Enzymes

1
• Enzymes are biological catalysts produced by
the living cells and they catalyze several
reactions in the body.
• They are protein in nature.
• They are specific in its action (i.e. each
enzyme can catalyze only one type of
reaction).
• They are required in very small quantities.
• The loss of catalytic activity was observed
when they are subjected to heat or strong
acids or bases or organic solvents.
2
• The enzymes mainly catalyze the metabolic
pathways in the body.
• The deficiency of the enzyme leads to inborn
errors of metabolism.
• Most of the enzymes are produced by the
cells of a particular tissue and function within
that cell. Such en zymes are called
intracellular enzymes (Example: enzymes of
glycolysis, TCA cycle and fatty acid synthesis).

3
• On the other hand there are certain enzymes,
which are produced by the cells of a particular
tissue from where these are liberated for use
in the other tissues. Such enzymes are called
extracellular enzymes. (Example: various
proteolytic enzymes of gastrointestinal tract
as Trypsin and Chymotrypsin).
• The enzyme binds with its specific substrate
and forms an enzyme-substrate complex.
• At the end of the reaction the substrate is
converted into the product and the enzymes
remain unchanged. 4
• In enzymatic reactions, the molecules at the
beginning of the process, called substrates,
are converted into different molecules, called
products.
• Almost all chemical reactions in a biological
cell need enzymes.

5
• Like all catalysts, enzymes work by lowering the
activation energy for a reaction, thus increasing
the rate of the reaction. As a result, products are
formed faster and reactions reach their
equilibrium state more rapidly.
• Most enzyme reaction rates are millions of times
faster than those of comparable un-catalyzed
reactions.
• Enzymes are not consumed by the reactions they
catalyze, nor do they alter the equilibrium of
these reactions.
• Enzymes are known to catalyze about 4,000
6
• A few RNA molecules called ribozymes also
catalyze reactions, with an important example
being some parts of the ribosome.
• Enzyme activity can be affected by other
molecules.
1. Inhibitors are molecules that decrease enzyme
activity.
2. Activators are molecules that increase activity.
3. Many drugs and poisons are enzyme inhibitors.
4. Activity is also affected by temperature,
chemical environment (e.g., pH), and the
concentration of substrate. 7
• Some enzymes are used commercially, for
example, in the synthesis of antibiotics.
• Some household products use enzymes to
speed up biochemical reactions:
1. Enzymes in biological washing powders
break down protein or fat stains on clothes
2. Enzymes in meat tenderizers break down
proteins into smaller molecules, making the
meat easier to chew).

8
9
History of enzymes

10
• As early as the late 17th and early 18th centuries,
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.
• In the 19th century, when studying the
fermentation of sugar to alcohol by yeast, Louis
Pasteur came to the conclusion that this
fermentation was catalyzed by a vital force
contained within the yeast cells called
"ferments", which were thought to function 11
• In 1897, Eduard Buchner submitted his first paper on the
ability of yeast extracts that lacked any living yeast cells to
ferment sugar. In a series of experiments at the University
of Berlin, he found that the sugar was fermented even
when there were no living yeast cells in the mixture. He
named the enzyme that brought about the fermentation
of sucrose "zymase". In 1907, he received the Nobel Prize
in Chemistry "for his biochemical research and his
discovery of cell-free fermentation".
• Following Buchner's example, enzymes are usually named
according to the reaction they carry out. Typically, to
generate the name of an enzyme, the suffix -ase is added
to the name of its substrate (e.g., lactase is the enzyme
that cleaves lactose) or the type of reaction (e.g., DNA
polymerase forms DNA polymers). 12
• In 1926, James B. Sumner showed that the
enzyme urease was a pure protein and
crystallized it; Sumner did likewise for the
enzyme catalase in 1937.
• Northrop and Stanley (1930), who worked on
the digestive enzymes pepsin, trypsin and
chymotrypsin. These three scientists were
awarded the 1946 Nobel Prize in Chemistry.

