This document discusses enzymes and their role as biological catalysts. It defines enzymes as proteins that increase the rate of reactions by lowering the activation energy. Enzymes catalyze reactions in cells and have a specific three-dimensional structure that allows them to bind to substrates. The document also mentions that deficiencies in enzymes can cause metabolic diseases.

1. DEAR STUDENTS
ANSWER
“ WE ARE THE CATALYSTS OF
THE LIVING WORLD,
INCREASE THE RATE OF
REACTION THOUSAND
TIMES.WORK IN AQUOUS
MEDIUM. PROTEIN IN
NATURE. AND IN ACTION
SPECIFIC, ACCURATE; BIG IN
SIZE BUT WITH SMALL
ACTIVE SITE; HIGHLY
EXPLOITED FOR DISEASE

2. ENZYMES
DR.K.S.SODHI,M.
D.
PROFESSOR
BIO-CHEMISTRY
MMIMS&R MULLANA
AMBALA.
© 2007 Paul Billiet ODWS

3. The Chemicals of Living Cells
©The Wellcome Trust

4. HISTORY Of EnzYmES
As early as the late 1700s and early
1800s, the digestion of meat by
stomach secretions and the
conversion of starch to sugars by
plant extracts and saliva were
known. However, the mechanism by
which this occurred had not been
identified.

5. ENZYMOLOGY
Contribution of Scientists.
Definitions.
Mode of Action of Enzymes.
Factors Influencing Enzyme
Activity.
Enzyme Inhibition.
Regulation of Enzymes.
Diagnostic Importance of
Enzymes.
Therapeutic Use of Enzymes.

6. DEFINITIONS
HOLOENZYMES ( APOENZYMES+CO
ENZ.)
APOENZYMES; SINGLE
POLYPEPTIDECHAIN,MORE THAN ONE
CHAIN,MULTI-ENZYME COMPLEX.
Co-ENZYMES : Non Protein (VITAMINS)
METAL-ACTIVATED ENZYMES.
(Zn,Cu,Fe,Mg,K,Ca etc.)
ZYMASE: Active without modification
ZYMOGENS : Pro Enzymes eg.Trypsinogen

7.  ISO-ENZYMES : Physically distinct
perform same function.
 RIBOZYMES: Small ribonuclear
particles.
 ENDOENZYMES : Produced in the
cell. Function inside the cell.
 EXOENZYMES : Produced inside the
cell. Act outside the cell.

8.  METALLO ENZYMES : Contain metal ions
as essential component.
 HOUSE KEEPING ENZYMES : Levels of
Enzymes can not be controlled. Always
present in cell.
 ADAPTIVE ENZYMES : Regulated by
genes. Conc.increase or Decrease.
 KEY ENZYMES :Regulatory eg HMG-CO.A
 HYBRID ENZYMES :Produced by genetic
fusion.

9.  An additional non-
protein molecule COFACTORS
that is needed by
some enzymes to
help the reaction
 Tightly bound
cofactors are called
prosthetic groups
 Cofactors that are
bound and released
easily are called
coenzymes
Nitrogenase enzyme with Fe, Mo and ADP
 Many vitamins are cofactors
coenzymes

10. A GOOD TEACHER
IS ALWAYS A GOOD
CATALYST IN
STUDENTS LIFE.

11. DISTRIBUTION OF 17 HORSES
OLDMAN AND THREE
SONS.
DISTRIBUTION OF
HORSES.
ELDER ½
MIDDLE 1/3
LITTLE 1/9

12. Notice that without the enzyme it takes a lot more energy
for the reaction to occur. By lowering the activation
energy you speed up the reaction.

13. Energy In Reactions
 Energy is released or
absorbed whenever
chemical bonds are
formed or broken.
 Because chemical
reactions involve
breaking and forming
of bonds, they
involve changes in
energy.

14. Enzymes as Biological Catalysts
 Enzymes are
proteins that
increase the rate of
reaction by
lowering the energy
of activation
 They catalyze
nearly all the
chemical reactions
taking place in the
cells of the body
 Enzymes have
unique three-
dimensional shapes
that fit the shapes
of reactants
(substrates)

15. Thermodynamics

16. The energies of various stages of a
chemical reaction. Substrates need a large
amount of energy to reach a transition state ,
which then decays into products.
The enzyme stabilizes the transition state,
reducing the energy needed to form products.
As all catalysts, enzymes do not alter the
position of the chemical equilibrium of the
reaction. Usually, in the presence of an enzyme,
the reaction runs in the same direction as it
would without the enzyme, just more quickly.

17. For example, carbonic anhydrase
catalyzes its reaction in either direction
depending on the concentration of its
reactants.
(in tissues ;
high CO concentration)
2
in
lungs; low CO concentration).
2

18. Kinetics
Enzyme kinetics is the investigation of
how enzymes bind substrates and turn
them into products.
The enzyme (E) binds a substrate (S) and
produces a product (P).

19. In 1902 Victor Henri contribute
was to think of enzyme reactions in two
stages. In the first, the substrate binds
reversibly to the enzyme, forming the
enzyme-substrate complex.
This is sometimes called the
Michaelis complex.
The enzyme then catalyzes the
chemical step in the reaction and releases
the product.

20. In 1902 Victor Henri contribute
was to think of enzyme reactions in two
stages. In the first, the substrate binds
reversibly to the enzyme, forming the
enzyme-substrate complex.
This is sometimes called the
Michaelis complex.
The enzyme then catalyzes the
chemical step in the reaction and releases
the product.

21. Saturation curve for an enzyme
reaction showing the relation between the
substrate concentration (S) and rate ( v).

22. Enzyme rates depend on solution conditions
and substrate concentration.
Conditions that denature the protein abolish
enzyme activity, such as high temperatures,
extremes of pH or high salt concentrations.
while raising substrate concentration tends
to increase activity. Saturation happens
because, as substrate concentration increases,
more and more of the free enzyme is converted
into the substrate-bound ES form.

23. At the maximum velocity (Vmax) of the
enzyme, all the enzyme active sites are
bound to substrate, and the amount of ES
complex is the same as the total amount
of enzyme. However, Vmax is only one
kinetic constant of enzymes.
K m , : is the substrate concentration
required for an enzyme to reach one-half
its maximum velocity. Each enzyme has a
characteristic Km for a given substrate.
k : is the number of substrate

24. So The efficiency of an enzyme = kcat/Km.
This is also called the specificity
constant and incorporates the rate
constants for all steps in the reaction
(affinity and catalytic ability).

25. CO-ENZYMES
 Essential for Biological activity.
 Low molecular weight, Organic in nature
 Non protein in nature.
 .Combine loosely with Enzyme &separate
later.
 Thermos table.
 Help in group transfer.
 Bind to apoenzymes.
 GTP, NADP, FMN, FAD, Biotin, Lipoic
Acid, Pyridoxal Phosphate,etc. (Vitamins)
 Co-enzyme separate from apo-Enz after
reaction.
 Can be separated by Dialysis.

26. Co-Enzymes can be divided
into two groups.
A.Oxidoreductases.NADH.NAD
PH,FAD.
B. Transfer Groups.
Thiamine-Hydroxyl group.
Pyridoxal phosphate-Amino
group
Tetrahydrofolate-one Carbon
Biotin-Carbon dioxide .

27. Control Points of Gene Regulation
Transcription
RNA Processing DNA
DNA RNA Transport
5’ mRNA
RNA Degradation process 3’
ribosome mature
mRNA mRNA
Translation cap
5’ 3’
proteins tail
Activity
proteins
Proteolysis
Prokaryotics Post-translational
Eukaryotics
control
Juang RH (2004) BCbasics

29. Enzyme structure
 Enzymes are
proteins
 They have a
globular shape
 A complex 3-D
structure
Human pancreatic amylase
© 2007 Paul Billiet ODWS

30. STRUCTURE
1.MONOMERIC: Single Peptide.
2.OLIGOMERIC: Many peptide
Chains.
3.Multienzyme Complex:
Fatty Acid Synthase
LDH Complex.
Prostaglandin Synthase

31. ENZYMES UNITS
KINGARMSTRONG.
SOMOGY.
REITMAN FRANKEL.
SPECTROPHOTOMETRIC.
KATAL.
INTERNATIONAL UNIT.

32. ENZYMEZS ESTIMATED
FROM:
WHOLE BLOOD, SERUM,
PLASMA.
RED BLOOD CELLS.
C.S.F.
URINE.
SWEAT.
SALIVA.
SEMEN.
AMNIOTIC FLUID.
Tears.

