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ENZYMES
By:- K NABEEN PATRA
Contents
• Terminology
• Introduction
• Properties
• Nomenclature and classification
• Mechanism of action
• Kinetics
• Factors affecting
• Enzyme Inhibitions
• Applications
• Coenzymes and its functions
Terminology
ATP: Adenosine triphosphate
ADP: Adenosine diphosphate
NAD: Nicotinamide Adenine Dinucleotide
NADP: Nicotinamide Adenine Dinucleotide Phosphate
GDP: Guanosine diphosphate
GTP: Guanosine triphosphate
FMN: Flavin Mono Nucleotide
FAD: Flavin Adenine Dinucleotide
TPP: Thiamine Pyrophosphate
LDH: Lactate dehydrogenase
CPK: Creatinine phosphokinase
SGOT: Serum glutamic oxaloacetic transaminase
SGPT: Serum glutamic pyruvic transaminase
Importance
• Enzymes play an important role in Metabolism, Diagnosis,
and Therapeutics.
• All biochemical reactions are enzyme catalyzed in the
living organism.
• Level of enzyme in blood are of diagnostic importance e.g.
it is a good indicator in disease such as myocardial
infarction.
• Enzyme can be used therapeutically such as digestive
enzymes.
• Enzymes are biological catalyst that speed up the rate of
reaction.
• Enzymes regulate the rate of physiological process.
 When cells are injured, enzyme leak into plasma. The
measurement of activity of such enzymes in plasma is an
integral part of medical diagnosis.
 Enzymes are used as drugs. Eg. Streptokinase which can
solubilise the blood cloting.
 Asparaginase as anticancer agent.
 Enzymes are used as cleansing agent in detergent industry.
 Enzymes are used as bio-sensors.
 AIDS detection involve used of enzyme dependent ELISA
technique.
 The immobilised enzymes are used for diagnostic purpose.
Define enzymes
(Enzymes as Biological Catalysts)
• Enzymes are proteineous in nature except
Ribozyme.
• 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
Properties of enzymes
 Enzymes are required only in small amounts.
 They quicken the reactions without being
consumed or lost in the process.
 Enzymes are proteins. Like all proteins, enzymes
are colloidal in nature and precipitated by salt
solutions.
 They are inactivated by heat and alteration of pH.
 Enzymes have great specificity.
 Each enzyme catalyses one particular reaction.
Properties of enzymes
• Enzymes are protein in nature but not vice
versa.
• Enzymes are bigger molecules as compared to
substrate molecules.
• Molecular weight varies from some thousand to
some Millions.
• They increase the rate of reaction to 105
to 10-10
times.
Localisation of Enzymes
Enzymes may be present inside the cell or
outside the cell.
For example:
•Enzymes of Citric acid cycle are present in
the mitochondria.
•Enzymes of glycolytic pathway are present in
the cytoplasm.
•Enzymes for the transport of nutrients are
present in the cell membranes.
Activation energy or Energy of Activation
• All chemical reactions require some amount of
energy to get them started.
OR
• It is First push to start reaction.
This energy is called activation energy.
 Enzymes catalyse various biochemical reactions.
 Normally, for any reaction to occur, the reacting
molecules must come in contact.
 For this, the molecules must gain a minimum amount
of energy. This is called energy of activation.
 Normally, the energy of activation can be lowered by
increasing the temperature.
 But in the human body, the temperature is constant.
So, enzymes act by lowering the energy of activation at
normal body temperature.
Energy of activation
Classification of Enzymes
Enzymes are sometimes considered under two
broad categories:
Intracellular Enzymes:
They are functional within cells, where they are
synthesized.
Extracellular Enzymes:
These enzymes are active outside the cell; All the
digestive enzymes belongs to this groups.
Nomenclature and classification of Enzymes
• The names of most of the enzymes end with the three
letters –ase
• This is a suffix which is added to the name of the
substrate on which the enzyme acts.
• For example, sucrase catalyzes the hydrolysis of sucrose
• The name describes the function of the enzyme
For example, oxidases catalyze oxidation reactions
• Sometimes common names are used, particularly for the
digestion enzymes such as pepsin and trypsin
• Some names describe both the substrate and the function
• For example, alcohol dehydrogenase oxides ethanol
Enzymes Are Classified into six functional
Classes (EC number Classification) by the
International Union of Biochemists (I.U.B.).
on the Basis of the Types of
Reactions That They Catalyze
• EC 1. Oxidoreductases
• EC 2. Transferases
• EC 3. Hydrolases
• EC 4. Lyases
• EC 5. Isomerases
• EC 6. Ligases
The classification of enzymes can be remembered by using
the tip “OTHLIL”
Principle of the international
classification
Each enzyme has classification number
consisting of four digits:
Example, EC: (2.7.1.1) HEXOKINASE
• EC: (2.7.1.1) these components indicate the following
groups of enzymes:
• 2. IS CLASS (TRANSFERASE)
• 7. IS SUBCLASS (TRANSFER OF PHOSPHATE)
• 1. IS SUB-SUB CLASS (ALCOHOL IS PHOSPHATE
ACCEPTOR)
• 1. SPECIFIC NAME
ATP,D-HEXOSE-6-PHOSPHOTRANSFERASE (Hexokinase)
H O
OH
H
OHH
OH
CH2OH
H
OH
H H O
OH
H
OHH
OH
CH2OPO3
2−
H
OH
H
23
4
5
6
1 1
6
5
4
3 2
ATP ADP
Mg2+
glucose glucose-6-phosphate
Hexokinase
1. Hexokinase catalyzes:
Glucose + ATP  glucose-6-P + ADP
Oxidoreductases, Transferases and Hydrolases
Lyases, Isomerases and Ligases
Mechanism of Enzyme action
• The active site of the enzymes represents a small region at
which a substrate binds and participates in the catalysis.
It consists of two parts: 1. Catalytic site and 2. Binding site
 Catalytic site: It is the portion of enzyme that is responsible
for catalysis. It determine the reaction specificity.
 Binding site: It is the part of enzyme that binds the
substrates. It determines the substrate specificity.
• The active site of enzymes are present within the enzyme
molecules.
• The active site is 3-D and consists of few amino acid
residues.
• The active site is not rigid structure and shape. It is
rather flexible to promote the specific substrate
binding.
• The active site is contributed by the amino acids
such as serine, aspartate, histidine, cysteine, lysine,
arginine, glutamate, tyrosine etc.
• Which are far from each other in the linear
sequence of amino acids. During catalysis, they are
brought together.
• The substrates binds at the active site by weak non-
covalent bond. The enzymes are specific in their
function due to the existence of active site.
ACTIVE SITES
• Enzyme molecules contain a special pocket or
cleft called the active sites.
Mechanism of Action of Enzymes
• Enzymes increase reaction rates by
decreasing the Activation energy:
• Enzyme-Substrate Interactions:
‒Formation of Enzyme substrate
complex by:
‒Lock-and-Key Model
‒Induced Fit Model
Enzymes
Lower a
Reaction’s
Activation
Energy
Activation energy
for Catalyzed &
uncatalyzed
reaction
Lock-and-Key Model
• This theory was proposed by the German scientist
Imil-Fischer in 1894.
• According to this model, the active site is the rigid
portion of enzyme molecules and its shape is
complementary to the substrate molecule like
Lock and Key.
• The complementary shape of the substrate and
active site favour tightly bound enzyme-substrate
complex formation followed by catalysis.
Lock-and-Key Model
• In the lock-and-key model of enzyme action:
- the active site has a rigid shape
- only substrates with the matching shape can fit
- the substrate is a key that fits the lock of the
active site
In the lock-and-key model of enzyme action:
•This explains enzyme specificity.
•This explains the loss of activity when enzymes denature.
•This is an older model, however, and does not work for all
enzymes
Induced Fit Model
 This model was proposed by Kashland in 1958.
 According to this model, the active site is flexible.
 In the enzyme molecule, the amino acid residue that make up
the active sites are not oriented properly in the absence of
substrates.
 When the substrate combines with the enzyme, it induces the
conformational changes in the enzyme molecules in such a way
that amino acids that make active sites are shifted into correct
orientation to favour tightly bound enzyme substrate complex
formation followed by catalysis.
 The enzyme molecule is unstable in the induced conformations
and returns to its native conformations in the absence of
substrate.
Induced Fit Model
• In the induced-fit model of enzyme action:
- the active site is flexible, not rigid
- the shapes of the enzyme, active site, and substrate
adjust to 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
Enzymes
Enzyme-substrate complex
• Step 1:
• Enzyme and substrate combine to form
complex
• E + S ES
• Enzyme Substrate Complex
+
Enzyme-product complex
• Step 2:
• An enzyme-product complex is formed.
• EESS EPEP
EESS EPEPtransitiontransition
statestate
Product
• The enzyme and product separate
• EPEP EE ++ PP The product
is made
Enzyme is
ready
for
another
substrate.
EPEP
Enzyme Specificity
• Enzymes have varying degrees of specificity for
substrates
• Enzymes may recognize and catalyze:
- a single substrate
- a group of similar substrates
- a particular type of bond
- a particular type of optical isomer of the
substrate (optical specificity). E.g. D-amino acid
oxidase acts only on D- amino acid & L -amino
acid oxidase acts only on L- amino acid
Enzymes
ENZYME KINETICS
Michaelis curve:
The velocity of an enzyme reaction increases with an increase in substrate
concentration.
After a certain limit, it become constant.
So, further increase in substrate concentration has no effect.
 If the velocity of the reaction is plotted against substrate concentration, a typical
hyperbolic curve is obtained.
The explanation for such a behaviour of the enzyme is as follows:
At low concentration of the substrate, all the enzyme molecules are not utilised in
the formation of enzyme substrate complex.
 As the substrate concentration is increased, more and more enzyme molecules
are utilised. So, there is proportional increase in velocity.
Vmax is reached when all the enzyme molecules are saturated with the substrate
So, there is no increase in velocity. The curve is called Michaelis curve.
The mathematical equation is
called Michaelis menton
equation, which is as follows.
Vo = Vmax[S]/Km+[S]
Where Vo= Initial velocity
Vmax= Maximal velocity
[S]= Substrate concentration
Km= Michaelis constant.
Km is equal to 1/V. i.e. Km is
equivalent to substrate
concentration required to
produce half maximal velocity.
Significance of Km
1.It is an enzyme kinetic constant.
2.It indicates the substrate concentration required
for the enzyme to work efficiently.
3.Low Km indicates high affinity of enzyme towards
substrate. High Km indicates low affinity of enzyme
towards substrate. Hence Km and affinity are
inversely related.
4.Km is required when enzyme are used as drugs.