13
Chemical Nature of
Enzymes

14
• Most enzymes are protein in nature.
• Some enzymes require the presence of
certain additional organic or inorganic
substances and are conjugated proteins.
• Such enzymes are called as holoenzymes. The
protein part of the conjugated protein is
called apoenzymes.
• The non-protein part is called prosthetic
group.

15
16
• Several apoenzymes require the presence of
metal ions such as Mg2+ (as Hexokinase), Zn2+
(for the activity of carboxypeptidase). Such
inorganic ions are called cofactors.
• If the metal ion is the integral part of the
enzyme, such enzymes are called
metalloenzymes.

17
Active
Site

18
In biology, the active site is a small port in
an enzyme where substrate molecules bind and
undergo a chemical reaction.
The active site is usually found in a 3-D groove
or pocket of the enzyme, lined with amino acid
residues.
These residues are involved in recognition of
the substrate.
After an active site has been involved in a
reaction, it can be used again.
19
Mechanism of enzyme
action

20
• Enzymes can act in several ways, all of which
lower ΔG‡ (energy of activation).
• Lowering the activation energy by creating an
environment in which the transition state is
stabilized.
• Substrates need a lot 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.

21
1. Lock and key model
• Enzymes are very specific, and it was
suggested by the Nobel laureate organic
chemist Emil Fischer in 1894 that this was
because both the enzyme and the substrate
possess specific complementary geometric
shapes that fit exactly into one another.
• This is often referred to as "the lock and key"
model. However, while this model explains
enzyme specificity, it fails to explain the
stabilization of the transition state that
enzymes achieve. 22
2. Induced fit
• The favored model for the enzyme-substrate
interaction is the induced fit model.
• This model proposes that the initial
interaction between enzyme and substrate is
relatively weak, but that these weak
interactions rapidly induce conformational
changes in the enzyme that strengthen
binding.

23
24
 Michaelis constant Km
• As enzyme-catalysed reactions are saturable, their
rate of catalysis does not show a linear response to
increasing substrate. If the initial rate of the
reaction is measured over a range of substrate
concentrations (denoted as [S]), the reaction rate
(v) increases as [S] increases. However, as [S] gets
higher, the enzyme becomes saturated with
substrate and the rate reaches Vmax, the enzyme's
maximum rate.
• The Michaelis constant Km is experimentally
defined as the concentration at which the rate of
the enzyme reaction is half V .
25
26
Classification of
Enzymes

27
Enzymes are classified into six groups according to the
International Union of Biochemistry (IUB). They are:
Oxidoreductases (EC1):
• Enzymes involved in oxidation-reduction reactions.
• E.g. Lactate dehydrogenase, Glyceraldehyde-3-
phosphate dehydrogenase.
Transferases (EC2):
• Enzymes transfer a particular group from one
substrate to another. E.g. Alanine aminotransferase.
Hydrolases (EC3):
• Hydrolyze the substrate with the addition of water
molecule. E.g. Glucose 6-phosphatase, Amylase,
Pepsin. 28
Lyases (EC4):
• Catalyze the removal of a small molecule from
a large substrate without the addition of
water.
• E.g. Fumarase and Enolase.
Isomerases (EC5):
• They isomerize substrates.
Ligases (EC6):
• Synthesize substances by joining two
substrates with the utilization of energy. E.g.
Glutamine synthetase. 29
EC number

30
• The Enzyme Commission number (EC number)
is a numerical classification scheme for
enzymes, based on the chemical reactions
they catalyze. As a system of enzyme
nomenclature, every EC number is associated
with a recommended name for the respective
enzyme.
• Every enzyme code consists of the letters "EC"
followed by four numbers separated by
periods. Those numbers represent a
progressively finer classification of the
enzyme. 31
• For example, the tripeptide aminopeptidases
have the code "EC 3.4.11.4", whose components
indicate the following groups of enzymes:
• EC 3 enzymes are hydrolases (enzymes that use
water to break up some other molecule)
• EC 3.4 are hydrolases that act on peptide bonds
• EC 3.4.11 are those hydrolases that cleave off
the amino-terminal amino acid from a
polypeptide
• EC 3.4.11.4 are those that cleave off the amino-
terminal end from a tripeptide
32
Enzyme Specificity

