2. • What are Enzymes?
• Highly specific proteinous substances that are synthesized
in a living cell & catalyze or speed up the
thermodynamically possible reactions necessary for their
existence.
5. Classification of Enzymes :--
A] on the basis of reactions they catalyze:
1. Oxidoreductases :
They bring about biological oxidation & reduction
between two substrates.
e.g; Dehydrogenases, Oxidases, Hydroperoxidases,
Oxygenases, Hydroxylases
2. Transferases :
Catalyse transfer of some group or radical from
one molecule to another.
E.g. Transaminases, Transphosphorylases,
Transglycosidases
6. 3. Hydrolases:
These enzyme catalyze the hydrolytic cleavage reaction.
Eg. Protease, Lipase
4. Lyases:
Catalyse removal of groups from larger substrates by
mechanisms other than hydrolysis, leaving double bonds.
e.g. Carboxylyases, Aldehydelysases
7. 5. Isomerases:
Catalyze interconversion of isomers. eg.
Dextrose isomerase
6. Ligases/ Synthetases:
Catalyse the linking or synthesizing together
of 2 compounds. Forming C-S bonds, C-N
bonds, C-C bonds. E.g: Lyases, Isomerases,
Ligases / Synthetases
8. B] On the basis of site of action:
1. Exoenzymes / Extracellular enzymes:
• Secreted outside the cell
• Decompose complex organic matter like proteins,
fats, cellulose into simpler compound.
E.g.: protease, lipases, amylase.
2. Endoenzymes / Intracellular enzymes:
• Present inside the cell.
• These involved in synthesis of cell component,
food reserves & bioenergetics i.e. liberation of
energy from food stuff.
• E.g..: synthetases, phosphorylases
9. 3. Constitutive Enzymes:
• Produced in absence of substrate.
• Which are produced in constant rate, in constant
amount irrespective of metabolic state of
organism.
• Eg.: Enzymes of glycolytic series.
4. Induced Enzymes:
• Produced in presence of substrate .
• Micro-organism produce them in response to
presence of substrate in environment only &
hence are known induced enzyme.
• Eg.: hepatic microsomal enzymes.
10. 5. Zymogens / Proenzymes:
• Produced naturally in an inactive form which can be activated when
required.
• Enzymes like pepsin are created in the form of pepsinogen, an
inactive zymogen.
• Pepsinogen is activated when Chief cells release it into HCl which
partially activates it.
C] On basis of Substrate on which they act:
1. Amylolytic Enzyme/ Carbohydrases—
Enzyme which act only on carbohydrate are known as
amylolytic/carbohydrases.
i)Amylopsin/ Diastase ii)Zymase
iii)Ptyalin iv) Invertase
v) Hemicellulose vi) Hyaluronidase
vii)Emulsin viii)Maltase
11. 2. Proteolytic Enzyme/ Protease:
Enzyme that act on protein & hydrolyse them
i) Tripsin ii)Chymotrypsin
iii) Pepsin iv) Renin
v) Fibrinolysin vi)Eurokinase
vii)Streptokinase viii)Papain
3. Lypolytic Emzyme: Enzyme which act on fats /
Lipids.
i)Lypase ii)Steapsin
iii)Pancreatin iv)Fungal lipase
12. ❑ Explain the term Enzyme specificity with examples.
• One of the important characteristic of enzyme is that
they are highly specific in their action.
• Each enzyme is capable of bringing out only one or
small group of reactions.
1.Reaction Specificity:
• Different enzymes bring out different reactions on
same substrate.
• For Eg: Oxidase bring about oxidation of amino acid
• Decarboxylase bring about decarboxylation of amino
acid
2.Substrate Specificity
i) Absolute specificity:
• Particular enzyme acts on a particular substrate
Eg--Urease on Urea give ammonia & carbon dioxide
14. ii) Relative specificity:
• Group specificity: Particular enzyme acts on
particular chemical groupings
• Glycosidase on glycosides or Esterase on ester
linkages
3.Optical Specificity/ Stereospecificity
• Enzymes show absolute optical specificity for
atleast a portion of substrate molecule.
• L-amino acid oxidase & D- amino acid oxidase
act only on L & D –amino acids respectively.
15. • Active site of an enzyme
1. Lock & key model
2. Induce fit model / Koshland theory
• Portion of an enzyme to which the substrate binds &
gets converted into the product is called an active
site of an enzyme.
• In the active site , the amino acids are grouped
together in such a manner so as to enable the
enzymes to combine with substrate.
• Binding of substrate is explained by 2 models: Lock
& key model and Induce fit model
16.
17. A) It is like a Lock & Key.
• In this case the shape of active site of an enzyme
and that of substrate is complementary to each
other.