5.Use of enzyme in immunodiagnostics (ELISA)
require Km of the enzyme.
LINEWEAVER-BURK PLOT:
(Double reciprocal plot)
The drawbacks of Michaelis curve are:
1.Only an approximate but not an accurate value of Km
can be obtained.
2.It is difficult to determine Vmax accurately. It is
because, Vmax is only approached and never attained.
3.It is only a hyperbolic curve and not a straight line
graph. So interpolation of data is not possible.
These drawbacks are overcome by a straight line graph
called Lineweaver-Burk plot. It makes use of reciprocals
of Vo and [S] i.e. 1/Vo and 1/[S].
As we have already seen, Michaelis
Menton equation is
Vo= Vmax [S]/Km+[S]
Reciprocal of this equation is
1/Vo= Km+[S]/Vmax[S]
It can be rewritten as
1/Vo= Km/Vmax[S] + [S]/Vmax[S]
It can be simplified as:
1/Vo= Km/Vmax X 1/[S] + 1/Vmax
The above eqn. represent Y=mx+C,
straight line eqn. with slope of
Km/Vmax. Since 1/S & 1/V are
reciprocal of S & V respectively, It is
known as double reciprocal plot.
The above equation is called as
Lineweaver-Burk equation.
Now, a graph can be constructed by plotting 1/vo on the y-axis
and 1/[s] on the x-axis. This gives a straight line.
From this graph three points are very clear and they can be
easily determined:
1.Slope of the curve = Km/Vmax
2.1/Vmax is the intercept of this slope on 1/Vo. From this Vmax
can be calculated easily.
3.Extension of this to line to the negative side cuts the x-axis at a
point equal to -1/Km. from this Km can be easily calculated.
4. In addition to Km and Vmax values, type of inhibition is
determined using this plot.
5. Inhibition constant (Ki) of inhibitor is also determined using
this plot.
Significance of (Ki)
1.Ki indicates affinity of inhibitor towards
enzyme like Km, Ki is inversely related to
affinity.
2.Use of inhibitors as drugs require knowledge of
Ki. Since Ki and affinity are inversely related
inhibitors of low Ki are highly potent drugs.
Thermodynamics of enzymatic reactions
The enzyme catalysed reactions may be broadly grouped
into three types based on thermodynamic (energy)
considerations.
Isothermic reactions: The energy exchange between
reactants and products is negligible e.g. Glycogen
phosphorylase.
Glycogen + Pi  Glucose 1-phosphate
Exothermic (exergonic) reactions: Energy is liberated in
these reactions e.g. Urease
Urea  NH3 + CO2 + energy
Endothermic (endergonic) reactions: Energy is consumed
in these reactions e.g. Glucokinase.
Glucose + ATP  Glucose 6-phosphate + ADP
Factors influencing Enzyme actionFactors influencing Enzyme action
The activity of enzyme can be modified by the following factorsThe activity of enzyme can be modified by the following factors..
1.Concentration of enzymes
2.Concentration of substrate
3.Contact between enzyme and substrate
4.Concentration of Product
5.Temperature
6.pH
7. Oxidising agent
8.Radiations
9.Activators (Coenzymes & Cofactors )
10.Inhibitors
Concentration of Enzyme
• As the concentration of the enzyme is increased,
the velocity of the reaction proportionately
increases.
• This is true only when sufficient substrate
molecules are available for combination with the
enzyme.
• When all the substrate molecules are saturated
with the enzyme, further increase in enzyme
concentration has no effect.
Substrate Concentration and Reaction
Rate• The rate of reaction increases as substrate
concentration increases (at constant enzyme
concentration)
• Maximum activity occurs when the enzyme is
saturated (when all enzymes are binding substrate)
Contact between enzyme and substrate
 For the enzyme activity the contact between
Substrate and enzyme is necessary.
 The type of contact also influence the enzyme
activity.
Concentration of Product
• If the product are accumulated then the
enzyme activity is decreased.
Environmental factorsEnvironmental factors
• Optimum temperature The temp at which enzymatic reaction occur
fastest.
 Extreme Temperature are the most dangerousExtreme Temperature are the most dangerous
 High tempsHigh temps may denature (unfold)denature (unfold) the enzyme.enzyme.
• Velocity of an enzyme reaction increases with
increase in temperature up to a maximum and then
declines.
• A Bell shaped curve is usually observed.
• Temperature co-efficient or Q10 is defined as increase
in enzyme velocity when the temperature is increased
by 100
C.
• For majority of enzymes Q10 is 2 between 00
C to 400
C.
• The optimum temperature for most of the enzymes is
lies between 400
C to 450
C.
• Exception cases, Like Venom phosphokinase,
Urease, Adenylate kinase etc.
Environmental factorsEnvironmental factors
• pH also affects the rate of enzyme-
substrate complexes
–Most enzymes have an optimum pH of
around 6-8 (neutral)
• However, some prefer acidic or basic conditions
• pH influences the enzyme activity and a Bell
shaped curve is normally obtained.
• Each enzyme has an optimum pH at which the
velocity is maximum.
Oxidising agent
• In the presence of Oxidising substances,
the –SH group (Sulfahydryl) is oxidised to
form disulphide linkage, which inactivate
the enzyme activity.
• To counteract this, the antioxidants like
Cysteine and glutathione are used.
Radiation
• X-rays, UV rays, Beta and gamma rays
produce inactivation of enzymes.
• They act by forming peroxides which
oxidise the enzymes and make them
inactive.
Activators (Cofactors)
These are non protein molecules required for activity of
some enzymes. They may be involved in catalysis or in
structure maintenance. There are two types of cofactors.
A.Organic cofactors 1.Prosthetic groups and
2. Coenzyme
B. Inorganic cofactors (metal ions)
Prosthetic groups are covalently attached to the enzyme
and they undergo change during catalysis but return to
their native sites at the end of the reaction.
APOENZYME and HOLOENZYME
• The enzyme without its non protein moiety is
termed as apoenzyme and it is inactive.
• Holoenzyme is an active enzyme with its non
protein component.
Definition of
Cofactors
& Coenzymes
Factors affecting enzyme activity: Co-enzyme &
Cofactors (Activators )
Factors affecting enzyme activity: co-enzyme &
cofactors
Comparison of Cofactors &
Coenzymes
Effects of Metal ions on enzymes (Inorganic
Cofactors)Several enzymes are depends on the presence of metal ions
like K+, Mg++, Zn++ and Cu++ for their activity. Metals help in
enzyme action by:
•Maintaining structural confirmation of enzymes.
•Promoting enzyme-substrate complex formation.
Metal containing enzymes can be classified as:
•Metal activated enzymes
•Metallo-enzymes
•Metal-dependent enzymes
•Metal activated enzmes: in presence of metals, some enzymes
get activated i.e. their activity increases many folds.
Example: Cl- It activates amylase and ACE
Ca2+ It activates trypsin
Metallo-enzymes:
In these enzymes, the metal ion is very tightly bound and it is an
integral part of enzyme molecule.
They participates in catalysis.
Example: Iron required for Cytochrome oxidase, catalase,
Xanthine oxidase.
Copper is required for cytochrome oxidase, superoxide
dismutase.
Zinc is required for carboxy peptidase, alkaline phosphatase.
Metal-dependent enzymes:
Metal is loosely associated with enzyme molecule or it may be
required for enzyme substrate complex formation.
In the absence of metal, enzyme may not intract with substrate
molecule or with coenzyme molecule.
Example: Mg2+ needed by enzyme using ATP Hexokinase,
pyruvate kinase.
Ca2+ required for the activity of calpain a calcium dependent
protease.
Inhibitors
• Substances that decrease the catalytic activity
of enzyme are called inhibitors.
• They may be protein or non-protein inhibitors.
• Enzymes can be inhibited by a number of
substances e.g. heavy metals like silver and
mercury, oxidising agents like iodine.
• The decrease in enzyme activity is called as
enzyme inhibition.
ENZYME INHIBITION
• Enzyme inhibitors are substances which lower down
the rate of enzyme reaction.
• They produce their effect by acting on the coenzyme,
apoenzyme or prosthetic group.
• Also they can act by inhibiting the combination of
the substrate with the enzyme.
Enzyme inhibition is classified as:
• Competitive inhibition
• Non-competitive inhibition
• Feed Back Inhibition
• Allosteric inhibition
Enzyme Inhibitors
• Competive - mimic substrate, may block active site, but
may dislodge it.
• The rate of formation of the product from ES complex is
same as that of the absence of inhibitor.
• So, velocity (Vmax) is not altered, but Km increases
(affinity of enzyme towards substrate decreases).
E + S - [ES]  E + P
E + I - [EI]  No Product
Vmax is unchanged.
Km is altered.
• The competitive inhibitors are called as antagonist or
antimetabolites of the substrate with which they compete.
• The use of antimetabolite in the treatment of disease is
called as chemotherapy.
EXAMPLES OF COMPETITIVE INHIBITION
 Statin Drug As Example Of Competitive Inhibition:
• Statin drugs such as lipitor compete with HMG-
CoA(substrate) and inhibit the active site of HMG
CoA-REDUCTASE (that bring about the catalysis of
cholesterol synthesis).
• Sulfonamide also acts as competitive inhibitor
against PABA.
• The drugs like Captoprile, Lisinopril and Enalopril
inhibit ACE.
Enzyme Inhibitors
• Noncompetitive
Non Competitive Inhibition
• In this type of inhibition no, competition occurs between
substrate and inhibitor to bind at active site of enzyme.
• Inhibitor is not structurally related to substrate.
• In addition inhibitor binds to some other site of enzyme which
is far off from active site.
E + S  [ES] + I  [ESI] E + P (Slow)
E + I  [EI] + S  [EIS] E + P (Slow)
• Inhibitor can react with free enzyme as well as ES complex.
• Here the Vmax is decreased and Km (affinity) remains same
because no competition of substrate and inhibitor in non-
competitive inhibition.
• Most of non-competitive inhibitors are irreversible.
• They are referred as enzyme poisons.
EXAMPLES OF UNCOMPETITIVE INHIBITION
 Drugs to treat cases of poisoning by methanol or ethylene
glycol act as uncompetitive inhibitors.
 Tetramethylene sulfoxide and 3- butylthiolene 1-oxide are
uncompetitive inhibitors of liver alcoholdehydrogenase.
 Iodoacetate block the formation of 1,3-biphosphoglycerate
from glyceraldehyde-3-P by inhibiting the enzyme
glyceraldehyde-3-P dehydrogenase.
 Fluoride blocks the action of enolase which converts 2-
Phosphogluconate to phosphoenol pyruvate.