33. TISSUES
BRAIN,HEART,LIVER,KIDNEY,MUSCLE
BRAIN
MUSCLE
→ ←
←
HEART
→ ←
KIDNEY
←
LIVER
STOMACH
INTESTINE

34. PLASMA ENZYMES
 FUNCTIONAL PLAMSMA ENZYMES.
eg. LIPOPROTEIN LIPASE, BLOOD
CLOT DISSOLVING ENZYMES etc.
 NONFUNCTIONAL PLASMA
ENZYMES. eg: SGOT,
SGPT,AMYLASE,CPK,LDH,LIPASE,ACID
-PHOSPHATASE,ALKALINE PHOS.,
CERULOPLASMIN etc.

35. NATURE OF ENZYMES
 Soluble,Colloidal, Organic
Catalysts
 Formed by Living Cells ,Specific in
action, Protein In Nature ,Inactive
at Zero degree centigrade
,Destroyed by moist heat at 100
degree centigrade (Heat Labile),
Huge in size, small Active Site,
Used for Treatment.

36. DIFFERENCE
 BIO-CATALYST :
Enzymes, protein in nature except
ribozymes, More specific, more efficient
and slight change in structure alter its
action.
 CATALYST:
Inorganic, less sp., less efficient and no
change in structure.

37. Compartments of cell
DNA, RNA, protein overview
DNA
RNA
Mutations
Amino acids,
protein structure

38. COMPARTMENTATION
 MITOCHONDRIA: Enzymes of:
E.T.C, TCA Cycle, Beta Oxidation,
Urea Cycle, Pyruvate to Acetyle
Co-A.
CYTOSOL: Glycolysis, HMP Shunt,
Fatty Acid Synthesis, Glucogenesis
and Glycogenolysis.
NUCLEUS: DNA Synthesis, RNA
Synthesis and Histones etc.
LYSOSOMES : Next Slide

40. FUNCTIONS OF ENZYMES

1. Catalyse thousands of reactions.
 2. Digestive Enzymes help in igestion.
 3. Lysosomal Enzymes destroy in cell.
 4. Lysozymes are bacteriocidal, local
immunity
 (TEARS)
4. Detergents
5. Textile.
6. Leather Industry.

43. What is a Ribozyme?
1) Enzyme
2) Ribonucleic Acid
NOT PROTEIN Sid Altman Tom Cech
1989 Nobel Prize
In Chemistry

44. RIBOZYMES
Small ribonucleic particles.
Contain rRNA.
Highly substrate specific.
Used in Intron splicing,pre RNA
to RNA Peptidyl Transferase.
Many ribozymes have hair-pin
or hammer head shaped active
centre &require Divalent Mg++
Catalyse reaction on
phosphpdiester bonds of other

45. Ribozymes Have following
Drawbacks.
Not as efficient as protein
catalysts( In RNA there are 4
nucleotides, in amino acid are 20 in
number.
Act once only in chemical
event,protein enzymes are reused
several times.
Rate of catalytic activity is slower.
Synthatic Ribozymes are having
better catalytic activity(Cleave
infectious Virus)

46. ABZYMES
Artificially synthasized catalytic
antibodies against Enz. Sub.
Complex in transition state of
reaction. CATMAB (Catalytic
Monoclonal Antibody).
Sometimes natural abzymes are
found in blood,eg.antivasoactive
intestinal peptide autoantibodies.
Useful in diseases viz.abzyme
against gp120 envelop protein of HIV
may prevent virus entry in to the host

47. ANTIENZYME
Extracts of Intestinal Parasites
like Ascaris,contain
substances called
antizymes,which inhibit the
action of digestive enzymes
pepsin and Trypsin.This is
probably the reason why the
worms are not digested in the

48. Phe
Ribozyme vs. tRNA
folding

49. The Future of Ribozymes
In Vitro Molecular Evolution of RNA
+
High Throughput Screening
Ribozyme-Based Therapies

50. In Clinical Trial...
HIV Gene Therapy...
Bone Marrow Sample
Treat Stem Cells with Retroviral Vector
Encodes Gene for anti-HIV Ribozyme
Re-Implant Treated Cells

51. ACTIVE SITE OF
RIBONUCLEASES
It lies in a hydrophobic cleft.
7 th Lysine 41 st Lysine on one
side and 12 th Histidine and
119Histidine on the opposite
side.(URIDYLIC ACID)
Peptidyl transferase (chain
Elongation)
Removal of Introns.

52. The Substrate
 The substrate of an enzyme are the
reactants that are activated by the
enzyme
 Enzymes are specific to their
substrates
 The specificity is determined by the
active site
© 2007 Paul Billiet ODWS

53. PRODUCT
 Substrate in the presence of Enzyme is
converted in to product.
 The reaction can be Reversible or Ir-
reversible.
 The increase in product concentration can
cause inhibition and stop the reaction in
the forwaed direction.

54. ABBREVIATIONS
 ENZYME [E]
 SUBSTRATE [S]
 PRODUCT [P]
 Enz. Sub. Complex [ES]
 INHIBITOR [I]
 Enz.+Inh. Complex [EI ]
 Enz.+Sub.+Inh. [ESI]

55. Enzymes are Biological
Catalysts

56. Enzymes are proteins that:
Increase the rate of reaction by
lowering the energy of activation.
Catalyze nearly all the chemical
reactions taking place in the cells
of the body.
Have unique three-dimensional
shapes that fit the shapes of

57. Enzyme Deficiency
A variety of metabolic diseases are now
known to be caused by deficiencies or
malfunctions of enzymes.
Albinism, for example, is often caused
by the absence of tyrosinase, an enzyme
essential for the production of cellular
pigments.
The hereditary lack of phenylalanine
hydroxylase results in the disease
phenylketonuria (PKU) which, if untreated,
leads to severe mental retardation in children.

58. ACTIVE SITE
The active site :
Is a region within an enzyme
that fits the shape of molecules
called substrates .
Contains amino acid R groups
that align and bind the substrate.
Releases products when the
reaction is complete.

59. ACTIVE SITE OF ENZYME
Chymotrypsin
His(57)Asp(102)Ser(195)
Trypsin
Histidine,Serine
Phosphoglucomutase Serine
Carboxypeptidase
Histidine,Arginine,tyrosine
Aldolase Lysine

60. Active Site Avoids the Influence of Water
+
-
Preventing the influence of water sustains

61. Active Site Is a Deep Buried Pocket
Why energy required to reach transition state
is lower in the active site?
It is a magic pocket
+ (1) Stabilizes transition
(2) Expels water
CoE (1) (2)
(3) Reactive groups
(4) - (4) Coenzyme helps
(3)
Juang RH (2004) BCbasics

62. ACTIVE SITE
 Generally the active site is situated on
the cleft of the Enzyme.
 Binding of substrate to active site
dependends upon the presence of sp.
Groups or atoms at active site.
 During binding these groups,realign
themselves so as to fit the substrate.
 The substrate bind to active site by non
co-valent bonds.(Hddrophobic in nature)
 Amino acid that make or break bonds
called catalytic group.

63. ACTIVE SITE

66. Enzymes
Enzymes
provide a site
where reactants
can be brought
together to react.
Such a site
reduces the
energy needed
for a reaction to
occur.

67. All enzymes have an active site,
where substrates are attracted to.
 Enzymes are used over and over again.

68. The Enzyme Substrate complex
 When enzymes function
the active site interacts
with the substrate.
 The active site shape
matches the substrates
shape.
 Once the substrate and
active site meet a change
in shape of the active site
causes a stress that
changes the substrate
and produces an end
product.

69. Which one will fit ?

70. ENZYME SPECIFICITY
Enzymes may recognize and catalyze:
 A single substrate.
 A group of similar substrates.
 A particular type of bond.