33
1. Stereospecificity:
The group of enzyme catalyzes either L or D
isomer.
2. Reaction Specificity:
One enzyme catalyzes only one type of
reaction.
3. Substrate Specificity:
A. Absolute Specificity: Glucokinase acts on
glucose only.
B. Group Specificity: Hexokinase catalyzes
hexoses. 34
4. Bond Specificity:
Refers to the action of proteolytic enzymes, which act on
peptide bonds of proteins.
5. Dual Specificity:
There are two types of dual specificity:
The enzyme act on two substrates by one reaction types.
e.g. xanthine oxidase enzyme acts on xanthine and
hypoxanthine (two substrates) by oxidation (one reaction
type).
The enzyme act on one substrate by two different
reaction types. e.g. isocitrate dehydrogenase enzyme acts
on isocitrate (one substrate) by oxidation followed by
decarboxylation (two different reaction types) to produce
α-ketoglutrate as a reaction product. 35
FACTORS AFFECTING
ENZYME ACTIVITY

36
1. Effect of pH:
Each enzyme has an optimum pH at which the
activity of the enzyme is maximum.
Either decreased or increased pH causes a
decrease in enzyme activity. Examples of
optimum pH are:
Pepsin has an optimum pH at (I-2).
Optimum pH for amylase is (6.8).
Optimum pH for ALP is (9.0).
Optimum pH for ACP is (5.0).
37
38
2. Effect of Temperature:
The temperature at which the enzyme activity is more is
called optimum temperature.
The enzyme activity was duplicated for every increase in
temperature by 10 degrees till reaching to the maximum
velocity for enzyme activity at the optimum temperature.
After this point the enzyme activity decreases with the
increment of temperature.
At 100oC the enzyme will be denatured. While, at zero
degree the enzyme will be kept inactivated and when the
temperature increased the activity of enzyme will be
increased.
Optimum temperature of enzymes in the human body is
37oC. 39
40
3. Effect of Substrate Concentration:
At low substrate concentration enzyme
molecules are free initially and the ES complex
(ES= enzyme-substrate) formation is
proportional to the substrate concentration.
At higher concentration all the enzyme
molecules are saturated with substrate. There
will be no change in the activity further.
Hence in an enzyme reaction system more
substrate is taken than the requirement.
41
42
4. Effect of Enzyme Concentration:
The velocity of the enzyme reaction is directly
proportional to the enzyme concentration.

5. Enzyme activators

6. Enzyme inhibitors

43
Enzyme Inhibitors

44
Enzyme inhibitors were classified into reversible and
irreversible inhibitors

I. Reversible inhibitors
Reversible inhibitors bind to enzymes with non-
covalent interactions (hydrogen bonds, hydrophobic
interactions and ionic bonds).
Multiple weak bonds between the inhibitor and the
active site combine to produce strong and specific
binding.
Reversible inhibitors generally do not undergo
chemical reactions when bound to the enzyme and
45
Classification of reversible inhibitors
A. Competitive inhibition
In competitive inhibition, the substrate and inhibitor
cannot bind to the enzyme at the same time. This usually
results from the inhibitor having an affinity for the active
site of an enzyme where the substrate also binds.
The substrate and inhibitor compete for access to the
enzyme's active site.
This type of inhibition can be overcome by sufficiently high
concentrations of substrate (Vmax remains constant).
Km will increase as it takes a higher concentration of the
substrate to reach the Km point.
Competitive inhibitors are often similar in structure to the
46
Example:
Malonate is a competitive inhibitor of succinate
for succinate dehydrogenase

47
B. Non-competitive inhibition
The binding of the inhibitor to the enzyme
reduces its activity but does not affect the
binding of substrate.
The extent of inhibition depends only on the
concentration of the inhibitor.
Vmax will decrease due to the inability for the
reaction to proceed as efficiently, Km will
remain the same as the actual binding of the
substrate will still function properly.
48
C. Uncompetitive inhibition
In uncompetitive inhibition, the inhibitor binds
only to the substrate-enzyme complex.
This type of inhibition causes Vmax to decrease
and Km to decrease.