 Heavy metals like Hg2+
, Ag+
, Pb2+
and Arsenic are also
enzyme poisons. They interact with –SH Sulfhydryl groups at
the active site of the enzyme.
• Non-competitive inhibitors are used as pesticides. Eg,
DDT, Melathione, Parathione are inhibitors of the
enzyme Choline esterase that catalyses hydrolysis of
Ach.
• CN-
inhibit cytochrome oxidase (cyanide).
• EDTA inhibits metalloenzymes by forming complex
with metal ion.
• Tubers, bananas and beans contain inhibitors to
trypsin, chymotrypsin and elestase.
• Di-isopropyl fluro phosphate (DFP) is a non-
competitive inhibitor used as nerve gas in World War
II. It causes constriction of larynx, pain in eyes and
mental confusion.
Difference between Competetive and Non-
Competetive inhibition
SI.
No
Competetive inhibition Non-Competetive inhibition
1 There is structural similarity
between the inhibitor and
substrate
No structural similarity
between the inhibitor and
substrate
2 Inhibition is reversed by
increasing the concentration
of the substrate
Increase in substrate
concentration does not
reverse enzyme inhibition
3 Inhibitor does not bind with
ES complex
Inhibitor binds with ES
complex
4 Vmax remains constant Vmax decreases
5 Km value increases Km value remains constant
Feed Back Inhibition
Inhibition of activity of enzyme of a biosynthetic pathway
by the end product of that pathway is called feedback
inhibition.
A + E1  B + E2  C + E3 D (product)
•By inhibiting E1 enzyme, D regulates its own synthesis.
Examples:
•Inhibition of Asparate trans carboxylase by CTP.
•Inhibition of HMG-CoA reductase by Cholestrol.
•Inhibition of ALA Synthase by Heme.
•Inhibition of Anthranilate synthetase by tryptophan.
Allosteric inhibition
• Allosteric site is a site other than the active site which
is present in the enzyme.
• The inhibitor binds to the allosteric site and produces
conformational changes in the enzyme.
• So, the substrate cannot bind with the enzyme and a
product cannot formed.
Examples:
• ATP is an allosteric inhibitor of hexokinase.
• ADP is an allosteric inhibitor of pyruvate
carboxylase.
INDUCED FIT OF ENZYME
CATALYSIS/INHIBITION
REGULATION OF ENZYME ACTIVITY
In biological system regulation of enzyme activities
occurs at different stages in one or more of the
following ways to achieve cellular economy.
•Allosteric regulation
•Activation of latent enzymes
•Compartmentation of metabolic pathways.
•Control of enzyme synthesis.
•Enzyme degradation
•Isoenzymes
Allosteric regulation and allosteric inhibition
• Some of the enzymes possess additional sites, known as
allosteric sites (Greek: allo-other) besides the active site.
• Such enzymes are known as allosteric enzymes. The
allosteric sites are unique places on the enzyme molecule.
Allosteric effectors:
• Certain substances referred to as allosteric modulators
(effectors or modifiers) bind at the allosteric site and
regulate the enzyme activity.
• 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.
INDUCED FIT OF ENZYME
CATALYSIS/INHIBITION
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 and Vmax.
K-class of allosteric enzymes: The effector changes the Km
and not the Vmax. e.g. Phosphofructokinase.
V-class of allosteric enzymes: The effector alters the Vmax
and not the Km. e.g. Acetyl CoA carboxylase.
Conformational changes in allosteric enzyme
• Most of the allosteric enzymes are oligomeric in nature.
• The subunits may be identical or different.
• 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.
• 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.
T R
Allosteric inhibitors favour T state whereas activators and
substrates favour R state.
Allosteric Activator (or) Substarte
Allosteric Inhibitor
• The substrate can bind only with the R form of the
enzyme.
• The concentration of 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.
The term Homotropic effect is used if the substrate
influences the substrate binding through allosteric
mechanism, their effect is always positive.
Heterotropic effect is used when an allosteric modulator
effects the binding of substrate to the enzyme.
Heterotropic interactions are either positive or negative.
Some enzymes with allosteric effectors
Enzymes Metabolic
pathway
Allosteric
Inhibitor
Allosteric
Activator
Hexokinase Glycolysis Glucose-6-
phosphate
-
Phosphofructokinase Glycolysis ATP AMP, ADP
Isocitrate
dehydrogenase
Krebs cycle ATP ADP, NAD+
Feedback regulation
The process of inhibiting the first step by the final product, in
a series of enzyme catalysed reactions of a metabolic
pathway is referred to as feedback regulation.
Example:
A + E1  B + E2  C + E3 D (product)
•A is the initial substrate, B and C are the intermediates and
D is the final end product, in a pathway catalysed by three
different enzymes (E1, E2, E3).
•The very first step (AB by the enzyme E1) is the most
effective for regulating the pathway, by the final end product
D. This type of control is often called negative feedback
regulation, since increased levels of end product will result in
its E1 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.
• Feedback inhibition or end product inhibition is a
specialised type of allosteric inhibition necessary to
control metabolic pathways for efficient cellular function.
Examples:
• Inhibition of Asparate transcarboxylase by CTP.
• Inhibition of HMG-CoA reductase by Cholestrol.
• Inhibition of ALA Synthase by Heme.
• Inhibition of Anthranilate synthetase by tryptophan.
Activation of latent enzymes
• Latent enzymes as such are inactive.
• Some enzymes are synthesised as proenzymes or
zymogens, which undergoes irreversible covalent
activation by the breakdown of one or more peptide
bonds.
• The following are examples of proenzymes and their
conversion to active forms.
Pepsinogen(inactive)  Pepsin (active)
Trypsinogen (inactive)  Trypsin (active)
• Certain enzymes exist in the active and inactive forms
which are interconvertible, depending on the needs of the
body.
• The interconversion is brought about by the
reversible covalent modifications, namely
phosphorylation and dephosphorylation, and
oxidation and reduction of disulphide bonds. E.g.
Glycogen phosphorylase is a muscle enzyme that
breaks down glycogen to provide energy.
• There are some enzymes which are active in
dephosphorylated state and become inactive when
phosphorylated e.g. Glycogen synthetase, acetyl CoA
carboxylase.
• A few enzymes are active only with sulfhydryl (-SH)
groups, e.g. Succinate dehydrogenase, Urease.
Compartmentation
• There are certain substances in the body (e.g., fatty acids,
glycogen) which are synthesized and also degraded.
• There is no point for simultaneous occurrence of both
the pathways.
• Generally, the synthetic (anabolic) and breakdown
(Catabolic) pathways are operative in different cellular
organelles to achieve maximum economy.
• For instance, enzymes for fatty acid synthesis are found
in the cytosol whereas enzymes for fatty acid oxidation
are present in the mitochondria.
• Depending on the needs of the body- through the
mediation of hormonal and other controls- fatty acids are
either synthesized or oxidised.
Control of enzyme Synthesis
• Most of the enzymes, particularly the rate limiting ones,
are present in very low concentration.
• Nevertheless, the amount of the enzyme directly controls
the velocity of the reaction, catalysed by that enzyme.
• Many rate limiting enzymes have short half-lives. This
helps in the efficient regulation of the enzyme levels.
There are two types of enzymes:
• Constitutive enzymes (house-keeping enzymes): the
levels of which are not controlled and remain fairly
constant.
• Adaptive enzymes: their concentrations increase or
decrease as per body needs and are well-regulated. The
synthesis of enzymes (proteins) is regulated by the genes.
Enzymes Induction and Repression
• The term induction is used to represent increased
synthesis of enzyme while repression indicates its
decreased synthesis.
• Induction or repression which ultimately determines the
enzyme concentration at the gene level through the
mediation of hormones or other substances.
Examples of enzyme Induction:
•The hormone insulin induces the synthesis of
Glycogensynthetase,Glucokinase,Phosphofructo
kinase and Pyruvate kinase.
• All these enzymes are involved in the
utilisation of glucose.
•The hormone cortisol induces the synthesis of
many enzymes e.g. Pyruvate Carboxylase,
tryptophan oxygenase and tyrosine
aminotransferase.
Examples of enzyme Repression:
•In many instances, substrate can repress the synthesis
of enzyme.
•Pyruvate carboxylase is a key enzyme in the synthesis
of glucose from non-carbohydrate sources like pyruvate
and amino acids.
• If there is sufficient glucose available, there is no
necessity for its synthesis.
•This is achieved through repression of pyruvate
carboxylase by glucose.
Enzyme degradation
• Enzymes are not immortal.
• There is a lot of variability in the half-lives of individual
enzymes.
• For some, it is in days, while for others in hours or in
minutes, e.g. LDH4: 5 to 6 days; LDH1: 8 to 12 hour;
amylase: 3 to 5 hours.
• In general, the key and regulatory enzymes are most
rapidly degraded.
• If not needed, they immediately disappear and, as and
when required, they are quickly synthesized.
• Through not always true, an enzyme with long half-life
is usually sluggish in its catalytic activity.
Isoenzymes
• Multiple forms of the same enzyme will also help
in the regulation of enzyme activity.
• Many of the isoenzymes are tissue specific.
• These are enzymes obtained from different
sources and have different physical and chemical
characteristics.
• But they catalyse the same chemical reaction.
• Although isoenzymes of a given enzyme catalyse
the same reaction, they differ in Km, Vmax or
both.
Example: Isoenzymes of LDH and CPK.
Explanation for the existence of iso-enzymes
Many possible reasons are offered to explain the presence
of iso-enzymes in the living systems.
•Isoenzyme synthesized from the different genes, example:
malate dehydrogenase of cytosol is different from that
found in mitochondria.
•Oligomeric enzymes consisting of more than one type of
sub units, example lactate dehydrogenase and creatine
phosphokinase.
•An enzyme may be active as monomer or oligomer,
example: glutamate dehydrogenase.
•In glycoprotein enzymes, difference in carbohydrate
content may be responsible for iso-enzymes, example:
alkaline phosphatase.
Isoenzymes of Lactate dehydrogenase (LDH)
• Lactate dehydrogenase (LDH) exists in blood in five
different isoenzyme forms i.e. LDH1, LDH2, LDH3,
LDH4 and LDH5. They can be separated by
electrophoresis.
 
• All these isoenzymes have different physical
characteristics. But all of them catalyse the same
reaction- oxidation of lactic acid to pyruvic acid.
 
Structure of LDH isoenzymes
• LDH is an oligomeric enzyme made up of four
polypeptide subunits, Two types of subunits
namely M (for muscle) and H (for heart) are
produced by different genes.
• M-subunits is basic while H subunits is acidic.
• The isoenzymes contain either one or both the
subunits giving LDH1 to LDH5.