71. MECHANISM OF
ACTION
 INDUCEFIT MODEL.
(KOSHLAND’S)
 LOCK AND KEY MODEL.
(FISHER’S TEMPLATE THEORY)

72. The Induced Fit
Hypothesis
 Some proteins can change their
shape (conformation)
 When a substrate combines with an
enzyme, it induces a change in the
enzyme’s conformation
 T he active site is then moulded
into a precise conformation
 Making the chemical environment
suitable for the reaction
 The bonds of the substrate are
stretched to make the reaction
© 2007 Paul Billiet ODWS

73. Induced-fit Model
In the induced-fit model of enzyme action:
 The active site is flexible, not rigid.
 The shapes of the enzyme, active site,
and substrate adjust to maximum the fit,
which improves catalysis.
 There is a greater range of substrate
specificity.

74. The Lock and Key
Hypothesis
 Fit between the substrate and the active
site of the enzyme is exact
 Like a key fits into a lock very precisely
 The key is analogous to the enzyme and
the substrate analogous to the lock.
 Temporary structure called the enzyme-
substrate complex formed
 Products have a different shape from
the substrate
 Once formed, they are released from the
active site
 Leaving it free to become attached to
another substrate
© 2007 Paul Billiet ODWS

75. Lock-and-Key Model
In the lock-and-key model of enzyme
action:
 The active site has a rigid shape.
 Only substrates with the matching shape
can fit.
 The substrate is a key that fits the lock of
the active site.
 Rigid structure could not explain
flexibility shown by enzymes

76. Acid-Base Catalysis
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.167
Specific Induced to transition state
Acid-base
Acid
Catalysis + catalysis +
N N
O H H
O
=
C H O O
=
+ C H
=
=
N C Both
H H C H N C C H
N C H H N C
H H O H O H
- O H H H H
O H H
H H O - O O - O
Base C C
catalysis
Slow Fast Fast Very Fast

77. Basic Mechanism of Catalysis
3 basic types 1) Bond Strain Conformational change
2) Acid-base transfer Chemical reaction
3) Orientation Space arrangement
Concert Carboxypeptidase A non-polar Metal protease
Carboxypeptidase B RK Exopeptidase
Carboxypeptidase Y non-specific
Sequential Chymotrypsin YFW Ser-protease
Trypsin RK Endopeptidase
Elastase GA
Juang RH (2004) BCbasics

78. Concerted Mechanism of Catalysis
Carboxypeptidase A
Active (248)
(270)
site Tyr
Glu 3 4 ACTIVE
pocket O - Site for
COO - H SITE specificity
H
+
H O-
C N R 5
N C C
2
Substrate O-
Juang RH (2004) BCbasics
peptide
chain
1 + COO - +
Arg (145)
His
(196)
Zn Glu C-terminus
(72) Check for
His (69)
C-terminal

79. MICHAELI
S
CONSTAN
(Km)
T

81. Salient Features of Km
 Km is sub. Conc.at ½ the max. velocity
 It denotes that 50% of Enzyme mol.are
bound with sub.at particular sub. Conc.
 Km is independent of Enzyme conc.If
Enz. Conc. Is doubled, the Vmax will be
double but km will remain same.
 Km is signature of Enzyme.
 Affinity of Enz. Towards its substrate is
inversely related to the dissociation
constant(smaller the dissociation greater
the affinity.
 Km denotes affinity of enzme for
substrate.lesser the Km more the affinity.

82. MICHAELIS CONSTANT
(Km)
It is defined as the conc. Of the
substrate at which the reaction
velocity is half of the maximum
velocity.
Km is independent of enzyme
conc.
If an enzyme has a small value of
Km, it achieves maximal catalytic
efficency at low substrate conc.
SIGNIFICANCE
Glucokinase has high Km is low

83. Hexokinase has low Km, High
affinity for Glucose ie glucose will
be provided to the vital organs
even at low glucose levels.
Lab. Significance: The sub. Conc.
Kept at saturation point at least 10
times the Km so that reaction
proceeds to completion.
Clinical Significance: The Km
value for the given enzyme may
differ from person to person and
explains various responses to
drugs/chemicals.

84. Names of Enzymes
The name of an enzyme:
 Usually ends in –ase.
 Identifies the reacting substance. For
example,
sucrase catalyzes the reaction of
sucrose.
 Describes the function of the enzyme.
For example, oxidases catalyze
oxidation.
 Could be a common name, particularly
for the digestion enzymes such as
pepsin and trypsin .

85. The top-level classification is:
 EC 1 Oxidoreductases : catalyze
oxidation/ reduction reactions .
 EC 2 Transferases : transfer a functional
group (e.g. a methyl or phosphate group).
 EC 3 Hydrolases : catalyze the hydrolysis of
various bonds .
 EC 4 Lyases : cleave various bonds by
means other than hydrolysis and oxidation.
 EC 5 Isomerases : catalyze isomerization
changes within a single molecule.
 EC 6 Ligases : join two molecules with
covalent bonds.

86. CLASSIFICATION
 I.U.C.B.
 1.OXIDO-REDUCTASE .transfer of hydrogen
or addition of oxygen.Eg.LDH
 2.TRANSFERASE. Eg.Aminotransferase.
 Hexokinase.
 3.HYDROLASE .Cleave bond adding water
 Eg. Acetyl choline esterase.
 4.LYASE .Cleave without adding water
(Aldolase)
 5.ISOMERASE.
 6.LIGASE. Acetyl co-A
carboxylase,Glu.Synthatase,PRPP Synthatase.

87. Classification of Enzymes:
Oxidoreductases and Transferases

88. Classification: Hydrolases and
Lyases

91. FACTORS AFFECTING
ENZYME
1.SUBSTRATE
CONCENTRATION.
2.ENZYME
CONCENTRATION.
3.TEMPERATURE.
4.pH.
5.EFFECT OF PRODUCT
CONC.
6.PRESENCE OF

92. FACTORS ………………..
9.EFFECT OF CLOSE
CONTCT.
10.OXIDATION OF
ADD.GROUPS.
11.EFFECT OF LIGHT.
12.EFFECTS OF
RADIATIONS.
13.PRESENCE OF
REPRESSOR

93. Substrate concentration: Non-enzymic
reactions
Reaction
velocity
Substrate concentration
 The increase in velocity is proportional
to the substrate concentration

94. Substrate Concentration
 The rate of
reaction
increases as
substrate
concentration
increases (at
constant enzyme
concentration).
 Maximum activity
occurs when the
enzyme is

95. Substrate concentration: Non-enzymic reactions
Reaction
velocity
Substrate concentration
 The increase in velocity is proportional to the
substrate concentration

96. Substrate concentration: Enzymic reactions
when[ s] conc. Is increased velocity increases in
the initial phase (Vmax.),but flatten afterward.
Vmax
Reaction
velocity
Substrate concentration
 Faster reaction but it reaches a saturation
point when all the enzyme molecules are
occupied.
 If you alter the concentration of the enzyme
© 2007 Paul BillietV max will change too.
then ODWS

97. Enzyme Concentration
 The rate of
reaction increases
as enzyme
concentration
increases (at
constant substrate
concentration).
 At higher
enzyme
concentrations,
more substrate
binds with
enzyme.

98. EFFECT OF
CONC.PRODUCT
 At Equilibrium as per law of mass
action,the reaction rate is slowed down,it
can slow,stopped or reversed.
 A —E1—B —E2—≠— C —E3—D .
 Increase in conc. Of D will cause feed
back Inhibition.

100. The effect of temperature
 For most enzymes the optimum
temperature is about 30°C
 Many are a lot lower,
cold water fish will die at 30°C because
their enzymes denature
 A few bacteria have enzymes that can
withstand very high temperatures up to
100°C
 Most enzymes however are fully
denatured at 70°C
© 2007 Paul Billiet ODWS

101. Affects of temperature on an enzyme
 If temp to high or to
low the enzyme will
not fit. No reaction
will occur.

102. Temperature and Enzyme
Action
Enzymes :
 Are most active at an
optimum temperature
(usually 37°C in
humans).
 Show little activity at
low temperatures.
 Lose activity at high
temperatures as
denaturation occurs.

103. The effect of temperature
Q10 Denaturation
Enzyme
activity
0 10 20 30 40 50
Temperature / °C

104. The effect of temperature
 Q10 (the temperature coefficient ) = the
increase in reaction rate with a 10°C rise in
temperature.
 For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or triples with
every 10°C rise in temperature)
 Enzyme-controlled reactions follow this rule as
they are chemical reactions
 BUT at high temperatures proteins denature
 The optimum temperature for an enzyme
controlled reaction will be a balance between
the Q10 and denaturation.

