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Title: Enzyme
Description: Enzyme - classification and functions

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Enzymes

Enzyme Classification
 Enzymes are classified according to the type
of reaction they catalyse
...


 According to the International Union of Biochemistry
and Molecular Biology (IUBMB) system, enzymes are
classified into seven major classes
 Enzymes are classified according to the type of reaction
they catalyse
...


The seven classes as per IUBMB are as follows:
1
...
EC-2: Transferase
3
...
EC-4: Lyase
5
...
EC-6: Ligase
7
...


Enzymes in this category include :
 Dehydrogenases
 Reductases
 Oxidases
 Peroxidases
...
from one
molecule to another

Enzymes in this category include :

 Amino transferase or transaminase
 Kinase: catalyzes the transfer of phosphate groups
 Transcarboxylase
...


Enzymes in this category are:
 All digestive enzymes like:
- α-amylase,
- pepsin,
- trypsin,
- chymotrypsin, etc
...


In cases where reverse reaction is important, then
synthase, (not synthetase of group EC-6) is used
in the name
...
They are called epimerases, isomerases or
mutases, depending on the type of isomerism involved
...


EC-7: Translocases (A new EC Class)

Translocases catalyze the movement of ions or
molecules across membranes or their separation
within membranes
...


Examples are:
 Enzymes catalyzing the translocation of:
Hydrons (H+), inorganic cations, inorganic anions, amino
acids and peptides, and carbohydrates and their derivatives
...


Zymogen OR Proenzyme
 Enzymes found in in an inactive (precursor)
form, called zymogen or Proenzyme
...


 For example,
- prothrombin,
- proelastase
- chymotrypsinogen,
- trypsinogen,
- pepsino- gen

Cofactors (Coenzyme And Activator)
 Some enzymes require an additional non-protein
component for its activity
...




Organic compounds, called coenzymes

 Enzymes without its cofactor is referred to as
an apoenzyme
 The complete catalytically active enzyme is
called holoenzyme
...


MECHANISM OF ENZYME ACTION

Formation of an enzyme-substrate (ES) complex is
the first step in enzymatic catalysis which is
subsequently converted to product and free enzyme
...

The active site of an enzyme is the region that
binds the substrate and which contains the
specific amino acid residues
...
Lock and key model or rigid template model of
Emil Fisher
...
Induced fit model or hand-in-glove model of
Daniel E Koshland

Lock and Key Model or Rigid Template Model
of Emil Fisher
 Enzyme is pre-shaped and the active site has a
rigid structure, complementary to that of the
substrate
...


Representation of Fisher’s lock and key model
...


Induced Fit Model or Hand-in-glove Model of
Daniel Koshland
Enzymes are flexible
Shapes of active site can be modified by the
binding of the substrate
...


Conformational change in enzyme induces
reciprocal changes in its bound substrate that
alters their orientation and configuration and
strains the structure of the bound substrate
...

Intrinsic binding energy converts substrate into
product
...

 Enzymes are highly specific both in the reaction catalyzed
and in their choice of substrates
...


Types of Specificity

1
...


Reaction specificity

3
...


Absolute substrate specificity

ii
...


Broad substrate specificity
...
g
...


Relative substrate specificity
Enzyme acts on more than one substrate
...


Chymotrypsin acts on several proteins by
hydrolyzing peptide bonds attached to aromatic
amino acids
...


α-amylase, cleaves α-(1→4) glycosidic bonds
of carbohydrates
...


Broad substrate specificity
 Enzyme acts on more than one structurally
related substrates
...


Reaction Specificity
Enzyme is specific to a particular reaction but
not to substrate (s) and catalyzes only one type
of reaction
...


Stereo Specificity


L-lactate dehydrogenase will act only on
L-lactic acid and not D-lactic acid
...




Salivary α-amylase acts on the α-1,4
glycoside linkage and is inactive on β-1,4
glycoside bond

Factors Affecting The Velocity Of
Enzyme Reaction



Substrate concentration



Enzyme concentration



pH i
...
H+ ion concentration



Temperature



Product concentration



Activators and coenzymes



Time



Physical agents

Effect of Substrate Concentration

V0

:

initial velocity

Vmax :

maximum velocity

Km

:

1/2 Vmax = Michaelis Menten constant

[S]

: substrate concentration

Effect of Enzyme Concentration

Effect of Hydrogen Ion Concentration pH
 Each enzyme has an optimum pH, i
...
a pH at which
the enzyme activity is maximum
...

