Enzyme Inhibition and Types of Reversible Enzyme Inhibitors.ppt

Useful to students

1. •A chemical reaction is
a process that converts one or
more
substances (known as reagents, reactants, or
substrates)
to another type of substance (the product).
•Catalyst increase
the rate at which chemical reactions
occur
without being consumed or permanently altered
themselves.

•Enzyme is
protein that act as catalyst within living
cells.
Enzymes are biological catalysts and are protein
in
nature with an exception of ribozyme. German
physiologist

Wilhelm Kuhne (1837–1900) coined
the
term
enzyme.
•Substrate is
the substance on which an enzyme acts.

2. •The enzyme substrate complex is
a temporary
molecule
formed when an 
enzyme comes
into
perfect
contact with its 
substrate.

The substrate causes
a conformational change, or
shape
change, when the 
substrate enters
the active
site.

•Active site is
the area of the 
enzyme capable
of
forming
weak bonds with the 
substrate.
•Vmax represents
the maximum rate achieved by
the
system, at saturating substrate concentration.
•The
Michaelis constant 
Km is
the substrate
concentration
at which the reaction rate is half
of Vmax.

3. Enzyme Inhibition
•The
molecule which acts directly on an enzyme to lower
its
catalytic rate of an enzyme is known as Inhibitor.
•A
variety of small molecules exists which can reduce the
rate
of an enzyme-controlled reaction. They are called
enzyme
inhibitors
.
The process of enzyme-controlled
reaction
is called as enzyme inhibition.
•Inhibition
is a normal part of the regulation of enzyme
activity
within cells.
•The
inhibitor may be organic or inorganic in nature.
•Can prevent formation of ES complex or
prevent ES breakdown to E + P.

4. •Inhibitors slow down the rate of a reaction.
Sometimes this is a necessary way of making sure
that the reaction does not proceed too fast, at other
times, it is undesirable
E + S <-> ES -> E + P
E + I <-> EI
Ki = [E][I]/[EI]
•Enzyme inhibition broadly divided into three
categories:
1. Reversible inhibition
2. Irreversible inhibition
3. Allosteric inhibition

6. Reversible inhibition
•The Inhibitors binds non-covalently
with enzyme and the enzyme inhibition
can be reversed if the inhibitor is
removed.
•The reversible inhibition is further sub-
divided into:
1. Competitive inhibition
2. Non- Competitive inhibition
3. Un- Competitive inhibition
4. Mixed inhibition

7. Types of Reversible Enzyme
Inhibitors

8. Competitive inhibition:
The inhibitor which closely resembles the real
substrate is regarded as substrate analogue.
The inhibitor competes with substrate and binds
at the active site of the enzyme but does not
undergo any catalysis.
As long as the competitive inhibitor holds the
active site, the enzyme is not available for the
substrate to bind.
The inhibition could be overcome by a high
substrate concentration.
The rate of reaction will be closer to the
maximum when there is more ‘real’ substrate,
e.g. Arabinose competes with glucose for the
active sites on glucose oxidase enzyme.

9. Competitive inhibition:

10. Competitive Inhibitors look like substrate
NH
2
C
O
HO
NH
2
S
O
H2N
O
PABA Sulfanilamide
Para-Aminobenzoic Acid (PABA) precursor to
folic acid in bacteria
O
2
C-CH
2
-CH
2
-CO
2
-------> O
2
C-CH=CH-CO
2
succinate fumarate
Succinate dehydrogenase
O
2
C-CH
2
-CO
2
Malonate

11. Competitive Inhibition (CI)
•CI binds free enzyme
•Competes with substrate for enzyme binding site.
•Raises Km without effecting Vmax (remains unchanged)
•Can relieve inhibition with more Substrate

13. Non-competitive inhibition:
The inhibitor binds at a site other than the
active site on the enzyme surface.
This binding impairs the enzyme function.
The inhibitor has no structural resemblance
with the substrate.
These molecules are not necessarily anything
like the substrate in shape.
They bind with the enzyme, but not at the
active site.
This binding does change the shape of the
enzyme though, so the reaction rate
decreases.

14. Non-competitive inhibition:
The inhibitor does not interfere with the
enzyme – substrate binding, but the catalysis
is prevented.
The inhibitor generally binds with the enzyme
as well as ES complex.
Heavy metal ions (Ag
+
, Pb
2+
, Hg
2+
etc.) can non-
competitively inhibit the enzymes by binding
with cysteinyl sulfhydrl groups.