Significance of differential catalytic activity
• LDH1 is predominately found in heart muscle and is
inhibited by Pyruvate- the substrate.
• Hence, pyruvate is not converted to lactate in cardiac
muscle but is converted to acetyl CoA which enters citric
acid cycle.
• LDH5 is mostly present in skeletal muscle and the
inhibition of this enzyme by pyruvate is minimal, hence
pyruvate is converted to lactate.
• Further LDH5 has low Km while LDH1 has high Km for
pyruvate.
• The differential catalytic activities of LDH1 and LDH5 in
heart and skeletal muscle, respectively, are well suited for
the aerobic and anaerobic prevailing in these tissues.
Isoenzymes of creatine phosphokinase
• Creatine kinase (CK) or creatine phosphokinase
(CPK) catalyses the inter-conversion of
phosphocreatine (or creatine phosphate) to creatine.
• CPK exists as three isoenzymes. Each isoenzyme is a
dimer composed of two subunits-M (muscle) or B
(brain) or both.
Isoenzyme Subunit Tissue of origin
CPK1 BB Brain
CPK2 MB Heart
CPK3 MM Skeletal muscle
The estimation of the enzyme CPK2 is the earliest reliable
indication of myocardial infarction.
Isoenzymes of Alkaline Phosphatase
Isoenzymes of Alcohol dehydrogenase
Lysozyme
• It is an enzyme present in animals.
• In humans, it is present in tears, nasal secretions,
saliva, plasma and gastric juice. The intestinal mucosa
produces a high amount of lysozyme.
Function:
• It hydrolyses acetyl amino polysaccharides of bacterial
cell wall.
• The bacteriolytic action of lysozyme protects the body
against bacteria which are inhaled or ingested with
food.
COENZYMES
 Coenzymes are non-protein organic compounds present in
enzymes and associated with them.
 Coenzymes accelerate enzyme action.
 Coenzymes are often regarded as the second substrates or co-
substrates, since they have affinity with the enzyme
comparable with that of the substrates.
 Coenzymes undergo alterations during the enzymatic
reactions, which are later regenerated.
 This is in contrast to the substrate which is converted to the
product.
 Coenzymes participate in various reactions involving transfer
of atoms or groups like hydrogen, aldehyde, keto, amino, acyl,
methyl, carbon dioxide etc.
 Coenzymes play a decisive role in enzyme function.
Coenzymes from B-complex vitamins:
•Most of the coenzymes are the derivatives of water soluble B-
complex vitamins.
•In fact, the biochemical functions of B-complex vitamins are
exerted through their respective coenzymes.
Non-vitamin Coenzymes:
•Not all coenzymes are vitamin derivatives.
•There are some other organic substances, which have no relation
with vitamins but function as coenzymes.
•They may be considered as non-vitamin coenzymes e.g.
ATP,CDP, UDP etc.
Nucleotide Co-enzymes:
• Some of the coenzymes possess nitrogenous base, sugar and
phosphates.
•Such co-enzymes are regarded as nucleotides. NAD+
, NADP+
,
FMN, FAD, co-enzyme A, UDPG etc.
Co-enzymes not related to B-complex vitamins
Coenzyme Abbreviation Biochemical functions
Adenosine
triphosphate
ATP Donate phosphate, adenosine and
adenosine monophosphate (AMP)
moieties.
Cytidine
diphosphate
CDP Required in phospholipid synthesis as
carrier of Choline and ethanolamine.
Uridine
diphosphate
UDP Carrier of monosaccharides (glucose
and galactose), required for glycogen
synthesis.
S- Adenosyl
methionine
SAM Donates methyl group in biosynthetic-
reactions.
Co-enzymes do not decide enzyme specificity
• A particular coenzyme may participate in catalytic
reactions along with different enzymes.
• For instance, NAD+
acts as coenzyme for lactate
dehydrogenase and alcohol dehydrogenase.
• In both the enzymatic reactions NAD+
is involved in
hydrogen transfer.
• The specificity of the enzyme is mostly dependent on
the apo-enzyme and not on the co-enzyme.
Attachment of coenzyme to Enzyme
 Coenzyme is a non protein substance.
 It is attached to the enzyme, which is protein substance.
 When the attachment is weak (so that dissociation can
occur) the non protein part is called as coenzyme.
 If the attachment is firm, the non-protein part is called as
prosthetic group.
Apoenzyme: It is the protein part of the enzyme to which
the coenzyme (or prosthetic group) is attached.
Haloenzyme: It is the complete enzyme, which consists of
apoenzyme and coenzyme ( or prosthetic group).
Haloenzyme = Apoenzyme + Coenzyme or prosthetic
group
They differ from enzymes in the following
aspects:
•They are non-protein in nature
•They have low molecular weight
•They are heat stable
•They can be separated by dialysis
•They are generally derived from vitamins
Classification of Coenzymes
Coenzymes can be broadly classified as:
i) Group transferring coenzymes and
ii) Hydrogen transferring coenzymes.
Group transferring coenzymes: TPP, Biotin, Coenzyme A
Hydrogen transferring coenzymes: NAD, NADP, FAD, FMN.
Association with vitamins
Many coenzymes form the constituents of vitamin B complex.
Also, the coenzymes are associated with these vitamins.
The following is the list of vitamins and their associated
coenzymes:
Vitamins Coenzymes
Pantothenic acid CoA
Vitamin B12 Cobamide
Niacin NAD, NADP
Riboflavin FMN, FAD
Thiamine TPP
Functions of coenzymes
• The important function performed by coenzymes is to
transfer hydrogen or groups.
• The coenzymes accept the atom or a group from the
substrate and transfer them to other molecules.
The following is the list of coenzymes and the function
performed:
Coenzymes Functions
NAD, NADP Hydrogen transfer
FAD, FMN Hydrogen transfer
TPP Acetyl transfer
Biotin Carboxyl group transfer
Coenzyme A Acyl group transfer
Co-Enzyme A (CoA)
• It is the coenzyme form of pantothenic acid.
• It is composed of ATP, pantothenic acid and mercaptoethyl
amine.
• It is a group transferring coenzyme.
• The reaction of co-enzyme A is due to the presence of
sulphydryl group (SH group).
• It accepts acetyl group to form acetyl CoA which takes part in
a number of metabolic reactions.
CoA is required for:
• Conversation of α-ketoglutarate to succinyl CoA in citric acid
cycle.
• Oxidation of fatty acids
Acetyl CoA
 It is the coenzyme form of pantothenic acid.
 It is formed by the transfer of acetate to coenzyme A.
Acetyl CoA takes part in the following metabolic
reactions:
• It is utilised in citric acid cycle
• It combines with choline to form acetyl choline
• It is the starting material for cholesterol synthesis
• It is the starting material for the synthesis of ketone
bodies.
• It is used in the synthesis and elongation of fatty acids.
• It is used for the synthesis of steroid hormones.
Coenzyme forms of Vitamin B & their
functions
Vitamin Activated form- (coenzyme ) Type of catalysis Enzyme using co -
enzyme
Thiamine Thiamine Pyrophosphate(TPP ) Aldehyde or Keto
Group
Trans- Ketolase
Riboflavin Flavin Mono Nucleotide (FMN ) Hydrogen or Electron L -Amino oxidases
Riboflavin Flavin Adenine Dinucleotide
(FAD )
Hydrogen or Electron D -Amino oxidases
Niacin Nicotinamide Adenine
Dinucleotide (NAD )
Hydrogen or Electron LDH
Niacin Nicotinamide Adenine
Dinucleotide Phosphate (NADP )
Hydrogen Or Electron G-6 P-D
Lipoic Acid Lipoic Acid Hydrogen Or Electron Pyruvate
Dehydrogenase
Complex
Coenzyme forms of Vitamin B & their functions
Vitamin Activated form- (coenzyme ) Type of catalysis Enzyme using
coenzyme
Pyridoxine Pyridoxal Phosphate Amino Group
Transfer
Alanine
Transaminase
Pantothenic
Acid
Coenzyme A Acyl Group
Transfer
Thio Ketolase
Folic Acid Tetra Hydro Folate (TFH4 ) One Group
Transfer- formyl,
Methyl
Formyl
Transferase
Biotin Biotin CO2 Pyruvate
Carboxylase
Cobalamine Methyl Cobalamine Methyl Malonyl
Co A Mutase
VITAMINS AND COENZYMES
Vitamin Coenzyme Reaction type Coenzyme class
SOURCE: Compiled from data contained in Horton, H. R., et al. (2002). Principles of Biochemistry , 3rd edition. Upper Saddle River, NJ: Prentice Hall.
B 1 (Thiamine)
B 2 (Riboflavin)
B 3 (Pantothenate)
TPP Oxidative decarboxylation Prosthetic group
FAD Oxidation/Reduction Prosthetic group
CoA - Coenzyme A Acyl group transfer Cosubstrate
B 6 (Pyridoxine) PLP
Transfer of groups to and
from amino acids
Prosthetic group
Read more: http://www.chemistryexplained.com/Ce-
Co/Coenzyme.html#ixzz3oL0qkSi1
B 12 (Cobalamin) 5-deoxyadenosyl cobalamin Intramolecular rearrangements Prosthetic group
Niacin Folic acid Biotin
NAD +
Tetrahydrofolate Biotin
Oxidation/Reduction
One carbon group transfer
Carboxylation
Cosubstrate
Prosthetic group
Prosthetic group
Factors affecting enzyme activity: Cofactors
(activators )
Cofactor –inorganic ion Enzymes
Fe 2 ,Fe3⁺ ⁺ Peroxidase
Cu++ Cytochrome oxidase
Mg++ Hexokinase
Ni++ Urease
Mn++ Arginase
K+ Pyruvate Kinase
Zn++ DNA Polymerase
Mo++ Nitrate Reductase
Se Glutathione Peroxidase
Ca++ Lipase
Cl- Salivary Amylase
DIAGNOSTIC APPLICATIONS OF
ENZYMES
• Some diseases can be diagnosed by the estimation
of blood level of certain enzymes.
• Under normal conditions, the blood levels of
these enzymes are low.
• But in certain diseases, the blood levels are more.
• It is due to release of these enzyme form damaged
tissues or organs.
• So, estimation of these enzymes in blood helps in
the diagnosis of diseases.
The following is the list of enzymes whose levels are
increased in disease conditions shown
Enzyme Disease that can be diagnosed
Amylase Acute pancreatitis
Alkaline phosphatase Rickets
Creatinine phosphokinase Myocardial infarction
Glutamic oxaloacetic
transaminase(GOT)
Myocardial infarction
Glutamic pyruvic transaminase
(GPT)
Liver diseases
Isocitrate dehydrogenase(IDH) Hepatitis, Liver metastasis
Lactate dehydrogenase(LDL) Myocardial infarction
Lipase Acute pancreatitis
Amylase
• It is an enzyme secreted in the pancreas.