105. Optimum pH Values
 Most enzymes of the body have an
optimum pH of about 7.4.
 In certain organs, enzymes operate at
lower and higher optimum pH values.

106. The effect of pH
 Extreme pH levels will produce
denaturation
 The structure of the enzyme is changed
 The active site is distorted and the
substrate molecules will no longer fit in it
 At pH values slightly different from the
enzyme’s optimum value, small changes
in the charges of the enzyme and it’s
substrate molecules will occur
 This change in ionisation will affect the
binding of the substrate with the active
© 2007 Paul Billiet ODWS

107. How pH affects an enzyme
 Ifthe pH is to high or
low the enzyme will
not work, because its
shape will change.

109. The effect of pH
Optimum pH values
Enzyme
activity Trypsin
Pepsin
1 3 5 7 9 11
© 2007 Paul Billiet ODWS
pH

110. pH and Enzyme Action
Enzymes :
 Are most active at
optimum pH.
 Contain R groups
of amino acids with
proper charges at
optimum pH.
 Lose activity in low
or high pH as
tertiary structure is
disrupted.

111. Optimum pH Values
 Most enzymes of the body have an
optimum pH of about 7.4.
 In certain organs, enzymes operate at
lower and higher optimum pH values.

112. ENZYME ACTIVATION BY
INORGANIC IONS
In the presence some inorganic ions
some enzymes show higher activity
eg.Chloride ion activate salivary
amylase,Ca. activates lipases.
Proenzymes in to enzymes.
Coagulatio factors are seen in blood
as zymogen.
Compliment cascade,these activities
needed occasionly.

113. Enzyme Inhibition
Competitive Inhibtion.
Non-Competitive Inhibition.
Un-competitive Inhibition.
Suicide Inhibition.
Allosteric Inhibition
Key Enzymes
Feedback Inhibition.
Inducors.Glucokinase is induced by
Insulin.
Repression (Heme is reprossor of
ALA Synthase.

114. Enzyme Inhibition (Mechanism)
I Competitive I Non-competitive I Uncompetitive
Substrate E
Cartoon Guide
S S E I
S X
E S I I
I
Compete for S I
Inhibitor active site Different site
E + S ← ES → E + P
→ E + S ← ES → E + P
→ E + S ← ES → E + P
→
Equation and Description
+ + + +
I I I I
↓↑ ↓↑ ↓ ↑ ↓↑
EI EI + S →EIS E IS
[I] binds to free [E] only, [I] binds to free [E] or [ES] [I] binds to [ES] complex
and competes with [S];
complex; Increasing [S] can only, increasing [S] favors
increasing [S] overcomes
not overcome [I] inhibition. the inhibition by [I].
Inhibition by [I].
Juang RH (2004) BCbasics

115. Competitive Inhibition
Product Substrate Competitive Inhibitor
Succinate Glutarate Malonate Oxalate
C-OO- C-OO- C-OO- C-OO- C-OO-
C-H H-C-H H-C-H H-C-H C-OO-
C-H H-C-H H-C-H C-OO-
C-OO- C-OO- H-C-H
C-OO-
Succinate Dehydrogenase

116. Sulfa Drug Is Competitive Inhibitor
Domagk (1939)
Para-aminobenzoic acid (PABA)
Bacteria needs PABA for
H2N- -COOH the biosynthesis of folic acid
Folic Tetrahydro-
Precursor acid folic acid
Sulfa drugs has similar
H2N- -SONH2 structure with PABA, and
inhibit bacteria growth.
Sulfanilamide
Sulfa drug (anti-inflammation)

117. Enzyme Inhibitors Are Extensively Used
● Sulfa drug (anti-inflammation)
Pseudo substrate competitive inhibitor
● Protease inhibitor Alzheimer's disease
Plaques in brain contains protein inhibitor
● HIV protease is critical to life cycle of HIV
HIV protease (homodimer): subunit 1 subunit 2
↑inhibitor is used to treat AIDS Asp Asp Symmetry
→ Human aspartyl protease: domain 1 domain 2 Not
symmetry
(monodimer) Asp Asp

118. Signal Transduction Network (Ras vs. P53)Signal
Cell function are controlled by protein interactions Receptor
Cell membrane
Cytosol Regulator protein Ras
Signal protein Effector
Transcription enzyme
Ribosome
Inhibitor
Apoptosis
Nucleus
P53 E2F
mRNA Transcription
factor Target gene
Transcription
mRNA Cell division ON
Juang RH (2007) BCbasics

119. GP kinase Phosphatase
GP b
GP kinase P
GP a
Glycogen synthase
Glucagon
Glycogen
Glycogen synthase P
A PKA
Protein phosphatase-1
Protein phosphatase-1 P
Protein phosphatase inhibitor-1
active Protein phosphatase inhibitor-1 P
inactive

120. Classification of Proteases
Family Example Mechanism Specificity Inhibitor
2+
Metal Carboxy- Zn
H196 Non- EDTA
E72 H69
Protease peptidase A polar EGTA
Aromatic DFP
S195-O-
Serine Chymotrypsin
H57 TLCK
Protease Trypsin D102 Basic TPCK
C25-S-
Cysteine Non- PCMB
Papain H195 specific Leupeptin
Protease
Aspartyl Pepsin D215 Non-
H2O
specific Pepstatin
Protease Renin D32
Juang RH (2004) BCbasics

123. vo
Sigmoidal Curve Effect
Noncooperative
(Hyperbolic)
Positive effector
ATP (ATP)
Sigmoidal curve CTP brings sigmoidal
curve
Cooperative
back to hyperbolic
(Sigmoidal)
Negative effector
(CTP)
keeps
vo
Exaggeration of
sigmoidal curve
yields a drastic Consequently,
zigzag line that Allosteric enzyme
shows the On/Off can sense the
point clearly concentration of
the environment and
adjust its activity
Off On
[Substrate] Juang RH (2004) BCbasics

125. INDUCTION
Induction is effected through the
process of derepression.
The inducer will relieve the repression
on the operator site.
In the absence of glucose,the
enzymes of Lactose metabolism will
increase thousand times.
Insulin is Inducer of Hexokinase
Enzyme.
Barbiturates induce ALA Synthase.

126. REPRESSION
Inhibition and repression
reduce the Enzyme Velocity.
In case of Inhibition the
Inhibitor act directly on the
Enzyme.
Repressor acts at the gene
level,effect is noticed after a
lag period of Hours or Days.

127. CO VALENT MODIFICATION
Activity of Enzyme can be
increased or decreasd by
co-valent modifications
Eg.Either addition of group
or Removal of group
Zymogen activation by
partial proteolysis is an Eg.
Of co-valent modification

128. ADP RIBOSYLATION
 It is a type of co-valent modification.
 ADP-Ribose from NAD is added to
enzyme/Protein.
 ADP Ribosylation of Alfa Sub unit of G Protein
leads to Inhibition of GTPase activity;hence G
protein remains active.
 Cholera toxin & Pertussive toxin act through
ADP-Ribosylation.
 ADP Ribosylation of Glyeraldehyde 3P-
Dehydrosense,result in inhibition of glycolysis.

129. Regulation of Enzyme Activity
Inhibitor Proteolysis
or proteolysis
o I I x x o
S I I S
inhibitor
Feedback regulation Phosophorylation
o R x x P o
S R S P
(+)
regulator
effector phosphorylation
Signal transduction
Juang RH (2004) BCbasics
A or A
x o
+
Regulatory cAMP or
S
subunit calmodulin
(-)

130. REGULATION OF ENZYMES
‘’The action of enzymes can
be activated or inhibited so
that the rate of enzyme
productin responds to the
physiologcal need of the cell
done to achieve cellular
economy’’

131. 1. Allosteric Regulation.
2. Activation of Latent
Enzyme.
3. Comprtmentation of
Enzymes of different
Pathways.
4. Control of Enzyme Synthesis.
5. Enzyme Degradation.

132. CHANGE IN CATALYTIC
EFFICIENCY OF ENZYME
 Catalytic effeciency is regulated is modulated by
 A. Allosteric Regulation.
 B. Covalent modification .
 A. ALLOSTERIC REGULATION: Here the site is
different from substrate binding site, this site is
called ALLOSTERIC SITE.
 Low molecular wt. substances bind at site other
than catalytic site,these are called ALLOSTERIC
MODULATORS.Location is called allosteric site.