15. Non-competitive Inhibition (NI)
•NI can bind free E or ES complex
•Lowers Vmax, but Km remains the same
•NI’s don’t bind to S binding site therefore don’t effect
Km
•Alters conformation of enzyme to effect catalysis but not
substrate binding

16. Uncompetitive Inhibition (UI)
•UI does not bind with enzyme
•Only Binds with ES complex,
•Prevents ES from proceeding to E + P or back to E + S.
•Lowers Km & Vmax values of the enzyme, but ratio of
Km/Vmax remains the same
•Occurs with multisubstrate enzymes

17. Mixed inhibition:
Mixed inhibition occurs when the inhibitor binds at a
separate site from the substrates active site to either the
free enzyme or the enzyme substrate complex. The inhibitor
can or doesn’t always resemble the structure of the
substrate.
Mixed inhibition is a type of enzyme inhibition in which
the inhibitor may bind to the enzyme whether or not the
enzyme has already bound the substrate but has a
greater affinity for one state or the other.
It is called "mixed" because it can be seen as a
conceptual "mixture" of competitive inhibition, in which
the inhibitor can only bind the enzyme if the
substrate has not already bound, and uncompetitive
inhibition, in which the inhibitor can only bind the enzyme
if the substrate has already bound.

18. Mixed inhibition:
If the ability of the inhibitor to bind the
enzyme is exactly the same whether or not the
enzyme has already bound the substrate, it is
known as a non-competitive inhibitor. 
Non-competitive inhibition is sometimes
thought of as a special case of mixed inhibition.
In mixed inhibition, the inhibitor binds to an
allosteric site, i.e. a site different from
the active site where the substrate binds.
However, not all inhibitors that bind at
allosteric sites are mixed inhibitors

19. Mixed inhibition may result in either:
A decrease in the apparent affinity of the
enzyme for the substrate (Km value appears to
increase) -- seen in cases where the inhibitor
favours binding to the free enzyme. More
closely mimics competitive binding.
An increase in the apparent affinity of the
enzyme for the substrate (Km value appears to
decrease) -- seen in cases where the inhibitor
favours binding to the enzyme-substrate complex.
More closely mimics uncompetitive binding.
In either case the inhibition decreases the
apparent maximum enzyme reaction rate (Vmax)

24. Irreversible inhibition:
•The inhibitors bind covalently with the enzymes
and inactive them, which is irreversible.
•These molecules bind permanently with the
enzyme molecule and so effectively reduce the
enzyme concentration, thus limiting the rate of
reaction.
•These inhibitors are usually toxic substances.
•For example, cyanide irreversibly inhibits the
enzyme cytochrome oxidase found in the electron
transport chain used in respiration. If this cannot
be used, death will occur.
•Iodoaetate is an irreversible inhibitor of the
enzymes like papain and glyceraldehyde 3-
phosphate dehydrogenase.

27. Irreversible Inhibitors
H
3C O P
O
SC
C
H
O
O CH
2CH
3
C O CH
2CH
3
O
S
CH
3
CH
2
HC
CH
3
O
CH
3
P
F
O
O C
CH
3
H
CH
3
Diisopropyl fluorophosphate
(nerve gas)
H
3C O P
O
S
S
CH
3
NO
2
parathion
malathion
•Organophosphates
•Inhibit serine hydrolases
•Acetylcholinesterase inhibitors

29. •Some
of the enzymes possess additional sites
known
as allosteric sites (Greek: allo –
different/other;
steric - shape) besides the active
site.
Such enzymes are known as
allosteric

enzymes.

•Allosteric
sites are unique places on the enzyme
molecules.

•The
term allosteric has been introduced by the two
Noble
laureates,
Monod
and Jacob
,
to denote an
enzyme
site, different from the active site, which
non
competitively binds molecule other than the
substrate
and may influence the enzyme activity.

30. Classes
of allosteric enzymes
•Enzymes
that are regulated by allosteric mechanism are
referred
to as allosteric enzymes. They are divided into
two
classes based on the influence of allosteric effector
on

Km & Vmax.
1.K-Class
of allosteric enzymes:
The
effector changes the
Km and not the Vmax.
Double reciprocal plots Similar
to
competitive inhibition are obtained.
Eg.