• Normally its concentration in blood is very low.
• It’s level increases in acute pancreatitis.
•So estimation of amylase in blood can provide
diagnosis for acute pancreatitis.
Alkaline phosphatase
• It is formed in the bone.
• Rickets and osteomalacia can be diagnosed by an
increase in blood level of this enzyme.
Acid phosphatase
 It is present mainly in the prostate gland.
 An increase in it’s blood level helps in the diagnosis of
prostatic cancer.
Creatinine phosphokinase (CPK)
 It is an enzyme present in heart and skeletal muscle.
 An increase in it’s blood level helps to diagnose
myocardial infarction and muscular dystrophy.
Isocitrate dehydrogenase
•It is an enzyme of Krebs cycle.
•It’s level increases in hepatitis and malignancy of liver.
Lactate dehydrogenase(LDL)
• LDL level of serum increases in leukemia,
generalised cancer and acute hepatitis.
• Also the concentration of LDL, (isoenzyme) in serum
increases in myocardial infarction.
• So estimation of LDL helps in the diagnosis of these
diseases.
Lipase
• Plasma lipase levels are increased in acute
pancreatitis and carcinoma of pancreas.
• These diseases can be diagnosed by estimating
plasma lipase levels.
Transaminases
These are enzymes involved in transamination reactions.
The two important transaminases present in serum are: 1.
Serum glutamic oxaloacetic transaminase (SGOT). 2.
Serum glutamic pyruvic transaminase (SGPT).
SGOT
•The level of SGOT increases in myocardial infarction.
• So, myocardial infarction can be diagnosed by estimating
SGOT.
SGPT
• It’s level increases in hepatic damage.
• So, estimation of SGPT helps in the diagnosis of liver diseases
like infective hepatitis and obstructive jaundice.
The following are the applications of enzymes in the
fields of medicine and pharmacy:
•Drugs acting through enzymes
•Therapeutic uses of enzymes
•Use of enzymes in the manufacture of bulk drugs
Drugs acting through enzymes:
•A number of drugs act by inhibiting enzymes.
•Enzymes act on drugs and convert them into inactive
metabolites.
Drug + Enzyme  Inactive metabolite
Drug + Enzyme inhibitor---- No inactive metabolite.
So the duration of action of the drug is increased.
Medicinal significance of Enzymes
• In presence of compounds which inhibit the enzyme
(i.e. enzyme inhibitor), the formation of inactive
metabolite is prevented. So the drug remains inactive
form. So the duration of action of the drug is
prolonged.
• For example, acetylcholine is inactivated by the
enzyme acetyl cholinesterase. This enzyme is
inhibited by anticholinesterase like physostigmine. So
in presence of physostigmine, the action of
acetylcholine is increased.
• Similarly, Allopurinol used for the treatment of gout
acts by inhibiting the enzyme xanthine oxidase. This
enzyme is required for the conversation of Xanthine
to uric acid.
Therapeutic uses of Enzymes
Enzymes are used for the treatment of number of
diseases. The following are the few examples:
•Enzymes like pepsin, papain and amylase are
administered for improving digestion.
•The enzyme hyaluronidase is used for diffusion of a
number of drugs.
•The enzymes streptokinase and urokinase are used for
dissolving blood clot.
•The enzymes trypsin is used for liquefying the lens. So
it is used in the treatment of cataract.
•The enzyme asparaginase is used for the treatment of
cancer.
Use of enzymes in the manufacture of Bulk
drugs
Many enzymes are used in pharmaceutical industry for the
manufacture of bulk drugs.
The following are a few examples:
•The enzyme penicillin acylase is used for the production of
6-amino penicillanic acid from penicillin G. 6-amino
penicillanic acid is needed for the synthesis of several Beta
lactam antibiotics.
•The enzyme glucose oxidase is needed for the production
of fructose syrup.
•Amylase is needed for the production of dextrin.
•The enzyme papain is used in the production of protein
hydrolysate.
Thank you

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Enzymes

  • 2. Contents • Terminology • Introduction • Properties • Nomenclature and classification • Mechanism of action • Kinetics • Factors affecting • Enzyme Inhibitions • Applications • Coenzymes and its functions
  • 3. Terminology ATP: Adenosine triphosphate ADP: Adenosine diphosphate NAD: Nicotinamide Adenine Dinucleotide NADP: Nicotinamide Adenine Dinucleotide Phosphate GDP: Guanosine diphosphate GTP: Guanosine triphosphate FMN: Flavin Mono Nucleotide FAD: Flavin Adenine Dinucleotide TPP: Thiamine Pyrophosphate LDH: Lactate dehydrogenase CPK: Creatinine phosphokinase SGOT: Serum glutamic oxaloacetic transaminase SGPT: Serum glutamic pyruvic transaminase
  • 4. Importance • Enzymes play an important role in Metabolism, Diagnosis, and Therapeutics. • All biochemical reactions are enzyme catalyzed in the living organism. • Level of enzyme in blood are of diagnostic importance e.g. it is a good indicator in disease such as myocardial infarction. • Enzyme can be used therapeutically such as digestive enzymes. • Enzymes are biological catalyst that speed up the rate of reaction. • Enzymes regulate the rate of physiological process.
  • 5.  When cells are injured, enzyme leak into plasma. The measurement of activity of such enzymes in plasma is an integral part of medical diagnosis.  Enzymes are used as drugs. Eg. Streptokinase which can solubilise the blood cloting.  Asparaginase as anticancer agent.  Enzymes are used as cleansing agent in detergent industry.  Enzymes are used as bio-sensors.  AIDS detection involve used of enzyme dependent ELISA technique.  The immobilised enzymes are used for diagnostic purpose.
  • 6. Define enzymes (Enzymes as Biological Catalysts) • Enzymes are proteineous in nature except Ribozyme. • 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
  • 7. Properties of enzymes  Enzymes are required only in small amounts.  They quicken the reactions without being consumed or lost in the process.  Enzymes are proteins. Like all proteins, enzymes are colloidal in nature and precipitated by salt solutions.  They are inactivated by heat and alteration of pH.  Enzymes have great specificity.  Each enzyme catalyses one particular reaction.
  • 8. Properties of enzymes • Enzymes are protein in nature but not vice versa. • Enzymes are bigger molecules as compared to substrate molecules. • Molecular weight varies from some thousand to some Millions. • They increase the rate of reaction to 105 to 10-10 times.
  • 9. Localisation of Enzymes Enzymes may be present inside the cell or outside the cell. For example: •Enzymes of Citric acid cycle are present in the mitochondria. •Enzymes of glycolytic pathway are present in the cytoplasm. •Enzymes for the transport of nutrients are present in the cell membranes.
  • 10. Activation energy or Energy of Activation • All chemical reactions require some amount of energy to get them started. OR • It is First push to start reaction. This energy is called activation energy.
  • 11.  Enzymes catalyse various biochemical reactions.  Normally, for any reaction to occur, the reacting molecules must come in contact.  For this, the molecules must gain a minimum amount of energy. This is called energy of activation.  Normally, the energy of activation can be lowered by increasing the temperature.  But in the human body, the temperature is constant. So, enzymes act by lowering the energy of activation at normal body temperature. Energy of activation
  • 12. Classification of Enzymes Enzymes are sometimes considered under two broad categories: Intracellular Enzymes: They are functional within cells, where they are synthesized. Extracellular Enzymes: These enzymes are active outside the cell; All the digestive enzymes belongs to this groups.
  • 13. Nomenclature and classification of Enzymes • The names of most of the enzymes end with the three letters –ase • This is a suffix which is added to the name of the substrate on which the enzyme acts. • For example, sucrase catalyzes the hydrolysis of sucrose • The name describes the function of the enzyme For example, oxidases catalyze oxidation reactions • Sometimes common names are used, particularly for the digestion enzymes such as pepsin and trypsin • Some names describe both the substrate and the function • For example, alcohol dehydrogenase oxides ethanol
  • 14. Enzymes Are Classified into six functional Classes (EC number Classification) by the International Union of Biochemists (I.U.B.). on the Basis of the Types of Reactions That They Catalyze • EC 1. Oxidoreductases • EC 2. Transferases • EC 3. Hydrolases • EC 4. Lyases • EC 5. Isomerases • EC 6. Ligases The classification of enzymes can be remembered by using the tip “OTHLIL”
  • 15. Principle of the international classification Each enzyme has classification number consisting of four digits: Example, EC: (2.7.1.1) HEXOKINASE
  • 16. • EC: (2.7.1.1) these components indicate the following groups of enzymes: • 2. IS CLASS (TRANSFERASE) • 7. IS SUBCLASS (TRANSFER OF PHOSPHATE) • 1. IS SUB-SUB CLASS (ALCOHOL IS PHOSPHATE ACCEPTOR) • 1. SPECIFIC NAME ATP,D-HEXOSE-6-PHOSPHOTRANSFERASE (Hexokinase)
  • 17. H O OH H OHH OH CH2OH H OH H H O OH H OHH OH CH2OPO3 2− H OH H 23 4 5 6 1 1 6 5 4 3 2 ATP ADP Mg2+ glucose glucose-6-phosphate Hexokinase 1. Hexokinase catalyzes: Glucose + ATP  glucose-6-P + ADP
  • 20. Mechanism of Enzyme action • The active site of the enzymes represents a small region at which a substrate binds and participates in the catalysis. It consists of two parts: 1. Catalytic site and 2. Binding site  Catalytic site: It is the portion of enzyme that is responsible for catalysis. It determine the reaction specificity.  Binding site: It is the part of enzyme that binds the substrates. It determines the substrate specificity. • The active site of enzymes are present within the enzyme molecules. • The active site is 3-D and consists of few amino acid residues.
  • 21. • The active site is not rigid structure and shape. It is rather flexible to promote the specific substrate binding. • The active site is contributed by the amino acids such as serine, aspartate, histidine, cysteine, lysine, arginine, glutamate, tyrosine etc. • Which are far from each other in the linear sequence of amino acids. During catalysis, they are brought together. • The substrates binds at the active site by weak non- covalent bond. The enzymes are specific in their function due to the existence of active site.
  • 22. ACTIVE SITES • Enzyme molecules contain a special pocket or cleft called the active sites.
