoring the tumor.
B) Acid Phospatase(ACP):
 ACP is present in all the cell, lysosomes except RBC.
 Also present in Bone marrow, Spleen, Milk, Platelets, and Prostrate
gland.
 optimal activity ranges PH 4-6.
 Enzyme is unstable above PH 7 at 370 c.
Normal Range: 2.5 to 12 IU/L