Phosphofructokinase
2.V-Class
of allosteric enzymes:
The
effector changes the
Vmax and not the Km.
Double reciprocal plots resemble
that
of non-competitive inhibition.
Eg.

Acetyl
CoA carboxylase

31. Properties of Allosteric enzymes
•Allosteric
enzymes have one or more allosteric site.
•Allosteric
sites are binding sites distinct from an enzyme active site or
substrate
binding site.
•Molecule
that can bind to allosteric sites are called allosteric effector or
allosteric
modulator.

•Allosteric
modulators bind at the allosteric site and regulate the enzyme
activity.

•They
modify enzyme activity by causing a reversible change in the
structure
of the enzymes active site. This in turn affects the ability of the
substrate
to bind to the enzyme.

32. •Binding
to allosteric sites alter the activity of the
enzyme,
this is called cooperative binding.
•The
enzyme activity is increased when a positive (+)
allosteric
effector binds at the allosteric site known as
activator
site. On the other hand, a negative (-)
allosteric
effector binds at the allosteric site called
inhibitor
site and inhibits the enzyme activity.
•Allosteric
enzymes display sigmoidal plot of V vs [S] ₀
(Initial
Reaction Rate Vs. Concentration of Substrate).

•Allosteric
enzymes are an
exception to the Michaelis-
Menten model.
Because they have more than two
subunits
and active sites, they do not obey the
Michaelis-Menten
kinetics.

34. Kinetic Properties of Allosteric Enzymes
•Most
of the allosteric enzyme are
oligomeric

(oligo
-"a few" + -mer - "parts") in nature.
•The
subunits may be identical or different.
•The kinetic properties
of allosteric enzymes are
often
explained in terms of a conformational
change.
•The
non covalent reversible binding of the
effector
molecule at the allosteric site brings
about
a conformational change in the active site
of
the enzyme, leading to the inhibition or
activation
of the catalytic activity.

37. •Allosteric
enzymes exist in two conformational
states
– the
T
(Tense or taut) and the
R
(Relaxed).
•The kinetic properties
of allosteric enzymes are
often
explained in terms of a conformational
change between
a low-activity, low-affinity
"tense"
or T state and a high-activity, high-
affinity
"relaxed" or R state.

38. Model of Allosteric Regulation
•Two
main model have been proposed to describe the
mechanistic
basis of enzyme allostery:
•They
are:
1.
Concerted (MWC) model
 by

Monod,


Wyman,
and Changeux


2.

Sequential
model
 by

Koshland,


Nemethy,
and Filmer
 
•Both
postulate that protein subunits exist in one of
two conformations,
tensed (
T)
or relaxed (
R),
and that
relaxed
subunits bind substrate more readily than those
in
the tense state. The two models differ most in their
assumptions
about subunit interaction and the
preexistence
of both states.

39. Concerted model
•The
concerted model of allostery, also referred to as the
symmetry
model or MWC model, postulates that
enzyme
subunits are connected in such a way that a
conformational
change in one subunit is necessarily
conferred
to all other subunits
.
Thus,
all
subunits must
exist
in the same conformation
.
The model further
holds
that, in the absence of any ligand (substrate or
otherwise),
the equilibrium favors one of the
conformational
states, T or R. The equilibrium can be
shifted
to the R or T state through the binding of
one ligand (the
allosteric effector or ligand) to a site
that
is different from the active site (the allosteric site).

40. According to the concerted model:
•In
the concerted model, allosteric enzymes exist
in
two conformational states – the
T
(Tense or
taut)
and the
R
(Relaxed).
•The

T
and
R
states are in equilibrium


Allosteric
activator or Substrate
TR


Allosteric
inhibitor
•Allosteric
inhibitors favour
T
state whereas
activators
and substrates favour
R
state. The
substrate
can bind only with the
R

form
of the
enzyme.

41. •The
concentration of the enzyme molecule in the
R
state increases as more substrate is added,
therefore
the binding of the substrate to the
allosteric
enzyme is said to be cooperative.

43. Sequential
model
•The
sequential model of allosteric regulation
holds
that subunits are not connected in such a
way
that a conformational change in one induces
a
similar change in the others. Thus, all enzyme
subunits
do not necessitate the same
conformation.
Moreover, the sequential model
dictates
that molecules of a substrate bind via
an induced
fit protocol.
•In
general, when a subunit randomly collides
with
a molecule of substrate, the active site, in
essence,
forms a glove around its substrate.