133. Examples of Second
messengers are
cAMP,cGMP and calcium
etc.
These can change the
enzyme conformation that
may alter either Km or
Vmax.
Based on this effect they
are classified in to two

134. cAMP Controls Activity of Protein Kinase A
Regulatory A
A Active kinase
subunits A cAMP
A
A C
R C R
Alberts et al (2002) Molecular Biology of the Cell (4e) p. 857, 858
A
A
R C Catalytic
R C
A
subunits
Nucleus CREB
Activation
P C
Gene
CREB expression
DNA ON

136. EXAMPLE OF 2 nd
MESSENGER
GLYCOGEN GLYCOGEN
BREAKDOW SYNTHESIS.
N.

137. A.Activator A.Inhibitor .
 Allosteric  AllostericInhibitor .
Activator .  Glucose-6-P,ATP
 Hexokinase: ADP  Glucose-6-P,ATP
 Isocit.Dehydr. ADP  ATP, NADH.
 Glu.Dehy. ADP
 Pyruvate Carboxylase  ADP
 Acetyl CoA

138.  HOMOTROPIC EFFECT : If the
effector
 Substace is substrate itself it is
called homotropic effect.
 HETEROTROPIC EFFECT : Effector
molecule is a substance other than
substrate.
 SECOND MESSENGER:Binding of
many hormones to their surface
receptor induce a change in
enzyme catalysed reaction by
inducing the release of allosteric
effector.These effector substances
are called as 2 nd messenger
 Hormone is first messenger.
Cont……

139. CONFERMATIONAL
CHANGES IN
ALLOSTERIC ENZYMES .
 Most of Enzymes are oligomeric,
binding of effector moecule at the
allosteric site brings a chage in the
active site of enzyme leading to
inhibition or activation.
 Allosteric Enzyme exhist in two
states.
 A. Tense (T)
 B. Relaxed (R )
 Both are in equlibrium.

140. Allosteric Enzyme ATCase
Active relaxed form
Carbamoyl Aspartate Carbamoyl aspartate
phosphate
COO- COO-
- - -
- - -
O CH2 O CH2
+
=
=
H2N-C-O-PO32- HN-C-COO- H2N-C- N-C-COO-
ATCase
-
-
HH HH
Quaternary structure ATP
Feedback
CCC Catalytic subunits inhibition
CTP CTP CTP
R R R
CTP
Regulatory subunits CTP CTP CTP
R R R
CCC Catalytic subunits Nucleic acid
Inactive tense form
Juang RH (2004) BCbasics
metabolism

141. The switch: Allosteric
inhibition
Allosteric means “other
site”
Active site
E
Allosteric
site
© 2008 Paul Billiet ODWS

142. The allosteric site the enzyme
“on-off” switch
Active
site
E Allosteric
Substrate site empty Conformational E
fits into change Inhibitor
the active molecule
Substrate
site is present
cannot fit
The inhibitor into the
molecule is active site Inhibitor fits
absent into allosteric
site
© 2008 Paul Billiet ODWS

143. The Reception and Transduction of Signals
Gilman, Rodbell (1994) G-protein-linked Receptor
Glucagon
Adenylate cyclase
+ Signal
   
-GDP    
GTP GTP GDP GDP
+GTP
G protein
A Glycogen breakdown
The third group: Insulin Enzyme-linked Receptoz
Ion-channel-linked Receptor 
+ Signal
Juang RH (2007) BCbasics
Glycogen Activation P kinase P 
Synthase P P
Protein
active Phosphatase
P P
Glycogen
SH2
Synthase Glycogen domain

144. vo
Sigmoidal Curve Effect
Noncooperative
(Hyperbolic)
Positive effector
ATP (ATP)
Sigmoidal curve CTP brings sigmoidal
curve
Cooperative
back to hyperbolic
(Sigmoidal)
Negative effector
(CTP)
keeps
vo
Exaggeration of
sigmoidal curve
yields a drastic Consequently,
zigzag line that Allosteric enzyme
shows the On/Off can sense the
point clearly concentration of
the environment and
adjust its activity
Off On
[Substrate] Juang RH (2004) BCbasics

145. FEED BACK INHIBITION
 Enzyme is inhibited by end product of
reaction.
 A-B-C-D-E-F……….P.
 P product will inhibit the enzyme which
converts A in to B.

147. COVALENT
MODIFICATIONS
 Two well known processes
 A. PHOSPHORILATION.
 B. PARTIAL PROTEOLYSIS.
 A. Phosphorilation-
dephosphorilation:many enzymes are
regulated by ATP dependent
phosphorilation.Eg. Of
Serine,Threonine,and tyrosine,catalysed
by protein kinases.

148. PARTIAL PROTEOLYSIS
 Some enzymes are secreted as inactive
precursors called Proenzymes or
Zymogens.
 This convertion takes place as a selective
proteolysis.
 It is ir-reversible process
 Pepsinogen to pepsin
 Trypsinogen to trypsin.

149. Hexokinase have low KM High
affinity for Glucose ie glucose
will provide to the vital organs
even at low glucose levels.
Lab. Significance: The sub.
Conc. Kept at saturation point
at least 10 times the Km so
that reaction proceeds to
completion.
Clinical Significance: The Km
value for the given enzyme
may differ from person to

150. INHIBITORS

151. Inhibitors
 Inhibitors are chemicals that reduce the rate of
enzymic reactions.
 The are usually specific and they work at low
concentrations.
 They block the enzyme but they do not usually
destroy it.
 Many drugs and poisons are inhibitors of
enzymes in the nervous system.
© 2007 Paul Billiet ODWS

152. The effect of enzyme
inhibition
 Reversible inhibitors: These can be
washed out of the solution of enzyme by
dialysis.
There are two categories
A. Competitive Inhibition.
B. Non Competitive Inhibition.
© 2008 Paul Billiet ODWS

153. COMPETITIVE INHIBITION
 There is close structural resemblance of
Inhibitor with the Substrate.
 Example:
 1.Malonate ions Inhibit Succinate
Dehydrgenae.
 2.Xanthene Oxidase is inhibited by
Allopurinol.

154. The effect of enzyme
inhibition
2. Non-competitive: These are not influenced
by the concentration of the substrate. It inhibits
by binding irreversibly to the enzyme but not
at the active site
Examples
 Cyanide combines with the Iron in the enzymes
cytochrome oxidase
 Heavy metals, Ag or Hg , combine with –SH
groups.
These can be removed by using a chelating agent
© 2008 Paulsuch as EDTA
Billiet ODWS

155. The effect of enzyme
inhibition
 Irreversible inhibitors: Combine with
the functional groups of the amino acids
in the active site, irreversibly
Examples: nerve gases and pesticides,
containing organophosphorus, combine
with serine residues in the enzyme
acetylcholine esterase
© 2008 Paul Billiet ODWS

156. Enzyme Inhibition (Mechanism)
I Competitive I Non-competitive I Uncompetitive
Substrate E
Cartoon Guide
S S E I
S X
E S I I
I
Compete for S I
Inhibitor active site Different site
E + S ← ES → E + P
→ E + S ← ES → E + P
→ E + S ← ES → E + P
→
Equation and Description
+ + + +
I I I I
↓↑ ↓↑ ↓ ↑ ↓↑
EI EI + S →EIS E IS
[I] binds to free [E] only, [I] binds to free [E] or [ES] [I] binds to [ES] complex
and competes with [S];
complex; Increasing [S] can only, increasing [S] favors
increasing [S] overcomes
not overcome [I] inhibition. the inhibition by [I].
Inhibition by [I].
Juang RH (2004) BCbasics

157. Enzyme Inhibition (Plots)
I Competitive I Non-competitive I Uncompetitive
Vmax Vmax Vmax
vo vo
Direct Plots
Vmax’ Vmax’
I I I
Km Km’ [S], mM Km = Km’ [S], mM Km’ Km [S], mM
Vmax unchanged Vmax decreased
Both Vmax & Km decreased
Km increased Km unchanged
Double Reciprocal
1/vo I 1/vo I 1/vo
I
Two parallel
Intersect lines
at Y axis 1/ Vmax Intersect 1/ Vmax 1/ Vmax
at X axis
1/Km 1/[S] 1/Km 1/[S] 1/Km 1/[S]
Juang RH (2004) BCbasics