  • 23. Mechanism of Action of Enzymes • Enzymes increase reaction rates by decreasing the Activation energy: • Enzyme-Substrate Interactions: ‒Formation of Enzyme substrate complex by: ‒Lock-and-Key Model ‒Induced Fit Model
  • 25. Activation energy for Catalyzed & uncatalyzed reaction
  • 26. Lock-and-Key Model • This theory was proposed by the German scientist Imil-Fischer in 1894. • According to this model, the active site is the rigid portion of enzyme molecules and its shape is complementary to the substrate molecule like Lock and Key. • The complementary shape of the substrate and active site favour tightly bound enzyme-substrate complex formation followed by catalysis.
  • 27. Lock-and-Key Model • In the lock-and-key model of enzyme action: - the active site has a rigid shape - only substrates with the matching shape can fit - the substrate is a key that fits the lock of the active site
  • 28. In the lock-and-key model of enzyme action: •This explains enzyme specificity. •This explains the loss of activity when enzymes denature. •This is an older model, however, and does not work for all enzymes
  • 29. Induced Fit Model  This model was proposed by Kashland in 1958.  According to this model, the active site is flexible.  In the enzyme molecule, the amino acid residue that make up the active sites are not oriented properly in the absence of substrates.  When the substrate combines with the enzyme, it induces the conformational changes in the enzyme molecules in such a way that amino acids that make active sites are shifted into correct orientation to favour tightly bound enzyme substrate complex formation followed by catalysis.  The enzyme molecule is unstable in the induced conformations and returns to its native conformations in the absence of substrate.
  • 30. Induced Fit Model • In the induced-fit model of enzyme action: - the active site is flexible, not rigid - the shapes of the enzyme, active site, and substrate adjust to 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
  • 32. Enzyme-substrate complex • Step 1: • Enzyme and substrate combine to form complex • E + S ES • Enzyme Substrate Complex +
  • 33. Enzyme-product complex • Step 2: • An enzyme-product complex is formed. • EESS EPEP EESS EPEPtransitiontransition statestate
  • 34. Product • The enzyme and product separate • EPEP EE ++ PP The product is made Enzyme is ready for another substrate. EPEP
  • 35. Enzyme Specificity • Enzymes have varying degrees of specificity for substrates • Enzymes may recognize and catalyze: - a single substrate - a group of similar substrates - a particular type of bond - a particular type of optical isomer of the substrate (optical specificity). E.g. D-amino acid oxidase acts only on D- amino acid & L -amino acid oxidase acts only on L- amino acid
  • 37. ENZYME KINETICS Michaelis curve: The velocity of an enzyme reaction increases with an increase in substrate concentration. After a certain limit, it become constant. So, further increase in substrate concentration has no effect.  If the velocity of the reaction is plotted against substrate concentration, a typical hyperbolic curve is obtained. The explanation for such a behaviour of the enzyme is as follows: At low concentration of the substrate, all the enzyme molecules are not utilised in the formation of enzyme substrate complex.  As the substrate concentration is increased, more and more enzyme molecules are utilised. So, there is proportional increase in velocity. Vmax is reached when all the enzyme molecules are saturated with the substrate So, there is no increase in velocity. The curve is called Michaelis curve.
  • 38. The mathematical equation is called Michaelis menton equation, which is as follows. Vo = Vmax[S]/Km+[S] Where Vo= Initial velocity Vmax= Maximal velocity [S]= Substrate concentration Km= Michaelis constant. Km is equal to 1/V. i.e. Km is equivalent to substrate concentration required to produce half maximal velocity.
  • 39. Significance of Km 1.It is an enzyme kinetic constant. 2.It indicates the substrate concentration required for the enzyme to work efficiently. 3.Low Km indicates high affinity of enzyme towards substrate. High Km indicates low affinity of enzyme towards substrate. Hence Km and affinity are inversely related. 4.Km is required when enzyme are used as drugs. 5.Use of enzyme in immunodiagnostics (ELISA) require Km of the enzyme.
  • 40. LINEWEAVER-BURK PLOT: (Double reciprocal plot) The drawbacks of Michaelis curve are: 1.Only an approximate but not an accurate value of Km can be obtained. 2.It is difficult to determine Vmax accurately. It is because, Vmax is only approached and never attained. 3.It is only a hyperbolic curve and not a straight line graph. So interpolation of data is not possible. These drawbacks are overcome by a straight line graph called Lineweaver-Burk plot. It makes use of reciprocals of Vo and [S] i.e. 1/Vo and 1/[S].
  • 41. As we have already seen, Michaelis Menton equation is Vo= Vmax [S]/Km+[S] Reciprocal of this equation is 1/Vo= Km+[S]/Vmax[S] It can be rewritten as 1/Vo= Km/Vmax[S] + [S]/Vmax[S] It can be simplified as: 1/Vo= Km/Vmax X 1/[S] + 1/Vmax The above eqn. represent Y=mx+C, straight line eqn. with slope of Km/Vmax. Since 1/S & 1/V are reciprocal of S & V respectively, It is known as double reciprocal plot. The above equation is called as Lineweaver-Burk equation.
  • 42. Now, a graph can be constructed by plotting 1/vo on the y-axis and 1/[s] on the x-axis. This gives a straight line. From this graph three points are very clear and they can be easily determined: 1.Slope of the curve = Km/Vmax 2.1/Vmax is the intercept of this slope on 1/Vo. From this Vmax can be calculated easily. 3.Extension of this to line to the negative side cuts the x-axis at a point equal to -1/Km. from this Km can be easily calculated. 4. In addition to Km and Vmax values, type of inhibition is determined using this plot. 5. Inhibition constant (Ki) of inhibitor is also determined using this plot.
  • 43. Significance of (Ki) 1.Ki indicates affinity of inhibitor towards enzyme like Km, Ki is inversely related to affinity. 2.Use of inhibitors as drugs require knowledge of Ki. Since Ki and affinity are inversely related inhibitors of low Ki are highly potent drugs.
  • 44. Thermodynamics of enzymatic reactions The enzyme catalysed reactions may be broadly grouped into three types based on thermodynamic (energy) considerations. Isothermic reactions: The energy exchange between reactants and products is negligible e.g. Glycogen phosphorylase. Glycogen + Pi  Glucose 1-phosphate Exothermic (exergonic) reactions: Energy is liberated in these reactions e.g. Urease Urea  NH3 + CO2 + energy Endothermic (endergonic) reactions: Energy is consumed in these reactions e.g. Glucokinase. Glucose + ATP  Glucose 6-phosphate + ADP
  • 45. Factors influencing Enzyme actionFactors influencing Enzyme action The activity of enzyme can be modified by the following factorsThe activity of enzyme can be modified by the following factors.. 1.Concentration of enzymes 2.Concentration of substrate 3.Contact between enzyme and substrate 4.Concentration of Product 5.Temperature 6.pH 7. Oxidising agent 8.Radiations 9.Activators (Coenzymes & Cofactors ) 10.Inhibitors
  • 46. Concentration of Enzyme • As the concentration of the enzyme is increased, the velocity of the reaction proportionately increases. • This is true only when sufficient substrate molecules are available for combination with the enzyme. • When all the substrate molecules are saturated with the enzyme, further increase in enzyme concentration has no effect.
  • 47. Substrate Concentration and Reaction Rate• The rate of reaction increases as substrate concentration increases (at constant enzyme concentration) • Maximum activity occurs when the enzyme is saturated (when all enzymes are binding substrate)
  • 48. Contact between enzyme and substrate  For the enzyme activity the contact between Substrate and enzyme is necessary.  The type of contact also influence the enzyme activity.
  • 49. Concentration of Product • If the product are accumulated then the enzyme activity is decreased.
  • 50. Environmental factorsEnvironmental factors • Optimum temperature The temp at which enzymatic reaction occur fastest.  Extreme Temperature are the most dangerousExtreme Temperature are the most dangerous  High tempsHigh temps may denature (unfold)denature (unfold) the enzyme.enzyme.
  • 51. • Velocity of an enzyme reaction increases with increase in temperature up to a maximum and then declines. • A Bell shaped curve is usually observed. • Temperature co-efficient or Q10 is defined as increase in enzyme velocity when the temperature is increased by 100 C. • For majority of enzymes Q10 is 2 between 00 C to 400 C. • The optimum temperature for most of the enzymes is lies between 400 C to 450 C. • Exception cases, Like Venom phosphokinase, Urease, Adenylate kinase etc.
  • 52. Environmental factorsEnvironmental factors • pH also affects the rate of enzyme- substrate complexes –Most enzymes have an optimum pH of around 6-8 (neutral) • However, some prefer acidic or basic conditions
  • 53. • pH influences the enzyme activity and a Bell shaped curve is normally obtained. • Each enzyme has an optimum pH at which the velocity is maximum.
  • 54. Oxidising agent • In the presence of Oxidising substances, the –SH group (Sulfahydryl) is oxidised to form disulphide linkage, which inactivate the enzyme activity. • To counteract this, the antioxidants like Cysteine and glutathione are used.
  • 55. Radiation • X-rays, UV rays, Beta and gamma rays produce inactivation of enzymes. • They act by forming peroxides which oxidise the enzymes and make them inactive.
  • 56. Activators (Cofactors) These are non protein molecules required for activity of some enzymes. They may be involved in catalysis or in structure maintenance. There are two types of cofactors. A.Organic cofactors 1.Prosthetic groups and 2. Coenzyme B. Inorganic cofactors (metal ions) Prosthetic groups are covalently attached to the enzyme and they undergo change during catalysis but return to their native sites at the end of the reaction.
  • 57. APOENZYME and HOLOENZYME • The enzyme without its non protein moiety is termed as apoenzyme and it is inactive. • Holoenzyme is an active enzyme with its non protein component.
  • 58. Definition of Cofactors & Coenzymes Factors affecting enzyme activity: Co-enzyme & Cofactors (Activators )
  • 59. Factors affecting enzyme activity: co-enzyme & cofactors
  • 60. Comparison of Cofactors & Coenzymes
  • 61. Effects of Metal ions on enzymes (Inorganic Cofactors)Several enzymes are depends on the presence of metal ions like K+, Mg++, Zn++ and Cu++ for their activity. Metals help in enzyme action by: •Maintaining structural confirmation of enzymes. •Promoting enzyme-substrate complex formation. Metal containing enzymes can be classified as: •Metal activated enzymes •Metallo-enzymes •Metal-dependent enzymes •Metal activated enzmes: in presence of metals, some enzymes get activated i.e. their activity increases many folds. Example: Cl- It activates amylase and ACE Ca2+ It activates trypsin
  • 62. Metallo-enzymes: In these enzymes, the metal ion is very tightly bound and it is an integral part of enzyme molecule. They participates in catalysis. Example: Iron required for Cytochrome oxidase, catalase, Xanthine oxidase. Copper is required for cytochrome oxidase, superoxide dismutase. Zinc is required for carboxy peptidase, alkaline phosphatase. Metal-dependent enzymes: Metal is loosely associated with enzyme molecule or it may be required for enzyme substrate complex formation. In the absence of metal, enzyme may not intract with substrate molecule or with coenzyme molecule. Example: Mg2+ needed by enzyme using ATP Hexokinase, pyruvate kinase. Ca2+ required for the activity of calpain a calcium dependent protease.