44. •While
such an induced fit converts a subunit
from
the tensed state to relaxed state, it does not
propagate
the conformational change to adjacent
subunits.
Instead, substrate-binding at one
subunit
only slightly alters the structure of other
subunits
so that their binding sites are more
receptive
to substrate.
•To
summarize:
Subunits
need not exist in the same conformation
Molecules
of substrate bind via induced-fit protocol
Conformational
changes are not propagated to all
subunits

46. TYPES OF ALLOSTERIC REGULATION
•Homotropic
effect
•Heterotropic
effect
Homotropic
effect:
A
homotropic allosteric
modulator
is a substrate for its target enzyme, as
well
as a regulatory molecule of the enzyme's
activity.
It is typically an activator of the enzyme.
Their
effect is always positive.


For
example, O
2 and
Co are homotropic
allosteric
modulators of hemoglobin.
 

48. Heterotropic
effect:
A
heterotropic allosteric modulator is a regulatory molecule
that
is not the enzyme's substrate. It may be either an activator or an inhibitor of
the
enzyme.


For
example, H
+
,
CO
2,
and 2,3-bisphosphoglycerate are heterotropic allosteric
modulators
of hemoglobin.
The effectors do not bind in the active site

Activator: R state is stabilised
Inhibitors: T state is stabilised
Heterotrophic interactions are either
positive or negative.
 

49. Allosteric inhibition
The
effector may be different from the substrate, in this
case
effector is said to be heterotropic effector.




For
example the feedback mechanism.
Feedback inhibition
is a form of allosteric regulation in
which
the final product of a sequence
of enzymatic reactions
accumulates in abundance. With
too
much of this product produced, the final product
binds
to an allosteric site on the first enzyme in the series
of
reactions to inhibit its activity.

50. A
B C D E
•A
is the initial substrate, B,C and D are the intermediates
and
E is the end product, in a pathway catalyzed by four
different
enzymes (e
1,e2,e3,e4).
The very first step
(A
B by the enzyme e
1)
is the most effective for
regulating
the pathway by the final product E. This type
of
control is often called negative feedback regulation
since
increased levels of end product will result in its (e
1)

decreased
synthesis.
•This
is a real cellular economy to save the cell from the
wasteful
expenditure of synthesizing a compound which
is
already available within the cell.
e3e1 e4e2

51. •Feedback
inhibition or end product inhibition is a
specialized
type of allosteric inhibition necessary
to
control metabolic pathways of efficient cellular
function.
•Aspartate
transcarbamoylase (ATCase) is a good
example
of an allosteric enzyme inhibited by
feedback
mechanism.

52. Sigmoid
curve of Allosteric Enzyme
•Allosteric
enzymes give a sigmoidal curve instead of
hyperbola
when the velocity (v) versus substrate (S)
concentration
are plotted.
•Michaelis-Menten
kinetics do not apply to the Allosteric
enzymes.

53. APPLICATIONS
•Recently,
the combined use of physical techniques (for
example,
x-ray crystallography and solution small angle
x-ray
scattering or SAXS) and genetic techniques (site-
directed
mutagenesis or SDM) has enabled researchers to
investigate
more deeply the molecular basis of allostery.
•The
Escherichia coli enzyme aspartate
carbamoyltransferase
(ATCase) has established itself as
one
of the model system's for allosteric regulation.
•Long-range
allostery is especially important in cell
signaling.

•Pharmacology

55. •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.
•Not
altered or consumed during reaction.
•Reusable
•The
area on the enzyme where the substrate or
substrates
attach to it is called the
active site.
•Enzymes
are usually very large proteins and the
active
site is just a small region of the enzyme
molecule.
•In
the 
active site,
the substrate binds and the
chemical
reaction takes place. Amino acids of
the enzyme protein
will bind to the substrate.

56. •An enzyme attracts
substrates to its active site, catalyzes
the
chemical reaction by which products are formed, and
then
allows the products to dissociate (separate from
the enzyme surface).

