ENZYME

S
Lelly Yuniarti, S.Si., M.Kes.

1
 What is enzyme?
Itis a biological catalyst accelerating
chemical reactions without itself
undergoing any change.
Terminology; Substrate,
coenzyme(cofactors), Apoenzyme,
prosthetic group,
Holoenzyme = apoenzyme +
prosthetic group
Substrate specificity
2
 Definition of Enzyme
Protein that function in the acceleration of chemical
reaction in biological system.

Enzymes are central to every biochemical

process. Acting in organized sequences, they
catalyze the hundreds of stepwise reactions that
degrade nutrient molecules, conserve and
transform chemical energy, and make biological
macromolecules from simple
precursors.
3
 NATURE OF ENZYMES
They are biological catalyst
They act at very low concentration and
increase the rate of chemical reactions
Most of the enzymes (not all) are proteins.
Then, it can be destroyed by heat, by
strong acid, or by strong alkalies
Enzymes convert substrates into
products.

4
 SOME OTHRE POINTS OF
ENZYMES

1. They are produced by living cells. But, its

activities are not depend on the cells. Then,
enzymes can work ex vivo/in vitro.

2. The presence of non protein moieties (called:

co factors/co enzymes/prosthetic groups) is
required for the activation of the enzyme.
But some of the enzymes (E.G.: Pepsin &
trypsin) need no requirement of co enzyme.
5
Enzyme molecule consists of
apo enzyme, an non protein
moiety, co enzyme
Co enzyme
(Non protein)

Apo enzyme
(Protein)

Holo Enzyme

6
 Apo enzyme : an enzyme or a part
of an enzyme that consists of
polypeptide chains alone, lacking
required non protein co factors

Holoenzyme : an enzyme that

has all component parts, including
apo enzyme & co enzyme

7
 An enzyme molecule is often very
much larger than a molecule of its
substrate
Enzymes have molecular weights
ranging from 10,000 to millions,
whereas substrate are usually in the
hundreds
Mostly, anyone enzymes will act on
the substrate
It show specificity
8
 An enzyme molecule has an active
group (functional group) which attacks
the substrate molecule
This substrate molecule fit on the
enzyme at a particular site, called: the
active site or binding site or catalytic
site
Functional Group: One of the groups of
atoms that give rise to the characteristic
reactions of organic compounds

9
The Architecture of The Active Site

Activesite : the part of an enzyme in which

the substrate binds an at which the reaction
takes place.

The active site consists of a definite

arrangement of the amino acid side chains
which are responsible for the catalytic
activity of the enzyme.

An enzyme molecule consists of polypeptide

chains which usually form secondary, or
tertiary, or quaternary structure
10
11
Binding of a substrate
to an enzyme at the
active site

12
13
 The interaction between enzyme
& substrate can be explained by
two models:
I. Fischer Lock and Key Hypothesis:
The active site has rigid structural features
which are complementary to the substrate

II. Koshland Induced Fit Hypothesis:

The active site is flexible, possessing a
structure complementary to that of
substrate only when the substrate is bound
to the enzyme
14
 Fisher Lock Key Model

S
a b c
+
a c
b
E ES -
Complex

Active Site is Flexible 15

Koshland Induced Fit Model

s
u
b
s
t a b c
r
a +
t
e
ES -
Complex
a b c
Enzyme

Active Site is 16
 These
models explain some aspects of
enzyme specificity

That is:
Enzyme exhibit chemical and
stereochemical specificity

17
Enzymes exhibit specificity,
because:
The conformation of the complex
protein molecule
The uniqueness of its active site
The structural configuration of the
substrate molecule.

18
 Chemical Specificity
Can be divided into two types of specificity:
1. Group Specificity:
Enzyme act on several different, but closely
related substrates
E.g.: Alcohol dehydrogenases catalyse the
oxidation of a variety of alcohol
2. Absolute Specificity:
Enzyme act only on one particular substrate.
e.g.: Glucokinase catalyse the transfer of
phosphate from ATP only to glucose.
19
Stereochemical Specificity