• The substrate molecule fits into the active site of
enzyme just as key fits into a lock.
• Hence called as Lock & Key model.
• The shape of active site is rigid and
complementary to the shape of substrate
complex.
18. • B) Induce fit model/Koshland theory of
enzyme action.
1. In this case the shape of active site of an
enzyme is flexible so as to accommodate wide
variety of substrate molecules.
2. The shape of active site of enzyme is made
complementary to the substrate molecule.
19.
20. • Different factors affecting rate of enzyme
catalysed reaction:--
1. Hydrogen ion concentration/ effect of pH
2. Concentration of enzymes
3. Concentration of substrate
4. Temperature
5. Time
6. Products of reaction
7. Effect of light & radiation
8. Effect of inhibitors
9. Effect of Activators.
21. 1) Effect of Hydrogen ion concentration:
•Enzymatic reactions are influenced by varying H+ ion
concentration.
• Enzyme has characteristic pH at which their activity is
maximum. This characteristic pH is called optimum pH.
•The optimum pH is that pH at which a certain enzyme
causes a reaction to progress most rapidly.
•On either side of the optimum pH, the rate of reaction is
lower & at certain pH enzyme may be inactivated or
even destroyed.
•Buffers are used to keep enzyme at an optimum or at
least a favorable H+ ion concentration.
•Eg.: optimum pH of sucrase is 6.2 ; pepsin is 1.5- 2.5
22.
23. 2) Effect of enzyme concentration
• Velocity of enzyme catalyzed reactions is directly
proportional to the concentration of enzyme in
system.
• This is true in the beginning of reaction but as the
reaction continues substrate gets used up.
24. 3) Effect of temperature:
• Velocity of reaction increases as temp. increases upto
optimum temp.
• Optimum temperature is usually reached at around
37oC—45oC for animal enzymes.
• Above the optimum temperature, rate decreases.
• The temp at which enzyme show max. activity is
called optimum temp.
• Most of the enzymes get inactivated above 60oC is
called thermal denaturation.
• The time of exposure is also important factor.
• An enzyme may withstand higher temperatures for
short periods of time.
• Enzyme activity is maximum at optimum temperature.
25.
26. 4) Effect of Substrate concentration-
• Increase in the substrate concentration gradually
increases the velocity of enzyme reaction within the
limited range of substrate levels.
• A rectangular hyperbola is obtained when velocity is
plotted against the substrate concentration.
• Three distinct phases of the reaction are observed in
the graph, first in linear fashion, later the curve flattens
and becomes plateau.
• Thus, by increasing the concentration of the substrate
indefinitely, the velocity of the reaction cannot be
increased beyond a certain stage.
• The rate of the enzyme reaction is independent of the
concentration of substrate and enzyme.
27. • Initially rate is directly proportional to substrate
conc.
• If substrate conc. Is further increased , then
velocity of reaction is partly depends on
substrate conc.
• If substrate conc. Is even further increased, the
velocity of reaction remain unchanged.
• From graph following equation is obtained:
• Michaelis Menten Equation—
V = Vmax [S]
Km + [S]
28. Diagram of graph –
First oorder kinetic
Mix order kinetic
Zero order kinetic
29. 5) Effect of activators:
• Presence of activator increases enzyme activity.
• Eg- Cysteine HCl increases proteolytic activity of papain.
6) Effect of Inhibitor:
• Presence of enzyme inhibitor reduces enzyme action.
• Heavy metal are inhibitor for enzyme activity.
7) Effect of Light & radiation:
• Some enzyme may get deacticted by exposure to UV, beta,
gamma & X-rays.
• Eg- Salivary amylase is deactivated by exposure to UV rays.
30. • Enzyme Inhibition
• Definition: Compounds or agents which inactivate
the enzymes, & thus adversely affect the rate of
enzyme catalysed reaction are called as inhibitors &
this process is known as enzyme inhibition.
1) Competitive inhibition:
• In Competitive inhibition structure of inhibitor (I)
closely resembles with that of the substrate (S).
• The inhibitor thus competes with the substrate to
combine with an enzyme (E) forming the enzyme
inhibitor complex (EI) rather than (ES) complex.
• The degree of inhibition depends upon the relative
concentration of the substrate & the Inhibitor.
31. • Thus by increasing the substrate concentration &
keeping the inhibitor concentration constant the
amount of inhibition decreases & decrease in
substrate concentration result in increased enzyme
inhibition.
• In this type of inhibition, enzyme can either bind
with substrate (ES) or inhibitor (EI) but not both.