•The
combination formed by an 
enzyme and
its substrates
is
called the 
enzyme–substrate complex.
•Enzymes
increase the rate of chemical reactions by
lowering
the free energy barrier that separates the
reactants
and products

57. Enzymes
Lower a
Reaction’s

Activation
Energy

59. Enzyme-substrate
complex
Step
1:
•Enzyme
and substrate combine to form
complex
•E
+ S ES
•

Enzyme
Substrate Complex
+

60. Enzyme-product
complex
Step
2:
•An
enzyme-product complex is formed.
EES

S

EEPP
EESS EEPP
transition transition
statestate

61. Product
Step
3:
•The
enzyme and product separate
•EEPP



EE
+

+
PP
The product
is made
Enzyme is
ready
for
another
substrate.
EEPP

62. •There
are two theories that describe the formation
of
Enzyme substrate complex and dissociation of
products
from enzyme (separate from the enzyme
surface).
•They
are:
‒Lock
and Key Model
‒Induced
Fit Model

63. Lock
and Key Model
•In
1894, Emil Fischer proposed this 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
•This
is an older model, however, and does not work for all
enzymes

65. Induced
Fit Model
•In
1958, Daniel Koshland suggested a modification to the lock
and
key 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
maximumize
the fit, which improves catalysis
-
there is a greater range of substrate specificity
•This
model is more consistent with a wider range of enzymes

68. Active site:
area on the enzyme where the
substrate or substrates attach to is
called the active site.
Enzymes are usually very large proteins and
the active site is just a small region of the
enzyme molecule.
Enzyme molecules contain a special pocket or
cleft called the active sites.

70. Enzymes
Figure 5.3
Apoenzyme: protein
Inactive
Cofactor: Nonprotein component
NAD+, (NADH)
NADP+, (NADPH)
FAD
Coenzyme: Organic cofactor
Vitamins
Coenzyme A
Holoenzyme: Apoenzyme + cofactor
Active

71. Isoenzymes
•Different
enzyme molecules that catalyze the same
reaction
are called "isoenzymes," 'isozymes," or
"multiple
molecular forms."
•Isozymes
were first described by R. L. Hunter and
Clement
Markert in 1957.
•Their
discovery has encouraged further searches for
organ-specific
catalytic proteins.
•Occasionally,
several different enzyme molecules, all
of
which catalyze the same chemical reaction, have
been
isolated from a single tissue. Such families of en­
zymes
are called isoenzymes or isozymes.

72. Reasons for isoenzyme
•Synthesized
from different genes (malate
dehydrogenase
in cytosol versus in mitochondria)
•Oligomeric
forms of more than one type of
subunits
(lactate dehydrogenase)
•Different
carbohydrate content (alkaline
phosphatase)

73. •Isozymes
are homologous enzymes that catalyze the same
reaction
but differ in structure. The differences in the
isozymes
allow them to regulate the same reaction at
different
places in the specie. In particular they differ in
amino
acid sequences.
•They
display different kinetic parameters as well as
regulatory
properties. For example, isozymes have
different
K
M and
V
max values,
and can be distinguished
from
one another by biochemical properties such as
electrophoretic
mobility.
•Different
isoenzymes are often organ-specific and their
determination
may improve the specificity of enzyme tests.
Characteristics

74. •The
heterogeneity of some isoenzymes is due to
different
protein subunits which are coded for by
separate
genes.
•Isozymes
are encoded by different genes and expressed
in
a distinct organelle or at a distinct stage of
development.
• The
purpose of isozymes is to allow fine adjustment of
metabolism
to meet the need of different development
stages
and help the different tissues and organs
function
properly depending on their physiology make
up
and in what kind of environment which they
function.
•Isozymes
appear in specific regions of the body;
differing
in specifics organelles or tissues.

75. •The
level of the different isozymes in a certain organ
is
related to the level of oxygen supply.
•In
terms of kinetics, isoenzymes have the capability to
fine
tune their enzymatic rate constants K
M
 and
K
cat
.

This
adaptation allows for the proper use of the
enzyme
based on its environment (e.g. lactate
dehydrogenase
isozymes present in the heart and in
the
liver, where O
2
 is
abundant in heart but not so in
the
liver).
•Isozymes
are enzymes that have different structures
but
carry out the same tasks.
• A
biochemical assay is needed to differentiate
between
different isozymes.