  • 63. Inhibitors • Substances that decrease the catalytic activity of enzyme are called inhibitors. • They may be protein or non-protein inhibitors. • Enzymes can be inhibited by a number of substances e.g. heavy metals like silver and mercury, oxidising agents like iodine. • The decrease in enzyme activity is called as enzyme inhibition.
  • 64. ENZYME INHIBITION • Enzyme inhibitors are substances which lower down the rate of enzyme reaction. • They produce their effect by acting on the coenzyme, apoenzyme or prosthetic group. • Also they can act by inhibiting the combination of the substrate with the enzyme. Enzyme inhibition is classified as: • Competitive inhibition • Non-competitive inhibition • Feed Back Inhibition • Allosteric inhibition
  • 65. Enzyme Inhibitors • Competive - mimic substrate, may block active site, but may dislodge it.
  • 66. • The rate of formation of the product from ES complex is same as that of the absence of inhibitor. • So, velocity (Vmax) is not altered, but Km increases (affinity of enzyme towards substrate decreases). E + S - [ES]  E + P E + I - [EI]  No Product Vmax is unchanged. Km is altered. • The competitive inhibitors are called as antagonist or antimetabolites of the substrate with which they compete. • The use of antimetabolite in the treatment of disease is called as chemotherapy.
  • 67. EXAMPLES OF COMPETITIVE INHIBITION  Statin Drug As Example Of Competitive Inhibition: • Statin drugs such as lipitor compete with HMG- CoA(substrate) and inhibit the active site of HMG CoA-REDUCTASE (that bring about the catalysis of cholesterol synthesis). • Sulfonamide also acts as competitive inhibitor against PABA. • The drugs like Captoprile, Lisinopril and Enalopril inhibit ACE.
  • 69. Non Competitive Inhibition • In this type of inhibition no, competition occurs between substrate and inhibitor to bind at active site of enzyme. • Inhibitor is not structurally related to substrate. • In addition inhibitor binds to some other site of enzyme which is far off from active site. E + S  [ES] + I  [ESI] E + P (Slow) E + I  [EI] + S  [EIS] E + P (Slow) • Inhibitor can react with free enzyme as well as ES complex. • Here the Vmax is decreased and Km (affinity) remains same because no competition of substrate and inhibitor in non- competitive inhibition. • Most of non-competitive inhibitors are irreversible. • They are referred as enzyme poisons.
  • 70. EXAMPLES OF UNCOMPETITIVE INHIBITION  Drugs to treat cases of poisoning by methanol or ethylene glycol act as uncompetitive inhibitors.  Tetramethylene sulfoxide and 3- butylthiolene 1-oxide are uncompetitive inhibitors of liver alcoholdehydrogenase.  Iodoacetate block the formation of 1,3-biphosphoglycerate from glyceraldehyde-3-P by inhibiting the enzyme glyceraldehyde-3-P dehydrogenase.  Fluoride blocks the action of enolase which converts 2- Phosphogluconate to phosphoenol pyruvate.  Heavy metals like Hg2+ , Ag+ , Pb2+ and Arsenic are also enzyme poisons. They interact with –SH Sulfhydryl groups at the active site of the enzyme.
  • 71. • Non-competitive inhibitors are used as pesticides. Eg, DDT, Melathione, Parathione are inhibitors of the enzyme Choline esterase that catalyses hydrolysis of Ach. • CN- inhibit cytochrome oxidase (cyanide). • EDTA inhibits metalloenzymes by forming complex with metal ion. • Tubers, bananas and beans contain inhibitors to trypsin, chymotrypsin and elestase. • Di-isopropyl fluro phosphate (DFP) is a non- competitive inhibitor used as nerve gas in World War II. It causes constriction of larynx, pain in eyes and mental confusion.
  • 72. Difference between Competetive and Non- Competetive inhibition SI. No Competetive inhibition Non-Competetive inhibition 1 There is structural similarity between the inhibitor and substrate No structural similarity between the inhibitor and substrate 2 Inhibition is reversed by increasing the concentration of the substrate Increase in substrate concentration does not reverse enzyme inhibition 3 Inhibitor does not bind with ES complex Inhibitor binds with ES complex 4 Vmax remains constant Vmax decreases 5 Km value increases Km value remains constant
  • 73. Feed Back Inhibition Inhibition of activity of enzyme of a biosynthetic pathway by the end product of that pathway is called feedback inhibition. A + E1  B + E2  C + E3 D (product) •By inhibiting E1 enzyme, D regulates its own synthesis. Examples: •Inhibition of Asparate trans carboxylase by CTP. •Inhibition of HMG-CoA reductase by Cholestrol. •Inhibition of ALA Synthase by Heme. •Inhibition of Anthranilate synthetase by tryptophan.
  • 74. Allosteric inhibition • Allosteric site is a site other than the active site which is present in the enzyme. • The inhibitor binds to the allosteric site and produces conformational changes in the enzyme. • So, the substrate cannot bind with the enzyme and a product cannot formed. Examples: • ATP is an allosteric inhibitor of hexokinase. • ADP is an allosteric inhibitor of pyruvate carboxylase.
  • 75. INDUCED FIT OF ENZYME CATALYSIS/INHIBITION
  • 76. REGULATION OF ENZYME ACTIVITY In biological system regulation of enzyme activities occurs at different stages in one or more of the following ways to achieve cellular economy. •Allosteric regulation •Activation of latent enzymes •Compartmentation of metabolic pathways. •Control of enzyme synthesis. •Enzyme degradation •Isoenzymes
  • 77. Allosteric regulation and allosteric inhibition • Some of the enzymes possess additional sites, known as allosteric sites (Greek: allo-other) besides the active site. • Such enzymes are known as allosteric enzymes. The allosteric sites are unique places on the enzyme molecule. Allosteric effectors: • Certain substances referred to as allosteric modulators (effectors or modifiers) bind at the allosteric site and regulate the enzyme activity. • 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.
  • 78. INDUCED FIT OF ENZYME CATALYSIS/INHIBITION
  • 79. 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 and Vmax. K-class of allosteric enzymes: The effector changes the Km and not the Vmax. e.g. Phosphofructokinase. V-class of allosteric enzymes: The effector alters the Vmax and not the Km. e.g. Acetyl CoA carboxylase.
  • 80. Conformational changes in allosteric enzyme • Most of the allosteric enzymes are oligomeric in nature. • The subunits may be identical or different. • 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. • 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. T R Allosteric inhibitors favour T state whereas activators and substrates favour R state. Allosteric Activator (or) Substarte Allosteric Inhibitor
  • 81. • The substrate can bind only with the R form of the enzyme. • The concentration of 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. The term Homotropic effect is used if the substrate influences the substrate binding through allosteric mechanism, their effect is always positive. Heterotropic effect is used when an allosteric modulator effects the binding of substrate to the enzyme. Heterotropic interactions are either positive or negative.
  • 82. Some enzymes with allosteric effectors Enzymes Metabolic pathway Allosteric Inhibitor Allosteric Activator Hexokinase Glycolysis Glucose-6- phosphate - Phosphofructokinase Glycolysis ATP AMP, ADP Isocitrate dehydrogenase Krebs cycle ATP ADP, NAD+
  • 83. Feedback regulation The process of inhibiting the first step by the final product, in a series of enzyme catalysed reactions of a metabolic pathway is referred to as feedback regulation. Example: A + E1  B + E2  C + E3 D (product) •A is the initial substrate, B and C are the intermediates and D is the final end product, in a pathway catalysed by three different enzymes (E1, E2, E3). •The very first step (AB by the enzyme E1) is the most effective for regulating the pathway, by the final end product D. This type of control is often called negative feedback regulation, since increased levels of end product will result in its E1 decreased synthesis.
  • 84. • 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. • Feedback inhibition or end product inhibition is a specialised type of allosteric inhibition necessary to control metabolic pathways for efficient cellular function. Examples: • Inhibition of Asparate transcarboxylase by CTP. • Inhibition of HMG-CoA reductase by Cholestrol. • Inhibition of ALA Synthase by Heme. • Inhibition of Anthranilate synthetase by tryptophan.
  • 85. Activation of latent enzymes • Latent enzymes as such are inactive. • Some enzymes are synthesised as proenzymes or zymogens, which undergoes irreversible covalent activation by the breakdown of one or more peptide bonds. • The following are examples of proenzymes and their conversion to active forms. Pepsinogen(inactive)  Pepsin (active) Trypsinogen (inactive)  Trypsin (active) • Certain enzymes exist in the active and inactive forms which are interconvertible, depending on the needs of the body.
  • 86. • The interconversion is brought about by the reversible covalent modifications, namely phosphorylation and dephosphorylation, and oxidation and reduction of disulphide bonds. E.g. Glycogen phosphorylase is a muscle enzyme that breaks down glycogen to provide energy. • There are some enzymes which are active in dephosphorylated state and become inactive when phosphorylated e.g. Glycogen synthetase, acetyl CoA carboxylase. • A few enzymes are active only with sulfhydryl (-SH) groups, e.g. Succinate dehydrogenase, Urease.
  • 87. Compartmentation • There are certain substances in the body (e.g., fatty acids, glycogen) which are synthesized and also degraded. • There is no point for simultaneous occurrence of both the pathways. • Generally, the synthetic (anabolic) and breakdown (Catabolic) pathways are operative in different cellular organelles to achieve maximum economy. • For instance, enzymes for fatty acid synthesis are found in the cytosol whereas enzymes for fatty acid oxidation are present in the mitochondria. • Depending on the needs of the body- through the mediation of hormonal and other controls- fatty acids are either synthesized or oxidised.
  • 88. Control of enzyme Synthesis • Most of the enzymes, particularly the rate limiting ones, are present in very low concentration. • Nevertheless, the amount of the enzyme directly controls the velocity of the reaction, catalysed by that enzyme. • Many rate limiting enzymes have short half-lives. This helps in the efficient regulation of the enzyme levels. There are two types of enzymes: • Constitutive enzymes (house-keeping enzymes): the levels of which are not controlled and remain fairly constant. • Adaptive enzymes: their concentrations increase or decrease as per body needs and are well-regulated. The synthesis of enzymes (proteins) is regulated by the genes.
  • 89. Enzymes Induction and Repression • The term induction is used to represent increased synthesis of enzyme while repression indicates its decreased synthesis. • Induction or repression which ultimately determines the enzyme concentration at the gene level through the mediation of hormones or other substances.