49
50
51
II. Irreversible inhibitors
• Irreversible inhibitors usually covalently modify an
enzyme, and inhibition cannot therefore be reversed.
• Irreversible inhibitors often contain reactive functional
groups react with amino acid side chains in the enzyme
active sites to form covalent adducts.
• The side chain of amino acid may be hydroxyl or
sulfhydryl groups; these include the amino acids serine
(as diisopropylfluorophosphate (DFP)), cysteine,
threonine or tyrosine.
• Irreversible inhibitors are generally specific for one
class of enzyme and do not inactivate all proteins; they
do not function by destroying protein structure but by
specifically altering the active site of their target.
52
53
54
COFACTOR

55
These proteins are commonly enzymes, and
cofactors can be considered "helper molecules"
that assist in biochemical transformations.
Cofactors are either organic or inorganic.
They can also be classified depending on how
tightly they bind to an enzyme, with loosely-
bound cofactors termed coenzymes and tightly-
bound cofactors termed prosthetic groups.
An inactive enzyme, without the cofactor is
called an apoenzyme, while the complete
enzyme with cofactor is the holoenzyme.
56
• Cofactors can be divided into two broad
groups: organic cofactors, such as flavin or
heme, and inorganic cofactors, such as the
metal ions Mg2+, Cu+, Mn2+, or iron-sulfur
clusters.
• Organic cofactors are sometimes further
divided into coenzymes and prosthetic
groups.
• Prosthetic group emphasizes the nature of the
binding of a cofactor to a protein (tight or
covalent) and, thus, refers to a structural
property. 57
Ion Examples of enzymes containing this
ion
Cupric Cytochrome oxidase
Ferrous or Ferric Catalase, Cytochrome (via Heme)

Magnesium Glucose 6-phosphatase, Hexokinase, DNA-

polymerase
Manganese Arginase
Molybdenum Nitrate reductase
Nickel Urease
Selenium Glutathione peroxidase
Zinc Alcohol dehydrogenase, Carbonic 58
Organic cofactor
• Organic cofactors are small organic molecules (with
a molecular mass less than 1000 Da) that can be
either loosely or tightly bound to the enzyme in
case of tightly bound organic cofactor it is difficult
to remove without denaturing the enzyme, it can
be called a prosthetic group.
• Vitamins can serve as precursors to many organic
cofactors (e.g., vitamins B1, B2, B6, B12, niacin, folic
acid) or as coenzymes themselves (e.g., vitamin C).
• Many organic cofactors also contain a nucleotide,
such as the electron carriers NAD and FAD, and
coenzyme A, which carry acyl groups. 59
 Zymogens
(Proenzymes)

60
• A zymogen (or proenzyme) is an inactive enzyme
precursor.
• A zymogen requires a biochemical change (such as a
hydrolysis reaction revealing the active site, or
changing the configuration to reveal the active site)
for it to become an active enzyme.
• The biochemical change usually occurs in a lysosome
where a specific part of the precursor enzyme is
cleaved in order to activate it.
• The amino acid chain that is released upon activation
is called the activation peptide.
• The pancreas secretes zymogens partly to prevent the
enzymes from digesting proteins in the cells in which
61
• Fungi also secrete digestive enzymes into the
environment as zymogens. The external
environment has a different pH than inside the
fungal cell and these changes the zymogen's
structure into an active enzyme.
• The protein digesting enzymes (proteolytic
enzymes) of the gastrointestinal tract are produced
in the form of precursor. This is to prevent
unwanted degradation of body self-proteins.
• These precursor forms of enzymes (zymogen) are
converted into active form by HCI and trypsin.
Pepsinogen → pepsin (HCl activates the pepsinogen).
62
ISOENZYMES