Enzyme will act on substrate(s) with

specific stereochemical form

20
 The Naming and
The Classification of
Enzymes
The naming of enzymes
The names of enzymes usually indicate
the substrate involved (with the ending:
-ase)
The Classification of Enzymes
The E.C of I.U.B (Enzyme Committee of
International Union of Biochemistry)
divided enzymes into six main classes,
on the basis of the reaction catalyzed.
21
 Enzyme Class Type of Reaction
Catalyzed
1. Oxidoreductases Oxidation/Reduction reactions
2. Transferases Transfer of an atom or group between
two molecules
3. Hydrolases Hydrolysis reactions
4. Lyases Removal of a group from substrate
5. Isomerases Isomerations reactions
6. Ligases The synthetic joining of two molecules
coupled with the breakdown of
pyrophosphate bond in nucleotide
triphosphate

22
23
 Enzyme catalyzed
reactions
E + S ES E + P

1. The first step: the formation of ES-complex

2. The formation of ES leads to the formation
of P
3. The E is regenerated at the end of the
reactions
24
Enzymes are affected by
several factors.

These are:
1. Temperatures
2. pH
3. The concentration of the enzyme
4. The concentration of the substrate
5. The presence of inhibitors
6. The presence of the other factors
25
The Effect of Temperature

The rates of the enzymatic reactions

increase with increasing temperature,
until the enzyme is destroyed
(denatured) and catalytic activity is
lost.

26
The effect of temperature
Q10 (the temperature coefficient) = the
increase in reaction rate with a 10C rise in
temperature.
For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or triples with
every 10C 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.
27
2007 Paul Billiet ODWS
The effect of temperature

Denaturat
Q10 ion
Enzyme
activity

0 1 2 3 4 5
0 0 0 0 0
28
2007 Paul Billiet ODWS
Temperature / C
The effect of temperature
For most enzymes the optimum temperature
is about 30C
Many are a lot lower,
cold water fish will die at 30C because their
enzymes denature
A few bacteria have enzymes that can
withstand very high temperatures up to
100C
Most enzymes however are fully denatured at
70C
29
2007 Paul Billiet ODWS
 The Effect of pH
By changing of pH, the tertiary
structure of the enzyme is disrupted.
Then, the enzyme denatured.

30
 The effect of pH

Optimum pH values

Enzyme
activity Trypsin

Pepsin
1 3 5 7 9 11
2007 Paul Billiet ODWS pH 31
 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
enzymes optimum value, small changes in the
charges of the enzyme and its substrate
molecules will occur
This change in ionisation will affect the binding
of the substrate with the active site.
32
2007 Paul Billiet ODWS
 The Effect of Enzyme Concentration
If the amount of enzyme in a reaction
is doubled, the amount of substrate
converted to product is doubled.

33
The Effect of Substrate Concentration
Doubling of the substrate concentration,
doubles the reaction rates (while the
enzyme concentration is kept
constant)

34
 Substrate concentration: Non-enzymic
reactions

Reaction
velocity

Substrate concentration

The increase in velocity is proportional to the substrate

concentration
35
2007 Paul Billiet ODWS
 Substrate concentration: Enzymic
reactions

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 then 36
Vmax will change too.
37
 The Order of Reaction
1. A First Order Reaction
Is one is proceed at a rate proportional to
the concentration of one reactant.
2. A Second Order Reaction
--------------- to the concentration of two.
reactants
3. A Zero Order Reaction
is one whose rate is independent of the
concentration of any of the reactants.
38
Co Factors
Some enzymes (not all) need a non protein
compound in order to act. These compound
are called: Co factors.

Two Kinds of
enzyme catalyzed reactions

Without requiring Require the Co factors

presence of
Co factors
39
Co Factors
Can be divided into two groups:
1. The prosthetic groups
2. Co enzymes
3. Metal activators

If this compound is tightly bound to the enzyme

molecule, this non protein part is called the
prosthetic group.
If This compound is loosely bound to the
enzyme molecule, this part is called: the co
enzyme
40
Co Factors
Occurrenc Hard to Easy to
e Dissociate Dissociate
Organic Prosthetic group Co Enzyme

Non Prosthetic group Activators (E.g.:

Organic (E.g.: Heavy Heavy metals)
metals)