158. Competitive Inhibition
Product Substrate Competitive Inhibitor
Succinate Glutarate Malonate Oxalate
C-OO- C-OO- C-OO- C-OO- C-OO-
C-H H-C-H H-C-H H-C-H C-OO-
C-H H-C-H H-C-H C-OO-
C-OO- C-OO- H-C-H
C-OO-
Succinate Dehydrogenase

159. Applications of inhibitors
 Negative feedback : end point or end
product inhibition
 Poisons snake bite, plant alkaloids and
nerve gases
 Medicine antibiotics, sulphonamides,
sedatives and stimulants
© 2008 Paul Billiet ODWS

160. Enzyme pathways
Cell processes (e.g. respiration or photosynthesis) consist
of series of pathways controlled by enzymes
eA eB eC eD eF
A B C D E F
Each step is controlled by a different enzyme (eA, eB, eC etc)
This is possible because of enzyme specificity
© 2008 Paul Billiet ODWS

161. End point inhibition
 The first step (controlled by e A ) is often
controlled by the end product (F )
 Therefore negative feedback is possible
A eA B eB C eC D eD E eF F
Inhibition
 The end products are controlling their own rate
of production
 There is no build up of intermediates (B, C, D
and E)
© 2008 Paul Billiet ODWS

162. ATP is the end point
 This reaction lies near the beginning of
the respiration pathway in cells
 The end product of respiration is ATP
 If there is a lot of ATP in the cell this
enzyme is inhibited
 Respiration slows down and less ATP is
produced
 As ATP is used up the inhibition stops
and the reaction speeds up again
© 2008 Paul Billiet ODWS

163. The switch: Allosteric
inhibition
Allosteric means “other
site”
Active site
E
Allosteric
site
© 2008 Paul Billiet ODWS

164. Switching off
 These enzymes
have two
receptor sites
 One site fits the Inhibitor
substrate like Substrate molecule
other enzymes cannot fit into
 The other site fits the active site
Inhibitor fits into
an inhibitor allosteric site
molecule
© 2008 Paul Billiet ODWS

165. The allosteric site the enzyme
“on-off” switch
Active
site
E Allosteric
Substrate site empty Conformational E
fits into change Inhibitor
the active molecule
Substrate
site is present
cannot fit
The inhibitor into the
molecule is active site Inhibitor fits
absent into allosteric
site
© 2008 Paul Billiet ODWS

166. A change in shape
 When the inhibitor is present it fits into its
site and there is a conformational
change in the enzyme molecule
 The enzyme’s molecular shape changes
 The active site of the substrate changes
 The substrate cannot bind with the
substrate
© 2008 Paul Billiet ODWS

167. Negative feedback is
achieved
 The reaction slows down
 This is not competitive inhibition but it is
reversible
 When the inhibitor concentration
diminishes the enzyme’s conformation
changes back to its active form
© 2008 Paul Billiet ODWS

168. Phosphofructokinase
 The respiration pathway
accelerates and ATP (the final
product) builds up in the cell
 As the ATP increases, more and
more ATP fits into the allosteric
site of the phosphofructokinase
molecules
 The enzyme’s conformation
changes again and stops
accepting substrate molecules in
© 2008 Paul Billiet ODWS

172. Competitive Inhibition
Product Substrate Competitive Inhibitor
Succinate Glutarate Malonate Oxalate
C-OO- C-OO- C-OO- C-OO- C-OO-
C-H H-C-H H-C-H H-C-H C-OO-
C-H H-C-H H-C-H C-OO-
C-OO- C-OO- H-C-H
C-OO-
Succinate Dehydrogenase
Adapted from Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.49

173. Sulfa Drug Is Competitive Inhibitor
Domagk (1939)
Para-aminobenzoic acid (PABA)
Bacteria needs PABA for
H2N- -COOH
the biosynthesis of folic acid
Folic Tetrahydro-
Precursor acid folic acid
Sulfa drugs has similar
H2N- -SONH2 structure with PABA, and
inhibit bacteria growth.
Sulfanilamide
Sulfa drug (anti-inflammation)

174. Enzyme Inhibition (Plots)
I Competitive I Non-competitive I Uncompetitive
Vmax Vmax Vmax
vo vo
Direct Plots
Vmax’ Vmax’
I I I
Km Km’ [S], mM Km = Km’ [S], mM Km’ Km [S], mM
Vmax unchanged Vmax decreased
Both Vmax & Km decreased
Km increased Km unchanged
Double Reciprocal
1/vo I 1/vo I 1/vo
I
Two parallel
Intersect lines
at Y axis 1/ Vmax Intersect 1/ Vmax 1/ Vmax
at X axis
1/Km 1/[S] 1/Km 1/[S] 1/Km 1/[S]
Juang RH (2004) BCbasics

175. The effect of enzyme
inhibition
 Irreversible inhibitors :
Combine with the functional groups of the
amino acids in the active site, irreversibly.
Examples: nerve gases and pesticides,
containing organophosphorus, combine
with serine residues in the enzyme
acetylcholine esterase.
© 2007 Paul Billiet ODWS

176. The effect of enzyme
inhibition
 Reversible inhibitors : These
can be washed out of the solution of
enzyme by dialysis.
There are two categories.
© 2007 Paul Billiet ODWS

177. The effect of
enzyminhibition
1. Competitive :
These compete
with the
substrate E+I EI
molecules for
the active site.
Revers Enzyme
The inhibitor’s
action is ible inhibitor
proportional to reactio complex
its
concentration. n
Resembles the
substrate’s
structure
closely.
© 2007 Paul Billiet ODWS

178. CLINICAL APPLICATIONS OF
COMPETITVE INHIBITORS
DRUG ENZYME TRUE Clinical
SUB. App.
ALLOPURI XANTHINE HYPOXAN GOUT
NOL OXIDASE THENE
SULFONA Dihydro PABA ANTIBIOTI
MIDE pteroate C
Synthase
ETHANOL Al.Dehy. METHANO METHANO
L L
POISONIN
G

179. NON-COMPETITIVE
2. Non-competitive: These are not
influenced by the concentration of the
substrate. It inhibits by binding
irreversibly to the enzyme but not at the
active site.
Examples
 Cyanide combines with the Iron in the
enzymes cytochrome oxidase.
 Heavy metals, Ag or Hg, combine with –
SH groups.
These can be removed by using a
chelating agent such as EDTA.
© 2007 Paul Billiet ODWS

182. Applications of inhibitors
 Negative feedback : end point or end
product inhibition
 Poisons snake bite, plant alkaloids and
nerve gases.
 Medicine antibiotics, sulphonamides,
sedatives and stimulants
© 2007 Paul Billiet ODWS

183. ISOENZYMES
OR
ISOZYMES

184. ISOENZYMES
 Isoenzymes or Isozymes are physically
distinct form of same enzyme having
same specificity, but are present in
different tissues of same organism, in
different cell compartment.
 Useful for diagnosing diseases of different
organs.
 Homomultimer:All the units are same.
 Heteromultimer:Sub units are
different.These are produced by different
genes.

185. IDENTIFICATION OF
ISOZYMES
1.Agar gel or PAGE.They have
different mobility.
2.Heat stability.
3.Inhibitors.Isozymes may be
sensative to different
inhibitors.eg.tartrate labile.
4. Km value or substrate
specificity. Eg.Glucokinase has
high Km and Hexokinase has low
Km for Glucose.

186. 5.Co-Factors.Eg Mitochondrial
isocitrate dehydrogenase is NAD
dependent,Cytoplasmic isocitrate
dehydrogenase is NADP dependent.
6. Localisation: Lactate
DehydrogenaseH4 heart,M4 Muscles.
7.Specific antibodies identify
sp.Isozyme.

187. Isoenzy Composit Compositi Present in Elevated in
me ion on
name
LDH1 ( H 4) HHHH Myocardiu myocardial
m, RBC infarction
LDH2 (H 3 M 1 ) HHHM Myocardiu
m, RBC
LDH3 (H 2 M 2 ) HHMM Kidney,
Skeletal
muscle
LDH4 (H 1 M 3 ) HMMM Kidney,
Skeletal
muscle
LDH5 (M 4 ) MMMM Skeletal Skeletal
muscle, muscle
Liver and liver

188. Isoenzymes
 Isoenzymes
catalyze the
same reaction in
different tissues
in the body.
 Lactate
dehydrogenase,
which converts
lactate to
pyruvate, (LDH)
consists of five
isoenzymes.