76. •Isoenzymes
are enzymes that catalyze identical
chemical
reactions but are composed of different
amino
acid sequences.
•Isoenzymes
are produced by different genes and
are
not redundant despite their similar functions.
•They
occur in many tissues throughout the body
and
are important for different developmental and
metabolic
processes

77. Diferences in Isoenzymes
• Physicochemical


1.
Differences in secondary and tertiary structure


(folding
of polypeptide chains),


2.
Different degrees of polymerization to dimers,


tetramers,
etc.
•Immunochemical


1.
Different reactivity with specific antibodies.
•Chemical


1.
Variations in degree of deamination of carboxylamide


groups
or acetylation of amino groups.


2.
Variable combinations with carrier proteins,


carbohydrates,
coenzymes, prosthetic groups or lipids.

78. 3.
Different degrees of activation or inactivation by


hydrolytic
cleavage of terminal peptides, oxidation or


reduction
of coenzyme or sulfhydryl groups.


4.
Varying degrees of amino acid differences.
Among
the various isoenzymes the lactic dehydrogenases
have
been most extensively studied, and five different forms
have
been identified.
This
enzyme is used to catalyze the synthesis of glucose in
anaerobic
metabolism of glucose. The isozymes of this
enzyme
are divided into two forms, the H isozyme and the M
isozyme.

79. The
H isozyme is expressed more in the heart,
whereas
the M isozyme is expressed more frequently
in
the skeletal muscle. Both isozymes have two
polypeptide
chains, and each isozyme share 75% of
the
amino acid sequence for the chains. Both isozymes
metabolize
glucose, but the difference is that the H
isozymes
have a higher affinity for their substrates
than
the M isozyme does. Another difference is that
the
H isozyme functions better in aerobic
environments
such as the heart, whereas the M
isozyme
functions better in anaerobic environments
such
as the muscle, where strenuous activity may
deplete
the oxygen supplies.

81. Methods for Detection of Isoenzymes
•Physicochemical


I.
Separation by electrophoresis.


2.
Separation by chromatography
•Immunochemical


l.
Combination with or inhibition by specific


antibodies.

•Chemical


1.
Rate of reaction under various conditions of pH,


temperature,
inhibitors, coenzyme analogues, or


substrate
concentration.

82. Isoenzymes
can be differentiated from one another using gel
electrophoresis.
In gel electrophoresis, isoenzyme fragments
are
drawn through a thick gel by an electric charge. Each
isoenzyme
has a distinct charge of its own because of its
unique
amino acid sequence. This enables gel
electrophoresis
to separate the fragments into bands for
identification.

83. Significance of isoenzymes
Isozymes
in general can be used to meet the metabolic
needs
of different tissues and developmental stages.
Isozymes
may also be utilized to diagnose tissue damage
such
as damaged heart muscle cells during a heart attack or
myocardial
infarction. When heart muscle cells are damaged,
they
release the cellular material such as the H isozyme.
When
taking blood samples, if the H isozymes appear in
increased
levels, then there is a possibility that the heart cells
are
damaged.
Isoenzymes
are useful biochemical markers and can be
measured
in the bloodstream to diagnose medical conditions.

84. •Some clinically important isoenzymes are as follows:
1)Lactate
dehydrogenase (LDH): Lactate dehydrogenase
catalyzes
the reversible conversion of pyruvate and
lactate.
LDH is essential for anaerobic respiration. When
oxygen
levels are low, LDH converts pyruvate to lactate,
providing
a source of muscular energy.
2)Creatine
Kinase (CK, CPK) is an enzyme found primarily
in
the heart and skeletal muscles, and to a lesser extent in
the
brain but not found at all in liver and kidney. Small
amounts
are also found in lungs, thyroid and adrenal
glands.
Significant injury to any of these structures will
lead
to a measurable increase in CK levels. It is not found
in
red blood cells and its level is not affected by
hemolysis.

85. 3.Alkaline phosphatase (ALP )-is
an enzyme that removes

phosphate groups from organic or inorganic compounds in
the
body. It is present in a number of tissues including liver,
bone,
intestine,and placenta. The activity of ALP found in
serum
is a composite of isoenzymes from those sites and, in
some
circumstances, placental or Regan isoenzymes. The
optimum
pH for enzyme action varies between 9-10. It is a
zinc
containing metalloenzyme and is localized in the cell
membranes
(ectoenzyme). It is associated with transport
mechanism
in the liver, kidney and intestinal mucosa.
4. Aspartate amino Transferase (AST): It
is also called as
Serum
Glutamate Oxalo acetate Transaminase (SGOT).
5. Alanine amino transferase (ALT)-
Also called serum
Glutamate
pyruvate transaminase.