 The optimum pH differs from enzyme to enzyme
...
2



Trypsin = 8
...




The ionization state of the substrate
...

 Each enzyme shows the highest activity at a particular
temperature called optimum temperature
...


Effect of temperature on enzyme activity

Increase in velocity is due to the increase in the
kinetic energy
...


Low temperature also decreases enzyme activity
and enzymes may be completely inactive at
temperature of 0°C and below
...


Most of the body enzymes have the optimum
tempe- rature close to 37°C to 38°C and have
progressively less activity as the temperature
rises
...


Effect of Activators and Co-enzymes
In absence of activators and coenzymes, enzymes
become functionally inactive
...

The time required for the completion of an
enzyme reaction increases with changes in
temperature and pH from its optimum
...


 One of the key factors affecting the enzyme reaction rates
is the concentration of substrate [S]
...


Km provides a amount of the substrate required for
significant catalysis to occur
...


It is a measure of the affinity of the enzyme for its
substrate, a high Km indicates weak binding and a
low Km indicates strong binding with its substrate
...


Significance of Vmax
 The Vmax of a reaction is an index of the catalytic
efficiency of an enzyme
...


Lineweaver-Burk plot
(Double reciprocal plot)

ENZYME INHIBITION
Any substance that can diminish the velocity of
an enzyme reaction is called inhibitor
...
Reversible inhibitor
2
...


REVERSIBLE INHIBITOR

Reversible inhibitors bind to enzymes through
non-covalent bonds and the activity of the enzyme
is restored fully when the inhibitor is removed
from the system
...
Competitive or substrate analogue inhibitor
ii
...
Uncompetitive inhibitor
...

Chemical structure of inhibitor (I) resembles that
of substrate (S) and binds to enzyme at active
site, forming EI complex rather than ES-complex
...

 Isoniazide [Isonicotinic acid hydrazine (INH)]
It is an anti-tuberculosis drug, inhibits the biosynthesis of
NAD and restrict the growth of the organisms that cause
tuberculosis
...
It inhibits the vitamin K activity and inhibits the
formation of prothrombin
...


Drugs such an ibuprofen (anti-inflammatory drug),
statin (cholesterol lowing drug) are competitive
inhibitors of enzymes, that involved in the
prostaglandins and cholesterol synthesis
respectively
...

 Inhibitor is usually structurally different from the
substrate
...


 Noncompetitive inhibitor can bind free enzyme
(EI) or the enzyme substrate complex (EIS)
 However, EIS complex does not continue to form
product
...

 Noncompetitive inhibition cannot be overcome by
increasing the substrate concentration

Diagrammatic representation of noncompetitive inhibition
...




Trypsin inhibitors occur in soybean and raw egg
white, inhibit activity of trypsin
...


For non-competitive inhibition, the Km value is unchanged while
Vmax is lowered

Uncompetitive Inhibitor
 Uncompetitive inhibitor can bind only to the
enzyme-substrate (ES) complex
...

 Enzyme-substrate-inhibitor complex, ESI does
not continue to form any product
...

 Consequently Vmax cannot be attained, even at
high substrate concentration
...


Uncompetitive inhibitor decreases both Vmax and Km
...


IRREVERSIBLE INHIBITOR

An irreversible inhibitor binds with an enzyme
tightly covalently and forms a stable complex
...


Irreversible inhibitors can be divided into three
categories:
 Substrate analogue inhibitor or affinity labels
 Group specific inhibitors
 Suicide inhibitor or mechanism based
inactivation
...

 These substrate analogues possess a highly reactive
group which is not present in the natural substrate
...


Group Specific Irreversible Inhibitor
 These inhibitors react with specific R-groups
(side chain) of amino acid residues in the
active site of enzyme
...


 Iodoacetamide and heavy metals like, Pb2+, Ag+,
Hg2+, etc
...


Suicide Inhibitor or Mechanism Based inactivation
 These compounds are relatively unreactive until they bind
to the active site of a specific enzyme
...