189. Isoenz Composi Present Elevated
yme tion in in
name
CNS
CK-1 BB Brain
diseases
Myocar Acute
CK-2 MB dium/ myocardi
Heart al
infarction
Skeletal
CK-3 MM muscle,
Myocar

190. DIAGNOSTIC
SIGNIFICANC
E

191. DISORDERS DIAGNOSED BY
ENZYMES
1) Cardiac Disorders. 4) Bone Disorders.
2) Hepatic Disorders. 5) Pancreatic
Disorders.
3) Skeletal Muscle 6) Salivary gland
Disorders. diseae (Mumps)
7) Malignancies

192. Plasma enzymes are of two types:
1. A small group of enzymes secreted
into the blood by certain cells e.g.
the liver secretes zymogens (inactive
form of enzymes) of blood
coagulation.
2. FUNCTIONAL: Lipoprotein
lipase,Pseudocholine estrase,blood
coagulation.
3. NON FUNCTIONAL ENZYMES:

193. 2. A large group of enzymes are
released from cells during normal
cell turnover.
These enzymes function
intracellularly (inside cells) and
have no function in the blood.
In healthy individuals, the blood
levels of these enzymes are
constant, as the rate of release
from damaged cells into blood is
equal to the rate of removal of
enzymes from blood.

194. Elevated enzyme activity
in blood indicates tissue
damage (due to increased
release of intracellular
enzymes).

195. A. Plasma Enzymes as diagnostic
tools
 Diseases that cause tissue damage
result in increased release of
intracellular enzymes into the plasma.
 Determination of the level of these
enzymes is used for diagnosis of
heart, liver, skeletal muscle, etc.
 The level of these enzymes in plasma
correlates with the extent of tissue
damage.

196. CREATINE KINASE(CPK OR
CK)
 Found in Heart, Skeltol Muscles,Brain
small amounts are also found in lungs,
thyroid and Adrenal glands.
 Not found in RBC so haemolysis no effect.
 NORMAL SERUM LEVELS:
 10-50 IU/L at 30 degree Centigrade.

197. CREATINE
KINSE……………………
 RAISED LEVELS ARE FOUND IN :
 1.Myocardial Infarction.
 2. Crushing Muscular Injury.
 3. Damage to cardiac muscle (Any region)
 4. Brain Injury.
 5. Hypothyroidism.
 6. Hypokalemia
 Highest level in 3-6,peak 24-30 hours normaml
in 3days.

198. ISOENZYMES OF (CPK)
 1.BB (CPK1) Tissue. is of origin is
Brain,Maximum Electrophoretic mobility,
presence in blood is 0%.
 MB (CPK2) Found in heart muscles,
Intermediate electrophoretic mobility,
presence in blood is 0-3%.
 MM (CPK 3) Found in skeltol
muscles,Least electrophoretic mobility, in
blood its conc. Is 97-100 %.

199. Myocardial muscle is the only
tissue that contains high level
of CK2 (MB) isoenzyme.
Appearance of CK2(MB) in
plasma is specific for heart
infarction.
Following an acute myocardial
infarction,CK2appears in
plasma 4-8 hours following
onset of chest pain (peak is

201. ASPARTATE AMINO
TRANSFERASE
 AST
(SGOT)SERUM GLUTAMATE
OXALOACETATE TRANSFERASE
 NORMAL LEVELS: 0-41 IU/L
 Rises in 12 hours ,Peak levels 24 hours
 Returns to normal 3-5 days.

202. ALANINE TRANSAMINASE
(ALT)
 ALT Highest conc. In Liver and next is
skeltol muscles.
 Raised levels are found in liver diseases
and muscle disorders.
 Marked elevation are found in acute
hepatitis and other liver diseases.

203. The presence of increased levels
of some enzymes in plasma is
diagnostic to damage of a
particular tissue;
e.g. The enzyme alanine
aminotransferase (ALT) is
abundant in the liver and the
appearance of elevated levels of
ALT in plasma indicates damage
to the liver.

204. ALKALINE
PHOSPHATASE (ALP)
 Works at optimum pH 9.
 Highest conc. Are found in Liver,
Bone,Intestine and Placenta.
 Diagnosis of Bone and Liver Pathology
 Metastatic or Primary Malignant may
increase the enzyme activity.
 It has many Iso-enzymes.
 Cont…..

205. ISOENZYMES OF (ALP)
 1. Alfa-1 ALP : Biliary canaliculi raised activity
shows obstructive jaundice.
 2. Alfa-2 ALP Its levels rises in Hepatitis.
 3.Pre Beta ALP: Bone cells, Bone diseases
raised levels are found.
 4. Gama ALP :Found in Intestinal cells. Levels
rise in Ulcerative colitis.
 5. Distinct Type: levels rise in
Lymphomas,Decrease in Chronic myl.Leuk.
 6. Regan Isoenzyme:Cancer of lung,liver, Gut.

206. GAMA GLTAMYL
TRANSPEPTIDASE
(GGT)
 Itis a sensative indicator of liver diseases,
especially of alcoholism.
 There are no other serum enzyme
abnormalities.

207. ACID PHOSPHATE
 Exhists at pH 5-6.
 Diagnosis of Carcinoma of Prostate.
 Also found in RBC.
 Used as cancer Marker.

208. LACTATE
DEHYDROGENASE (LDH)
 Enzyme of anaerobic glycolysis.
 Liver,Myocardium, RBC.
 It is a tetramer made up of four units.
 These units can be separated by
electrolysis.
 There are two sub units (H&M)

209. Isoenzymes
 Isoenzymes
catalyze the
same reaction in
different tissues
in the body.
 Lactate
dehydrogenase,
which converts
lactate to
pyruvate, (LDH)
consists of five
isoenzymes.

210. LDH (ISOENZYMES)
 LDH 1 Tetramer of  Moves fastest at
four units.30% in S. pH8.6,myocardium
,RBC
 LDH  Myocardium RBC .
2. 35% in
Serum.
 LDH 3. 20%  Brain Kidney
 LDH 4 10%  Skeltol muscles ,Liver
 LDH 5 5%  ----- Do -----

211. AMYLASE / LIPASE
 Digestive enzymes,exocrine pancreas.
 Levels rise in Acute Pancreatitis.
 Patient present with severe abd. Pain.
 Lipase levels are raised in Intestinal
infarction,Pertonitis or Perforation.

212. CHOLINESTRASE
 Secreted by hepatic cells.
 Always present in serum.
 Metabolism of drugs cocaine and succinyl-
choline.

213. TRYPSIN
Raised levels of Trypsin in plasma
occurs during acute stage of
PANCREATITIS
Along with Amylase and Lipase.
It is a more reliable index of
Pancreatic disease rather than
Amylase/Lipase

214. Intracellular Distribution of
Diagnostic Enzymes
Liver Hea Pancre Saliva Bon Muscl Bilia Prosta
rt as ry e e ry te
Glands Trac
t
LD5 LD1 LPS AMS AL CK ALP ACP
ALT AST AMS P GGT
AST CK

215. DISORDERS DIAGNOSED BY
ENZYMES
1) Cardiac Disorders. 4) Bone Disorders.
2) Hepatic Disorders. 5) Pancreatic
Disorders.
3) Skeletal Muscle 6) Salivary gland
Disorders. diseae (Mumps)
7) Malignancies

216. NAME OF THE Conditions in which
ENZYME level of activity in
serum is elevated
Aspartate Amino Myocardial infarction,
transferase (AST) Liver disease
Serum glutamate- especially with liver
oxaloacetate cell damage
transaminase
(SGOT)
Alanine Amino Liver disease
transferase (ALT) especially with liver
Serum glutamate- cell damage

217. Isoenzymes
 Isoenzymes
catalyze the
same reaction in
different tissues
in the body.
 Lactate
dehydrogenase,
which converts
lactate to
pyruvate, (LDH)
consists of five
isoenzymes.