41
Some Important Points of Co-
Enzymes

1. They are organic compounds required

by enzymes for catalytic activity
2. They are often vitamins or
derivatives of vitamins
3. Sometimes they can act as catalysis
in the absence of the enzymes
4. They form an integral part of the
active site of the enzyme.
42
Co enzymes are often derivatives of B-
complex vitamins.
B-Complex Vitamins:
1. Thiamine (Aneurin, 8. Inositol
Vit. B1) 9. Choline
2. Lipoic Acid 10. Para Amino Benzoic
3. Riboflavin (Vit. B2) Acid (PABA)
4. Niacin (Nicotinic Acid) 11. Folic Acid
5. Pyridoxine (Vit. B6) 12. Cobamide (Vit. B12)
6. Pantothenic Acid 13. Carnithine
7. Biotin (Vit. H)

43
 From the structural and physiological
point of view, there is no similarity among
the member of B-complex vitamins
The only similarity is:
Almost all of them function as the co
enzyme.
Co enzyme function as group transfer
reagents:

D-G A-G D : Donor

Co. E
G : Group Transfer
Co.E - G A
D A : Acceptor
44
Therefore, Co enzymes can be classified
base on the group whose transfer they
facilitate:
1. For transfer of group other than hydrogen-sugar
phosphate:
CoA.SH (transfer acyl group)
Thyamine pyrophosphate (-- aldehyde group)
Biotin (CO2)
Cobamide coenzymes (--Alkyl group)
Lipoic acid (-- Acyl group)
2. For transfer of hydrogen:
NAD+, NADP+
FMN, FAD
Coenzymes Q
45
 Some co enzymes as
derivatives of B-complex
vitamins
Co Enzyme Derivative of
vitamin
Biocytin Biotin
Cobamide Cobalamine (B12)
FH4 Folic Acid
Nicotinamide Nicotinamide
Pyridoxal phosphate Pyridoxine (B6)
Thyaminpyro Thiamin (B1)
phosphate
Flavin Coenzymes Riboflavin (B2) 46
 NAD & NADP
+ +

Derivatives of nicotinic acid

Act on oxidation/reduction reactions
(that is on H+ and electron transfer) on
pyruvate metabolism, lipid metabolism,
glycolysis, etc
Mostly: NAD+ act on catabolic
metabolism
NADP+ act on anabolic metabolism

47
 Flavinnucleotide (FMN & FAD)
- They are derivative of riboflavin
- They act of oxidation reduction
reactions

Co enzyme A (CoA-SH)
- Contains panthotenic acid
- Act on several metabolic processes: bio
energetic metabolism, lipid metabolism,
carbohydrate metabolism, etc
48
 FH4 (Tetrahydrofolate)
- Derivatives of folic acid
- Acts on: single carbon metabolism and
erythropoesis

TPP (Thiamin Pyrophosphate)

- Derivatives of Thiamin
- Acts on: Oxydative-phosphorilation
process on bioenergetic metabolism, and
on HMP (Hexose Monophosphate) shunt

49
 Cobamide
Important co enzyme on:
erythropoesis, nucleate synthesis and
protein synthesis

PyridoxalPhosphate
- Derivatives of pyridoxine
- Acts on decarboxylation and
transamination process

50
 ENZYME ASSAY
We can measure the concentration of
the enzyme, indirectly, by its activity

The amount of activity is expressed as:

The amount of substrate converted to
product by a known amount of enzyme
in a known period of time.

51
The activity of enzyme is often expressed in
International Unit (I.U)

Itis defined as:

The amount of enzyme causing loss of
one micromole of the substrate per
minute under specified conditions

52
The activity of enzyme can be expressed
also in specific activity.

It is defined as:
Units of enzyme activity related to the
total protein content of the sample
being assayed.
(It is expressed as: one unit/mg
protein)

53
 INHIBITORS
The effect of Inhibitors on Enzyme
Action
There are two main classes of
inhibitors of enzyme action:
1. Irreversible inhibitors
2. Reversible inhibitors

54
1. Irreversible Inhibitors
When the enzyme is exposed to the
inhibitor, it forms a covalent bond which
is very difficult to be broken.
Then the EI-complex is permanently
inactive.
E + I EI E + P
E.g.: DIPF (Diisopropylphosphofluoridate)
potent nerve gas
55
2. Reversible Inhibitors
When the enzyme is exposed to the inhibitor,
it forms a loosely bond which easily
dissociates from the enzyme, leaving its
activity unimpaired.
E + I EI E+P

Reversible Inhibitors are subdivided into two

groups:
1. Competitive inhibitors
2. Non competitive inhibitors
56
Competitive Inhibitors
The inhibitors compete with the
substrate for the same active site on
the enzyme molecule and prevent
binding of substrate, or put differently,
the enzyme molecule recognizes the
inhibitors as substrate, but is unable to
convert it to product.