Vmax is unchanged KM is increased
32. I
I
I
I
I
I
No Product
1) E = Enzyme 5) EI = Enzyme inhibitor complex
2) S = Substrate
3) P = Product
4) I = Inhibitor
❌
33. I I
Product
1) E = Enzyme 5) EI = Enzyme inhibitor complex
2) S = Substrate 6) ES = Enzyme substrate complex
3) P = Product
4) I = Inhibitor
36. 2) Noncompetitive Inhibition:
• Here no competition occurs between the substrate
(S) & the inhibitor (I).
• The inhibitor has little or no structural resemblance
with the substrate & it binds with an enzyme at
place other than active site.
• This binding impairs the enzyme function. In this
inhibition the inhibitor generally binds with enzyme
as well as ES complex.
• In this type of inhibition both EI & ESI complex
formation take place.
• V max is lowered but KM value is unchanged
41. • Isoenzyme:
• The enzymes which have multiple molecular
forms in the same organism, catalysing the
same biochemical reaction are called as
Isoenzymes.
• e.g. lactate dehydrogenase exist in the blood
in five different isoenzyme forms i.e.
LDH1,LDH2, LDH3, LDH4, LDH5.
42. • Allosteric Enzyme (Regulatory enzyme) :-
• The enzyme that changes their conformation upon
binding of an allosteric modulator which result in an
apparent change in binding affinity at a different
ligand binding site.
• Allosteric modulator—
1) Positive Allosteric Modulator =they increase
activity of enzyme.
2) Negative Allosteric Modulator = they reduces
activity of enzyme.
43.
44. • Eg– Phosphofructokinase is an allosteric enzyme, which
convert
Fructose-6-Phosphate--------→ Fructose-1,6-diphosphate.
[F-6-P P- F - Kinase → F-1,6-DiP]
• ADP is positive allosteric modulator &
• ATP is negative allosteric modulator
• Co-factors:
• Some enzyme require non-protein moiety for
catalytic activity is called as co-factor.
• Some co-factor are derived from vitamin are called
co-enzyme.
• Even some enzyme require metal ion for their
catalytic activity and are called co-factor.
45. Feedback mechanism:
• In case of some enzyme, their activity is inhibited by
their product or successive metabolite. Such
inhibition is called feedback inhibition.
46. • Enzyme activity unit–
• It is defined as amount which causes
transformation of 1µ mole of substrate per
minute at 250
C, under optimum condition of
measurement.
47. • Pharmaceutical Importance of
Enzymes—
1) Medicinal significance of enzyme
2) Enzyme therapy
3) Manufacturing of bulk drug
4) Diagnostic uses of Enzymes.
48. 1) Medicinal application:
• Sulphanilamide an antimetabolite, is structurally
similar with that of PABA which is required for the
synthesis of folic acid competes with PABA & inhibit the
enzyme folic acid synthetase, & thereby inhibit the
reaction of invading bacteria.
• By using competitive inhibitors like ephedrine,
isoprenaline block the action of hormone adrenaline,
noradrenaline & 5- HT can be prolonged by blocking
the action of an enzyme monoamine oxidase which are
responsible for the catabolism of these hormones.
• Xanthine oxidase enzyme is involved in the conversion
of xanthine & hypoxanthine to uric acid.
---Allopurinol a competitive inhibitor of xanthine oxidase
is used to reduce the conversion of xanthine &
hypoxanthine to uric acid in the treatment of gout.
49.
50. 2) diagnostic applications of enzymes.
• Enzymes are normally confined within the cell.
• The little amount is present in body fluids like blood
& C.S.F. etc.
• Certain enzymes come into plasma due to leakage
from living cells or from dead or dying cells.
• Such enzymes are very useful for the diagnosis of
various diseases. They are called as ‘marker
enzymes’.
• Marker enzyme : The enzyme whose presence or
increased or decreased level in the blood,
cerebrospinal fluid or urine indicates the presence
of specific disease is known as marker enzyme.
• hence we can identify the disease.
51. For Eg—
1) The level of SGOT rises rapidly after a heart attack.
2) The level of SGPT increases in infectious hepatitis
3) Activity of Creatine-kinase increases in the plasma,
during infection in cardiac muscle.
52. 3) Manufacturing Applications:
Enzymes are very useful for manufacturing of drugs
Eg—
a) An enzyme ‘penicillin acylase’ is used for the
production of 6-aminopenicillanic acid from
Penicillin-G
b) For the preparation of digestants various enzymes
like papain, pepsin, trypsin are used.
c) Hyaluronidase is used in orthopaedic practice
d) Urokinase in cardiac diseases
e) Streptokinase in treatment of thrombosis.
f) Amylase is used in the production of dextrin.
53.
54. 4) Enzyme therapy:
• It has found that enzyme can be used in
treatment.
• Eg:- i) Asparaginase = treatment of tumors
ii)Galactosidase = treat lactose intolerance.