 Instead of being transformed into a normal product,
however, the inhibitor is converted to a very reactive
compound that combines irreversibly with the enzyme
leading to its irreversible inhibition
 These are also called mechanism based inactivation
because they utilize the normal enzyme reaction
mechanism to inactivate the enzyme

Example Suicide Inhibitor
Penicillin
Inactivates bacterial enzyme glycopeptidyl
transpeptidase involved in the formation of bacterial
cell wall
...


 Disulfiram (antabuse)
 Inhibits aldehyde dehydro- genaseenzyme resulting in
accumulation of acetaldehyde
...


Clinical Application of Enzyme Inhibitor

 Enzyme inhibitors have therapeutic applications
...


ALLOSTERIC ENZYME

Allosteric enzyme is a regulatory enzyme
...


Allosteric enzymes are those having other site in
addition to active site for binding of modulator
(regulatory metabolites)
...
Whereas those that increase
enzyme activity are called positive modulators
...

 Isoenzymes show different chemical and physical
properties like electrophoretic mobility and kinetic
properties
...


 For example:

1
...
Creatine kinase (CK)

Lactate Dehydrogenase (LDH)

 Lactate dehydrogenase is a tetrameric enzyme that
catalyzes the oxidation of L-lactate to pyruvate
...


 LDH has five isoenzymes:


LDH1



LDH2



LDH3



LDH4



LDH5
...


 LDH1 is the fastest moving fraction towards the anode
and LDH5 is the slowest moving isoenzyme of LDH
...
Significant elevation of LDH1 and LDH2 occurs within
24 to 48 hours after myocardial infarction
...
Predominant elevation of LDH2 and LDH3 occur in
leukaemia
...
LDH3 elevated in malignancy of many tissues
...
Elevation of LDH5 occurs after damage to the liver or
skeletal muscle
...

1

CK1 (BB) : present in the brain

2

CK2 (MB) : present only in Cardiac tissue

3

CK3 (MM) : present in skeletal muscle

Clinical Application
1
...
Increased level of CK2 occurs in myocardial infarction Cardiac
tissue is the only tissue which has mixed MB (CK2) isoenzyme
...
CK-MB isoenzyme starts to increase within 4 hours after an acute
myocardial infarction (AMI) and reaches a maximum within 24 hrs
...
Elevated levels of CK3 in serum occur in dystrophies and
myopathies
...


Diagnostic Use of Enzymes
The enzymes that are found in plasma can be
categorized into two major groups:
 Plasma specific enzyme
 Plasma nonspecific enzyme
...


These enzymes are clinically of interest when their
concentration decreases in plasma
...


 Estimation of plasma nonspecific enzymes is very
important for the diagnosis of several disease
...

 The plasma ALT normal value for adult is 10 to 40 U/L
...


 Aspartate transaminase (AST)

 It was known formerly as glutamate oxaloacetate
transminase (GOT)
...

 Increased AST level occurs after myocardial infarction
...


The plasma AST level starts increasing after 6 to 8 hours
after the onset of chest pain with peak values 18 to 24 hours
and the values fall to normal level by the fourth or fifth day
...

 Normal serum level for adults is 3-13 KA units/dl
...

 Very high levels may be noticed in obstructive
jaundice, bone diseases such as Paget’s disease, rickets,
osteomalacia, carcinoma of bone and
hyperparathyroidism

Acid phosphatase (ACP)
 It hydrolyzes phosphoric acid ester at pH 5 to 6
...
5 to 4 KA units/dL
...
ACP is therefore an
important tumor marker
...


 Normal serum value is 50-120 U/L
...


Creatine kinase (CK) : Refer isoenzyme
...


Enzymes as Tumor Marker
Elevated enzyme levels may signal the presence of
malignancy
...


Nonenzyme proteins includes :

 Myoglobin (Mb)
 Cardiac troponin T and I (cTnT and cTnI)
...


Enzyme Assays in Liver Diseases
1
...

ALT is the more liver-specific enzyme
...
Enzymes in cholestasis:
 Alkaline phosphatase
 5’-nucleotidase
 γ-glutamyl transferase
Title: Enzyme
Description: Enzyme - classification and functions