63
Isozymes (also known as isoenzymes) are enzymes that
differ in amino acid sequence but catalyze the same
chemical reaction.
These enzymes usually display different kinetic parameters
(e.g. different Km values), or different regulatory properties.
Catalyze the same reactions but are formed from
structurally different polypeptides.
They perform the same catalytic function.
Various isoenzymes of an enzyme can differ in three major
ways:
1. Enzymatic properties
2. Physical properties (e.g heat stability)
3. Biochemical properties such as amino acid
composition and immunological reactivities. 64
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

65
Isoenzyme Composition Present in Elevated in
name

LDH1 ( H 4) Myocardium, myocardial

HHHH RBC infarction

LDH2 (H3M1) HHHM Myocardium,

RBC

LDH3 (H2M2) HHMM Kidney, Skeletal

muscle

LDH4 (H1M3) HMMM Kidney, Skeletal

muscle

LDH5 (M4) MMMM Skeletal muscle, Skeletal muscle

Liver and liver diseases

66
Creatine Kinase (CK)
Creatine + ATP → phosphocreatine + ADP
• (Phosphocreatine – serves as energy reserve
during muscle contraction)
• Creatine kinase is a dimer made of two
monomers occurs in the tissues.
• Skeletal muscle contains M subunit, Brain
contains B subunits.
• Three different isoenzymes are formed.

67
Isoenzyme name Composition Present in Elevated in

CK-1 BB Brain CNS diseases

Myocardium/ Acute myocardial

CK-2 MB
Heart infarction

Skeletal muscle,
CK-3 MM
Myocardium
68
Allosteric modulation
• Allosteric sites are sites on the enzyme that
bind to molecules in the cellular environment.
• The sites form weak, noncovalent bonds with
these molecules, causing a change in the
conformation of the enzyme.
• This change in conformation translates to the
active site, which then affects the reaction
rate of the enzyme.
• Allosteric interactions can both inhibit and
activate enzymes and are a common way that
69
70
 Clinical
Enzymology

71
Serum enzymes increases may be due to:-
• Cell death - this results in a small short-lived increase
(e.g., following myocardial infarction).
• Increased cell membrane permeability in living cells
(due to hypoxia, inflammation, drugs/poisons, cellular
swelling) gives rise to a large protracted increase in
serum enzymes as there is ongoing enzyme synthesis
(e.g., Duchenne muscular dystrophy, acute viral
hepatitis).
• Increased synthesis in a specific cell type (e.g., gamma
glutamyl transferase in liver cells is induced by alcohol
or anticonvulsant drugs, alkaline phophatase in liver
cells is induced by obstruction, lactate dehydrogenase is
72
induced in neoplastic tissues).
CAUSES OF CELL DAMAGE OR DEATH

73
CREATINE KINASE (CK)

74
Important in tissues where significant metabolic
energy is stored as creatine phosphate.
Distribution : skeletal muscle, heart, brain.
Isoenzyme composition : CK is a dimer consisting of
sub-units M or B coded by two distinct genes.
Thus 3 possible isoenzymes - M M ; M B ; B B
Skeletal muscle - all MM; Heart - 80% MM, 20% MB;
Brain - all BB.
Normal pattern in serum - predominantly MM
present, with MB < 6% of total CK.

75
• Myocardial infarction
Early increase of total CK, specifically the MB
isoenzyme.
Total CK levels start to rise at about 6 hours , peak at
18 to 30 hours, and return to normal by 3 days.
If total CK raised, measurement of CK-MB is indicated.
CK-MB levels may be raised by 4 hours, are almost
certain to be raised by 12 hours, and may return to
normal by 24 hours after MI.
Used as a diagnostic test before 24 hours, and as a
prognostic indicator (amount of increase reflects
extent of cardiac damage).
76
• Skeletal muscle damage
e.g., trauma, surgery (especially in cardiac surgery), over-
exercise, convulsions, ischaemia, inflammation (myositis),
malignant hyperthermia, congenital muscular dystrophy.
increase of total CK, but MB isoenzyme not increased. In
neurogenic muscle disease, e.g., poliomyelitis and
Parkinsonism, CK levels are normal.
In Duchenne Muscular Dystrophy, CK elevation precedes
onset of symptoms by years, and falls as disease
progresses.
In chronic muscle disease there is reversion to the foetal
isoenzyme pattern (MB appears in skeletal muscle and
serum).
• Hypothyroidism is associated with high total CK levels.77
LACTATE DEHYDROGENASE (LDH, LD)

78
Important in the disposal of glycolytically generated
NADH, particularly when mitochondrial disposal is
impaired by hypoxia.