57
 Competitive inhibition can be overcome by a
high concentration of substrate sufficiently
58
Non Competitive Inhibitors
The inhibitors bind to a site other than
the substrate binding site on the
enzyme.
It result in changing the structure of
the enzyme.

59
 Non competitive inhibition cannot be overcome
by increasing the substrate concentration. 60
Uncompetitive Inhibitors
The inhibitors bind to a site other than the
substrate binding site on the ES complex

E+S ES E+P
+
I

EIS

E+P
61
Iso Enzymes
They are polymeric enzymes, which catalyze
the same chemical reaction, but migrate
differently on electrophoresis.
The mechanism for the formation of Iso
enzyme is:
The arrangement of subunits arising from two
different genetic loci in different combinations
to form the active polymeric enzyme.
Enzyme isoform (=isoenzyme) is an enzyme
that can exist in different forms in different
tissues.
62
Some Enzymes which posses
isoform
Lactate dehydrogenase (LDH)
Creatine Kinase (CK)
Phosphofructokinase

63
LDH
A tetrameric enzyme
Only two distinct subunits have been elucidated,
i.e.: H (heart type) and M (muscle type)
These two subunits are combined in five different
types:
Type Compositio Location
n
LDH1 HHHH Myocardium & RBC
LDH2 HHHM Myocardium & RBC
LDH3 HHMM skeletal muscle &
Kidney
64

LDH4 HMMM skeletal muscle&

CK
A dimer enzyme
Consists of two types of subunit, I.e.: M (muscle
type) and B (Brain type)
These two subunits are combined in three different
types:

Type Compositio Location

n
CK 1 BB Mostly in brain
CK 2 MB Only in myocardium
CK 3 MM Mostly in skeletal 65

muscle
Phosphofructokinase
A tetrameric enzyme
Consists of two types of subunit : M
(nuscle type) and L (liver type)
These two types are combined to form
five tetramer:
M4, M3L, M2L2, ML3, and L4

66
KINTETICS OF THE ENZYME
CATALYZED REACTIONS

NEXT LECTURE

67
 Enzymes: Clinical Application

Diagnosis of disease
Prognosis of disease
Enzymes as therapeutic agents

68
Clinical Diagnosis
The possibility that enzymes can be used as
markers for disease is based on:
1. Some enzymes are found only in specific
or in a limited number of tissues
2. Many enzymes arising from a variety of
sources can be detected in a plasma or
serum.
3. Therefore, an increase of any tissue
specific enzyme in the blood indicates
some kind of tissue damage.
69
4. Some enzymes are secreted into the
plasma and function there. (Called:
Functional Plasma Enzymes)
For this group:
Injury to the organ(s) producing these
enzymes will cause a decrease or even a
lost in activity
E.g.:
- Coagulation enzymes (Thrombin etc)
- Cholinesterase

70
5. Some are intracellular and function there
(Termed: Non Functional Plasma Enzymes)
For this group:
They are only found in plasma in significant
quantities when cells are damaged as a
consequent of the leaky cell membranes.
Thus, the assay of the enzyme activity in this
group will be related to the number of cells
damaged.
These enzymes are useful in clinical
diagnosis, both in the initial stages of the
diseases, and during the period of recovery
and repair.
71
Distribution of enzymes in
tissues and serum patterns
If the concentration of a particular enzyme in a
tissue is normally high, then a damage to a tissue
will cause release into plasma of a high
concentration of this enzyme.
And Vice Versa
The nature of the enzyme released as the
consequence of damage depends on its
subcellular localization
If the damage is minimal, only cytoplasmic
enzymes will leak out.
If the damage is extensively, mitochondrial
enzymes will also be released.
72
73
 Patterns of activities in human
organs

Anyone enzyme can be found in several

organs with unequal amount of
activities.
Examples:
1. Glutamate dehydrogenase can be
found in liver, brain, heart, erythrocyte,
etc. But, this enzyme is much more
active in liver than in heart or in lung.