218. Diagnostic Significance
Enzymes
 The levels of diagnostic
enzymes determine the
amount of damage in
tissues.

221. B. Isoenzymes and Heart
Diseases
 Isoenzymes (or isozymes) are a group of
enzymes that catalyze the same reaction.
 However, these enzymes do not have the
same physical properties (as they differ in
amino acid sequence).
 Thus, they differ in electrophoretic mobility.
 The plasma level of certain isozymes of the
enzyme Creatine kinase (CK) level is
determined in the diagnosis of myocardial
infarction.

222. CARDIAC MARKERS
 CPK (MB)
 LDH (1)
 CARDIAC TROPONIN (I)&(T)
 BRAIN NATRIURETIC PEPTIDE
 (Marker of Ventricular function)
 AST
 ALT

223. Abnormal Liver enzymes
and/or LFTs:
work-up and diagnosis
LIVER
MARKERS

224. Liver Tests
 AST, ALT
 Alkaline Phosphatase
 GGT
 Bilirubin
 Albumin True “liver function tests”
 Protime/INR

225. AST, ALT
Aspartate aminotransferase,
alanine aminotransferase
Enzymes that are in the
hepatocyte and function during
gluconeogenesis
Leak out of the hepatocytes in
times of injury and can be
measured in the serum
Normally present in serum at
levels ~30-40 U/L

226. Alkaline Phosphatase
 Exists in liver in membrane of hepatocyte
where it lines the canaliculus
 Liver > bone > intestine
 Placenta
 Normally changes with age
400
350
300
250
200
150
100
50
5 15 25 35 45 55 65 75 85

227. Other cholestatic
enzymes
 GGT: gamma-glutamyltransferase
Found in hepatocytes and biliary epithelial
cells
 5’ nucleotidase
 Both these enzymes can be used to
confirm alk phos elevation is coming from
liver
 GGT is also sensitive to alcohol ingestion

228. Bilirubin
 Breakdown product of heme
70-80% of normal production is from
breakdown of hemoglobin in senescent RBC
 Conjugation of bilirubin occurs in ER of
hepatocyte, and conjugated bilirubin is
then transported into bile (rate limiting
step)
 Almost 100% of bilirubin in healthy people
is indirect

229. Albumin
 Important plasma protein synthesized by
the liver
 Half-life 20 days
 Levels <3 mg/dL should raise the
suspicion of chronic liver disease
***not specific for liver disease
 Also reduced in heavy alcohol
consumption, chronic inflammation,
protein malnutrition

230. PROSTATE MAR
 PSA (prostate SP.ANTIGEN.
 ACP (Acid Phosphatase)

231. MUSCLE MARKER
 CK (MM)
 AST (Aspartate Amino Transferase)
 ALD (Aldolase)

232. BONE MARKER
 ALP (Alkaline Phosphatase)

233. 1.Cardiac Markers:
e.g. Acute Myocardial Infarction (AMI).
1) The myocardium becomes ischemic and
undergoes necrosis.
2) Cellular contents are released into the
circulation. Blood levels of the following
enzymes increase:
AST LD1 CK

234. 2. Hepatic Disorders
a) Hepatocellular Disorders:
(1) Viral hepatitis: Hepatitis B & Hepatitis C.
(2) Toxic hepatitis: caused by chemicals &
Toxins (e.g aflatoxin, Asp. flavus)
Increased levels of the following enzymes :
ALT AST LD5

235. b) Biliary tract disorders:
The plasma levels of the following
enzymes increase:
ALP GGT

236. 3. Skeletal Muscle Disorders
 Muscle dystrophy.
 Muscle trauma.
 Muscle hypoxia.
 Frequent I.M Injections.
 The plasma levels of the following enzymes
increase:
CK AST

237. 4. Bone Disorders:
1) Paget’s Bone Disease: caused by increased
osteoclastic activity.
2) Rickets
3) Osteomalacia:
The plasma levels of the following enzyme
increase:
ALP

238. 5. Acute Pancreatitis
The plasma levels of the following
enzymes increase:
Lipase AMS

239. 6. Salivary Gland Inflammation:
In Mumps:
The levels of α -Amylase (AMS)
is significantly increased

240. 7. Malignancies
a) Plasma (Acid phosphatase) ACP
levels increase in:
• Prostatic carcinoma.
• Bone metastatic carcinoma

241. b) Plasma levels of Alkaline
phosphatase (ALP) increase in:
• Pancreatic carcinoma.
• Bile duct carcinoma.
• Liver metastasis.

242. c) Plasma levels of Total Lactate
dehydrogenase (LDH) increase
in:
• Leukemia
• Lymphomas.
• Liver metastasis.

243. ENZYMES TUMOUR MARKERS


244. B. Isoenzymes and Heart
Diseases
 Isoenzymes (or isozymes) are a group of
enzymes that catalyze the same reaction.
 However, these enzymes do not have the
same physical properties (as they differ in
amino acid sequence).
 Thus, they differ in electrophoretic mobility.
 The plasma level of certain isozymes of the
enzyme Creatine kinase (CK) level is
determined in the diagnosis of myocardial
infarction.

245.  Many isoenzymes contain different
subunits in various combinations.
 CK occurs in 3 isoenzymes, each is a
dimer composed of 2 subunits (B &
M): CK1 = BB, CK2 = MB and
CK3 = MM, each CK isozyme shows a
characteristic electrophoretic
mobility.

246. Myocardial muscle is the only
tissue that contains high level
of CK2 (MB) isoenzyme.
Appearance of CK2(MB) in
plasma is specific for heart
infarction.
Following an acute myocardial
infarction,CK2appears in
plasma 4-8 hours following
onset of chest pain (peak is

247. Alkaline Phosphatase
1.Alfa1-ALP Liver
2.Alfa2-ALP Liver (Heat
Labile)
3.Pre Beta-ALP (BONES)
4.Gama ALP (Ulcerative
Colitis)
5.Regan ALP (Bronchogenic
cancer)

248. ENZYMES IN OTHER
BODY FLUIDS
Adenosine deaminase in
pleural fluid :Elevated in
Tuberculosis not in
Malignant effusion.
LDH; In CSF,Pleural fluid &
Ascitic Fluid.
Elevated levels in
Malignacy .

249. Enzymes as Therapeutic
Agents
 Dissolving Streptokinase,Urokinase.
 Asparaginase used in some
leukemias.
 Deoxyribonuclease is adminstered
via respiratory route to clear viscid
secretions in pt. of cystic fibrosis.
 Serratiopeptidase is used to
minimise edema in acute
inflamatory conditions.
 Hyaluronidase for hypovolumia
 Hemocoagulase used as hemostat.

250. ENZYMES USED IN
DIAGNOSTICS PROCEDURES
 Urease Urea.
 Uricase Uric Acid.
 Glucose Oxidase Glucose.
 Peroxidase Cholesterol.
 Hexokinase Glucose.
 Lipase Triglycerides.
 Alkaline phosphatase ELISA.
 Restriction endonuclease RFLP

251. Fungal Diastase &Pepsin
1.
Clinical Enzymology Questions
For a biological process to occur a free energy
overcome. Enzymes work in this process to:
a. Lower the free energy of activation
of activation must be
used as digestive enz.
b. Raise the free energy of activation
c. Enzymes have no effect on free energy o f activation
d. The effect on free energy of activation is dependent on the
enzyme in question
e. None of the above
2. CK-M and CK-B are examples of what type of enzyme?
a. Homogeneous enzymes
Ribozymes &Abzymes
b. Isoenzymes
c. Heterogeneous enzymes
d. Co-factors
e. None of the above
3. A 68-year-old male presents to the emergency room with acute mental
confusion. Upon questioning his family members they recall that for
the last several months he has been complaining of tingling and loss
of feeling in his hands and feet, difficulty walking, and vomiting.
Streptodornase; DNA
Which of the following co -factors is he most likely suffering from a
deficiency in?
a. Folic Acid coenzymes
b. Biotin
c. Flavin coenzymes
d. Thiamine pyrophosphate
e. B12 coenzymes
applied locally.
4. Which of the following type of enzyme reaction does not normally
require the use of a cofactor?
a. Oxidation-reduction reaction
b. Group Transfer reactions
c. Isomerizations
d. Hydrolytic reactions
Alpha-1-ant-trypsin;
Emphysema