Distribution: ubiquitous, including heart, skeletal muscle,

liver and RBCs - specificity is improved by isoenzyme
analysis.
LD is a tetramer consisting of sub-units H or M coded by
two different genes.

79
• Anoxic tissues (liver, muscle) express M subunit,
thus LD5 predominates.
• Erythrocyte precursors and heart express the H
subunit, hence LD1 predominates in these
tissues.
• Normal pattern in serum - predominantly LD2,
with slightly less LD1, and even less LD3, LD4 and
LD5.

80
• Myocardial infarction - late and long-lasting
increase in total LD. Total LD levels start to rise at
about 8 to 12 hours, peak at 24 to 48 hours and
return to normal by 10 days. The predominant
isoenzyme is LD1, which is present in greater
concentration than the normal LD2 (flipped
pattern).

• Liver damage, including viral or toxic hepatitis

(ethanol, paracetamol overdose, carbon
tetrachloride), cardiac failure (liver congestion) -
increase in total LD, exclusively due to LD5.
81
Hematological disorders - often isolated elevation in
total LD. Due to breakdown of circulating red cells or red
cell precursors in bone marrow.
• Intra-vascular (or in vitro) haemolysis, e.g., due to an
auto-immune disorder, prosthetic heart valve,
inherited enzyme deficiency (G6PD, PK) - both LD1 and
LD2 increased. Associated features include elevated
serum unconjugated bilirubin, increased urine and
stool urobilinogen, and decreased haptoglobin.
• Megaloblastic anaemia due to folate or vitamin B12
deficiency : failure of cell division leads to cell lysis and
enzyme release from the bone marrow - predominant
increase in LD1 (as in myocardial infarction). Extremely
high levels can be achieved. 82
Malignant tumours may manifest an isolated increase
in serum LD due to enhanced synthesis of glycolytic
enzymes by a wide variety of neoplasms (even
measured in aqueous humour to diagnose
retinoblastoma).
Typically centripetal isoenzyme pattern (LD2, LD3 and
LD4) due to expression of both subunits (H and M). An
exception is seen in germ cell tumours which show an
increase in LD1.

83
TRANSAMINASES

84
ASPARTATE TRANSAMINASE (AST) (aspartate +α-
ketoglutarate -----> oxaloacetate + glutamate).

ALANINE TRANSAMINASE (ALT) (alanine + α-

ketoglutarate -----> pyruvate + glutamate).

Wide distribution in tissues, including liver, skeletal

muscle, heart, kidney and RBCs.
Cofactor vitamin B6 (pyridoxine) - carries amino acid
intermediates.

85
Acute hepatitis - high, sustained elevations of both ALT and
AST (up to 100 x normal).
ALT higher especially in mild injury; precedes onset of
jaundice (allows early diagnosis.
Disproportionate AST elevation indicates liver cell necrosis
(involving mitochondria) or ethanol induced damage
(acetaldehyde formed during ethanol metabolism depletes
cytosolic pyridoxine, hence ALT activity selectively lost).
Myocardial infarction - AST elevation of 5 - 10 x normal.
AST levels start to rise at about 6 to 8 hours, peak at 18 to
24 hours and return to normal by 4 to 5 days. ALT hardly
increases at all.
AST always greater than ALT (de Ritis quotient AST/ALT > 5.
In liver disease this ratio is usually < 1). 86
ALKALINE PHOSPHATASE (ALP)