74
2. Lactate dehydrogenase is found in a
great amount in muscle and in liver,
but is very minimal in lung.

Therefore:
Determination of several enzymes will
be useful to provide information about
the site and extent of pathological
change.

75
Glutamate dehydrogenase activity in human organs

76
Lactate dehydrogenase activity in human organs

77
Examples of the use of serum
enzymes in diagnosis
Enzyme Disease
Glutamate Myocardial Infarction
oxaloacetate
transmainase (SGOT)
Glutamate pyruvate Viral Hepatitis
transaminase (SGPR)
Amylase, Lipase Acute pancreatitis
Creatine kinase Muscle disease
Acid phosphatase Prostate carcinoma
Alkali phosphatase Bone disease 78
 Clinical
Enzymolog
y
79
 Objectives
Listthe clinically important enzymes and
isoenzymes.

Statewhich 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

80
 Enzymes
Biological catalysis
Very efficient can increase reaction
rates at the order of x 10
All are proteins- so liable to
denaturation
Specific to substrates
Partly specific to tissues
Assay by measure of rate of specific
reaction catalyzed by that enzyme
81
 Measurement of serum
enzymes
Diagnostic enzymology
Enzymes are normally intracellular and LOW
concentration in blood
Enzyme release (leakage)in the blood indicates
cell damage (cell death, hypoxia, intracellular
toxicity)
Quantitative measure of cell/tissue damage
Fairly non invasive possible to do repeated tests
Organ specificity- but not absolute
specificity in spite of same gene content.
Most enzymes are present in most cells-
82

differing amounts
Information from enzymes
measurements in serum

Presence of disease
Organs involved
Aetiology /nature of disease:
differential diagnosis
Extentof disease-more damaged
cells-more leaked enzymes in blood
Time course of disease
83
 Enzymes routinely
measured
NAME OF THE ENZYME PRESENT IN

Aspartate Amino transferase (AST) Heart and Liver

Serum glutamate-oxaloacetate
transaminase (SGOT)

Alanine Amino transferase (ALT) Heart and Liver

Serum glutamate-pyruvate
transaminase (SGPT)

Alkaline Phosphatase (ALP) Bone, intestine and other

tissues
Acid Phosphatase (ACP) Prostate
glutamyl Transferase ( GT) Liver
Creatine kinase (CK) Muscle Including cardiac
muscle
Lactate Dehydrogenase (LDH) Heart, liver, muscle, RBC
Amylase Pancreas 84
 Isoenzymes
catalyse same reactions but are formed from
structurally different polypeptides.
They perform the same catalytic function.
Different isoenzymes may arise from different
tissues and their specific detection may give
clues to the site of pathology.
Various isoenzymes of an enzyme can differ in
three major ways:
- enzymatic properties
- physical properties (e.g heat stability)
- biochemical properties such as amino acid
composition and immunological reactivities.
85
 Measurement of enzyme
activity
Enzyme activity is
expressed in International
unit (IU)
It corresponds to the amount of
enzymes that catalyzes the conversion
of one micromole (mol) of substrate to
product per minute

86
 LACTATE DEHYDROGENASE
(LDH)
Pyruvate Lactate (anaerobic glycolysis)

LDH is elevated in myocardial infarction, blood

disorders

It is a tetrameric protein and

made of two types of
subunits namely H = Heart, M = skeletal muscle

It exists as 5 different isoenzymes with various

combinations of H and M subunits

87
Isoenzyme Composition Composition Present in Elevated in
name

LDH1 ( H 4) HHHH Myocardium, myocardial

RBC infarction

LDH2 (H3M1) HHHM Myocardium,

RBC

LDH3 (H2M2) HHMM Kidney,

Skeletal
muscle
LDH4 (H1M3) HMMM Kidney,
Skeletal
muscle
LDH5 (M4) MMMM Skeletal Skeletal muscle
muscle, Liver and liver
diseases
88
 CREATINE KINASE (CK)
Creatine + ATP phosphocreatine +
ADP
(Phosphocreatine serves as energy reserve during muscle
contraction)

Creatine kinase is a dimer made of 2

monomers
occurs in the tissues
Skeletal muscle contains M subunit,
Brain contains B subunits
Three different isoenzymes are formed
89
Isoenzyme
Composition Present in Elevated in
name