87
Located in membranes, specifically the brush borders
of the PCT of the kidney, the small intestinal mucosa,
both the sinusoidal and canalicular surfaces of the
hepatocyte, in osteoblasts in bone and in the
placenta.
Important in the formation of new bone by
osteoblasts.
Normal level in serum is determined by age and sex,
reflecting periods of active bone growth, i.e., very
high in young children.
Normal pattern in children is preponderance of bone
isoenzyme, while in adults liver isoenzyme
predominates. 88
Bone disease
Reflects increased osteoblastic activity i.e., bone
synthesis.
High levels of total ALP, due to increased bone
isoenzyme, seen in:
• active bone growth (young children, at puberty, healing
fractures).
• Primary bone tumours (osteogenic sarcoma).
• secondary tumours evoking a sclerotic response e.g.,
prostatic and breast metastases.
• Rickets (children) and osteomalacia (adults).
• long-standing primary or secondary hyperparathyroidism
(e.g., chronic renal disease where calcium is resorbed
from bone, leading to renal osteodystrophy) 89
Liver disease
Classic marker of cholestasis due to extra-hepatic
(gallstones) or intra-hepatic (drugs, inflammation)
obstruction.
Synthesis of liver isoenzyme induced by biliary
obstruction - differentiates obstruction from
hepatocellular damage. Elevated liver ALP without
jaundice suggests :
• Intermittent or incomplete obstruction (gallstone).
• Intra-hepatic space-occupying mass (tumour).

90
• Placental isoenzyme
is found in the serum in late pregnancy and
remains elevated a week or two after delivery.

91
GAMMA GLUTAMYL TRANSFERASE (GGT)

92
γ-glutamyl-N-donor + acceptor --> γ-glutamyl-N-
acceptor + donor.
Distribution :
It present in all cells except muscle.
Located in cell membranes and endoplasmic
reticulum of hepatocytes.
Role:
Synthesis of reduced glutathione (GSH) required for
drug detoxification e.g., paracetamol.
Normal level in serum derived from liver.

93
Hepatic synthesis of GGT is induced by biliary
obstruction.
Serum levels correlate with liver ALP and 5’
nucleotidase.
Very sensitive and specific marker of liver disease.
Hepatic synthesis of GGT is also induced by drugs
(especially barbiturates, antidepressants and
anticonvulsants) and alcohol.
Serum GGT is increased not just in patients with
alcoholic liver disease, but also in people who are
heavy drinkers.

94
ACID PHOSPHATASE (ACP)

95
• Distribution
Prostate, lysosomes of all cells, red blood cells (avoid
hemolysis). Number and genetics of isoenzymes not
known, but at least prostatic and red cell isoenzymes
exist.
Identify prostatic isoenzyme by L-tartrate inhibitable
activity.
ACP is temperature and pH labile. So, specimens for
enzyme assay should be submitted on ice.

96
• Prostatic CA. High activity of ACP, especially the
prostatic isoenzyme, indicates disseminated disease
(usually spreads to bone). CA-in-situ and benign
hypertrophy of the pr normal levels of ACP.
Recently, ACP assays have been largely replaced by
measurement of prostate-specific antigen.
• 2. Gaucher’s disease, bone destruction by infection
and neoplasia. Lysosomal isoenzyme - tartrate
insensitive, normal RIA result. ostate usually have

97
AMYLASE

98
Distribution
• Amylase secreted by the pancreas and salivary
glands; also present in Fallopian tubes and small
intestine.
• Two common isoenzymes occur, a pancreatic (P)
and salivary (S) type, which can be differentiated
using a wheat germ lectin which selectively inhibits
the S isoenzyme.

99
• Acute pancreatitis
an increase in total amylase, in the appropriate
clinical setting, is suggestive of acute pancreatitis.
In cases of an acute abdomen, determination of
isoenzyme type is seldom helpful - P isoenzyme is
increased not only in acute pancreatitis, but also in
perforated peptic ulcer and intestinal obstruction/
infarction
Amylase only remains elevated in the serum for 3 to 4
days, but remains elevated in the urine for longer (6
days). Levels of both amylase and lipase are normal in
chronic pancreatitis.
100