CK-1 BB Brain CNS diseases

Acute
Myocardium
CK-2 MB myocardial
/ Heart
infarction

Skeletal
CK-3 MM muscle,
Myocardium

90
 ALANINE TRANSAMINASE (ALT) AND
ASPARTATE TRANSAMINASE( AST)

- Oxoglutarate + L-aspartate - Oxoglutarate + L-alanine

Aspartate Alanine
aminotransferase aminotransferase
(AST) (ALT)
L- glutamate + oxaloacetate L - glutamate +
pyruvate
Alanine transaminase (ALT) and Aspartate transaminase (AST)
enzymes are the most abundantly present in the liver and is
elevated in blood as a result of leakage from damaged cells
Measurement of these transaminases is useful for the diagnosis of liver
diseases
In viral hepatitis the enzyme levels are increased 20-50 times above
the upper limit of the normal range
Alanine transaminase (ALT) increase is specific for liver damage
involving hepatocellular damage
91
Aspartate transaminase (AST) is moderately increased in Muscular
LEVELS OF ENZYMES IN DISEASES
INVOLVING LIVER DAMAGE

In viral hepatitis
Rapid rise in
transaminases (AST &
ALT) in serum occurs
even before bilirubin rise
is seen

92
LEVELS OF ENZYMES IN MYOCARDIAL
INFARCTION
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
HBDH

LDH

CK AST
CK-MB

93
 ALKALINE PHOSPHATASE (ALP)

Is a group of enzymes that have maximal activity

at a high pH 9.0-10.5
Widely distributed throughout the body
High levels are seen is liver, bone, placenta and
intestine and useful to assess hepatobiliary and bone
diseases
In hepatobiliary obstruction,hepatocytes lining the
biliary ducts induces the ALP synthesis.
High levels of ALP is indicative of extrahepatic
obstruction rather than intrahepatic obstruction
In bones, the enzyme is derived from osteoblasts.
Hence increased in bone diseases like rickets,
osteomalacia, neoplastic diseases with bone94
metastates and healing fractures
 ALKALINE PHOSPHATASE (ALP) conti

p-NPP + H2O ALP, Mg2+ p-NP (benzenoid form) + PO43-

pH 10.3 Colorless
Para nitro
phenylphosphate Rearrangement

p-NP (quinonoid form) + PO43-

Yellow

Color read at 405nm

The activity of the bone isoenzyme can be estimated
by heat treating a serum sample at 56oC. The bone
ALP is heat liable and is destroyed or heat
inactivated at this temperature.
Measurement of ALP before and after heat treatment95
gives a measure of bone ALP
 ACID PHOSPHATASE (ACP)
Is a group of enzymes that have maximal activity
at pH 5.0-6.0

It is present in prostate gland, liver, spleen and RBC.

The main source of ACP is prostate gland and so

can be used as a marker for prostate disease.

AMYLASE
Is the digestive enzymes from the pancreas and salivary
glands to digest complex carbohydrates.
Elevated in acute pancreatitis.
It is used as a marker to detect acute pancreatitis AND
appendicitis. 96
 glutamyltransferase ( GT)

Amino acid + Glutathione( GT) -glutamyl amino acid +

Cysteinylglycine

It is involved in aminoacid transport across the membranes.

Found mainly in biliary ducts of the liver, kidney and
pancreas.
Enzyme activity is induced by a number of drugs and in
particular alcohol.

-GT increased in liver diseases especially in obstructive

jaundice.
-GT levels are used as a marker of alcohol induced liver
disease and in liver cirrhosis.
97
 MEASUREMENT OF ENZYMES
Enzymes are measured
End point assay
Kinetic assay

Measurement of enzymes are affected by the presence of

inhibitors or activators.
Hence most of the enzymes are measured by coupled
assay.

A coupled assay is one in which a second enzyme is used

to act on the product of the enzyme of primary interest.
Second enzyme used NADH as coenzyme. The rate can be
followed by measuring oxidation of NADH which can be
done conveniently at 340nm.
98
Principle involved in AST estimation

- Oxoglutarate + L-aspartate
Aspartate
aminotransferase
AST
L- glutamate + oxaloacetate
+
NADH + H+
Malate
dehydrogenase
MDH
L-matate + NAD+

99
MEASUREMENT OF ENZYMES

100

NAME OF THE ENZYME
Aspartate Amino transferase (AST)
Serum glutamate-oxaloacetate
transaminase (SGOT)
Alanine Amino transferase (ALT)
Serum glutamate-pyruvate
transaminase (SGPT)
Alkaline Phosphatase (ALP)
Acid Phosphatase (ACP)
glutamyl Transferase ( GT)
Creatine kinase (CK)
Lactate Dehydrogenase (LDH)
Amylase
Conditions in which level of activity in
serum is elevated
Myocardial infarction, Liver disease especially
with liver cell damage
Liver disease especially with liver cell damage
Liver disease- biliary obstruction
Osteoblastic bone disease-rickets
Prostatic carcinoma
Liver disorder like liver cirrhosis
Myocardial infarction and skeletal muscle
disease(muscular dystrophy
Myocardial infarction, other diseases like liver
disease.some blood diseases
Acute pancreatitis
101
 SUMMARY
Enzymes are biological catalysts present in every cell of the body.
An enzyme will act on a specific substrate yielding a product.
An isoenzyme is a genetic variant produced largely within a specific tissue.
Isoenzyme patterns can give information about organ-specific disease.
Important enzymes in the investigation of heart disease are CK, LDH and AST.
Important enzymes in the investigation of liver disease are AST, ALT, alkaline
phosphatase and GGT.
Creatine kinase has three isoenzymes: CK-MM, CK-MB and CK-BB.
LDH has five isoenzymes.
Alkaline phosphatase can be used in the investigation of liver and bone
disease.
Increased levels of acid phosphatase are found in prostate cancer.
GGT is induced by alcohol and is useful in monitoring alcohol abuse.
Enzyme measurements should be performed using zero order kinetics, i.e.
using excess substrate.
Determinations of enzyme activity can be performed using an end-point or
kinetic method 102
The Fate Of The Plasma
Enzymes
Following release into the plasma, the
enzymes will be eliminated:
1. Excreted via urine (some enzymes with
small molecule weight, like amylase)
2. The majority of enzymes (with high
molecule weight) will be degraded via
normal catabolic pathways at very
different rates.

103
 Half Life Of Several Plasma
Enzymes
Enzyme Half Life
GOT Circa 20 h
GPT 50 h
GLDH 18 h
LDH 140 h
CPK 10 h
Choline Esterase 10 d
Lipase 5h 104
Relationship Between Genetic
Abnormalities and The Enzyme
Activities
Genetics defects could have significant effects on
the synthesis and activities of the enzymes, which
will emerge to the illness
Examples:
1. Deficiency of anti elastase will cause destructive
lung disease
2. Defect of activity of antihaemophilic factor (Factor
VII) will cause haemophilia
3. Defect of activity of tyrosine kinase will cause a
disturbance of the growth and differentiation of
the cells, which will lead to the pathogenesis of
carcinoma mammae.
105
 Enzyme Can Function As Therapeutic
Agents
Examples:
1. Streptokinase is applied in
thrombosis. Mode of action:
Streptokinase

Plasminogen
Plasmin

Fibrin More soluble components 106

2. Asparaginase is applied in leucemia.
Mode of action:
Asparaginase
Asparagin Decrease of asparagin
(Nutrition factor concentration
for tumor cells)

Decrease of asparagin concentration leads

to decrease of tumor cells viability
Another covalent modification
control
(Instead of Phosphorylation):
ADP-ribosylation
Nuleotidylation 107

Methylation
 Enzymes as therapeutic agents

Streptokinase, Urokinase
Blood clotting disorder, myocardial
infarction
L-asparaginase
Acute lymphocytic leukemia
Adenosine deaminase (PEG-conjugated)
Immunedeficiency disease
Interferon alfa-2a (PEG-conjugated)
Chronic hepatitis

108
 Note:
Not all of the enzymes are protein!
e.g.: RNA molecules (I.E. L-19 RNA) function
as ribonuclease and as RNA-polymearse.

L-19 RNA has characteristic as an enzyme:

1. Has a substrate specificity (I.E. on
oligoribonucleotide).
2. Has a saturation phenomenon
3. It follows michaelis menten kinetic.
109