  • 90. Examples of enzyme Induction: •The hormone insulin induces the synthesis of Glycogensynthetase,Glucokinase,Phosphofructo kinase and Pyruvate kinase. • All these enzymes are involved in the utilisation of glucose. •The hormone cortisol induces the synthesis of many enzymes e.g. Pyruvate Carboxylase, tryptophan oxygenase and tyrosine aminotransferase.
  • 91. Examples of enzyme Repression: •In many instances, substrate can repress the synthesis of enzyme. •Pyruvate carboxylase is a key enzyme in the synthesis of glucose from non-carbohydrate sources like pyruvate and amino acids. • If there is sufficient glucose available, there is no necessity for its synthesis. •This is achieved through repression of pyruvate carboxylase by glucose.
  • 92. Enzyme degradation • Enzymes are not immortal. • There is a lot of variability in the half-lives of individual enzymes. • For some, it is in days, while for others in hours or in minutes, e.g. LDH4: 5 to 6 days; LDH1: 8 to 12 hour; amylase: 3 to 5 hours. • In general, the key and regulatory enzymes are most rapidly degraded. • If not needed, they immediately disappear and, as and when required, they are quickly synthesized. • Through not always true, an enzyme with long half-life is usually sluggish in its catalytic activity.
  • 93. Isoenzymes • Multiple forms of the same enzyme will also help in the regulation of enzyme activity. • Many of the isoenzymes are tissue specific. • These are enzymes obtained from different sources and have different physical and chemical characteristics. • But they catalyse the same chemical reaction. • Although isoenzymes of a given enzyme catalyse the same reaction, they differ in Km, Vmax or both. Example: Isoenzymes of LDH and CPK.
  • 94. Explanation for the existence of iso-enzymes Many possible reasons are offered to explain the presence of iso-enzymes in the living systems. •Isoenzyme synthesized from the different genes, example: malate dehydrogenase of cytosol is different from that found in mitochondria. •Oligomeric enzymes consisting of more than one type of sub units, example lactate dehydrogenase and creatine phosphokinase. •An enzyme may be active as monomer or oligomer, example: glutamate dehydrogenase. •In glycoprotein enzymes, difference in carbohydrate content may be responsible for iso-enzymes, example: alkaline phosphatase.
  • 95. Isoenzymes of Lactate dehydrogenase (LDH) • Lactate dehydrogenase (LDH) exists in blood in five different isoenzyme forms i.e. LDH1, LDH2, LDH3, LDH4 and LDH5. They can be separated by electrophoresis.   • All these isoenzymes have different physical characteristics. But all of them catalyse the same reaction- oxidation of lactic acid to pyruvic acid.
  • 96.   Structure of LDH isoenzymes • LDH is an oligomeric enzyme made up of four polypeptide subunits, Two types of subunits namely M (for muscle) and H (for heart) are produced by different genes. • M-subunits is basic while H subunits is acidic. • The isoenzymes contain either one or both the subunits giving LDH1 to LDH5.
  • 97. Significance of differential catalytic activity • LDH1 is predominately found in heart muscle and is inhibited by Pyruvate- the substrate. • Hence, pyruvate is not converted to lactate in cardiac muscle but is converted to acetyl CoA which enters citric acid cycle. • LDH5 is mostly present in skeletal muscle and the inhibition of this enzyme by pyruvate is minimal, hence pyruvate is converted to lactate. • Further LDH5 has low Km while LDH1 has high Km for pyruvate. • The differential catalytic activities of LDH1 and LDH5 in heart and skeletal muscle, respectively, are well suited for the aerobic and anaerobic prevailing in these tissues.
  • 98. Isoenzymes of creatine phosphokinase • Creatine kinase (CK) or creatine phosphokinase (CPK) catalyses the inter-conversion of phosphocreatine (or creatine phosphate) to creatine. • CPK exists as three isoenzymes. Each isoenzyme is a dimer composed of two subunits-M (muscle) or B (brain) or both. Isoenzyme Subunit Tissue of origin CPK1 BB Brain CPK2 MB Heart CPK3 MM Skeletal muscle The estimation of the enzyme CPK2 is the earliest reliable indication of myocardial infarction.
  • 99. Isoenzymes of Alkaline Phosphatase
  • 100. Isoenzymes of Alcohol dehydrogenase
  • 101. Lysozyme • It is an enzyme present in animals. • In humans, it is present in tears, nasal secretions, saliva, plasma and gastric juice. The intestinal mucosa produces a high amount of lysozyme. Function: • It hydrolyses acetyl amino polysaccharides of bacterial cell wall. • The bacteriolytic action of lysozyme protects the body against bacteria which are inhaled or ingested with food.
  • 102. COENZYMES  Coenzymes are non-protein organic compounds present in enzymes and associated with them.  Coenzymes accelerate enzyme action.  Coenzymes are often regarded as the second substrates or co- substrates, since they have affinity with the enzyme comparable with that of the substrates.  Coenzymes undergo alterations during the enzymatic reactions, which are later regenerated.  This is in contrast to the substrate which is converted to the product.  Coenzymes participate in various reactions involving transfer of atoms or groups like hydrogen, aldehyde, keto, amino, acyl, methyl, carbon dioxide etc.  Coenzymes play a decisive role in enzyme function.
  • 103. Coenzymes from B-complex vitamins: •Most of the coenzymes are the derivatives of water soluble B- complex vitamins. •In fact, the biochemical functions of B-complex vitamins are exerted through their respective coenzymes. Non-vitamin Coenzymes: •Not all coenzymes are vitamin derivatives. •There are some other organic substances, which have no relation with vitamins but function as coenzymes. •They may be considered as non-vitamin coenzymes e.g. ATP,CDP, UDP etc. Nucleotide Co-enzymes: • Some of the coenzymes possess nitrogenous base, sugar and phosphates. •Such co-enzymes are regarded as nucleotides. NAD+ , NADP+ , FMN, FAD, co-enzyme A, UDPG etc.
  • 104. Co-enzymes not related to B-complex vitamins Coenzyme Abbreviation Biochemical functions Adenosine triphosphate ATP Donate phosphate, adenosine and adenosine monophosphate (AMP) moieties. Cytidine diphosphate CDP Required in phospholipid synthesis as carrier of Choline and ethanolamine. Uridine diphosphate UDP Carrier of monosaccharides (glucose and galactose), required for glycogen synthesis. S- Adenosyl methionine SAM Donates methyl group in biosynthetic- reactions.
  • 105. Co-enzymes do not decide enzyme specificity • A particular coenzyme may participate in catalytic reactions along with different enzymes. • For instance, NAD+ acts as coenzyme for lactate dehydrogenase and alcohol dehydrogenase. • In both the enzymatic reactions NAD+ is involved in hydrogen transfer. • The specificity of the enzyme is mostly dependent on the apo-enzyme and not on the co-enzyme.
  • 106. Attachment of coenzyme to Enzyme  Coenzyme is a non protein substance.  It is attached to the enzyme, which is protein substance.  When the attachment is weak (so that dissociation can occur) the non protein part is called as coenzyme.  If the attachment is firm, the non-protein part is called as prosthetic group. Apoenzyme: It is the protein part of the enzyme to which the coenzyme (or prosthetic group) is attached. Haloenzyme: It is the complete enzyme, which consists of apoenzyme and coenzyme ( or prosthetic group). Haloenzyme = Apoenzyme + Coenzyme or prosthetic group
  • 107. They differ from enzymes in the following aspects: •They are non-protein in nature •They have low molecular weight •They are heat stable •They can be separated by dialysis •They are generally derived from vitamins
  • 108. Classification of Coenzymes Coenzymes can be broadly classified as: i) Group transferring coenzymes and ii) Hydrogen transferring coenzymes. Group transferring coenzymes: TPP, Biotin, Coenzyme A Hydrogen transferring coenzymes: NAD, NADP, FAD, FMN.
  • 109. Association with vitamins Many coenzymes form the constituents of vitamin B complex. Also, the coenzymes are associated with these vitamins. The following is the list of vitamins and their associated coenzymes: Vitamins Coenzymes Pantothenic acid CoA Vitamin B12 Cobamide Niacin NAD, NADP Riboflavin FMN, FAD Thiamine TPP
  • 110. Functions of coenzymes • The important function performed by coenzymes is to transfer hydrogen or groups. • The coenzymes accept the atom or a group from the substrate and transfer them to other molecules. The following is the list of coenzymes and the function performed: Coenzymes Functions NAD, NADP Hydrogen transfer FAD, FMN Hydrogen transfer TPP Acetyl transfer Biotin Carboxyl group transfer Coenzyme A Acyl group transfer
  • 111. Co-Enzyme A (CoA) • It is the coenzyme form of pantothenic acid. • It is composed of ATP, pantothenic acid and mercaptoethyl amine. • It is a group transferring coenzyme. • The reaction of co-enzyme A is due to the presence of sulphydryl group (SH group). • It accepts acetyl group to form acetyl CoA which takes part in a number of metabolic reactions. CoA is required for: • Conversation of α-ketoglutarate to succinyl CoA in citric acid cycle. • Oxidation of fatty acids
  • 112. Acetyl CoA  It is the coenzyme form of pantothenic acid.  It is formed by the transfer of acetate to coenzyme A. Acetyl CoA takes part in the following metabolic reactions: • It is utilised in citric acid cycle • It combines with choline to form acetyl choline • It is the starting material for cholesterol synthesis • It is the starting material for the synthesis of ketone bodies. • It is used in the synthesis and elongation of fatty acids. • It is used for the synthesis of steroid hormones.
  • 113. Coenzyme forms of Vitamin B & their functions Vitamin Activated form- (coenzyme ) Type of catalysis Enzyme using co - enzyme Thiamine Thiamine Pyrophosphate(TPP ) Aldehyde or Keto Group Trans- Ketolase Riboflavin Flavin Mono Nucleotide (FMN ) Hydrogen or Electron L -Amino oxidases Riboflavin Flavin Adenine Dinucleotide (FAD ) Hydrogen or Electron D -Amino oxidases Niacin Nicotinamide Adenine Dinucleotide (NAD ) Hydrogen or Electron LDH Niacin Nicotinamide Adenine Dinucleotide Phosphate (NADP ) Hydrogen Or Electron G-6 P-D Lipoic Acid Lipoic Acid Hydrogen Or Electron Pyruvate Dehydrogenase Complex
  • 114. Coenzyme forms of Vitamin B & their functions Vitamin Activated form- (coenzyme ) Type of catalysis Enzyme using coenzyme Pyridoxine Pyridoxal Phosphate Amino Group Transfer Alanine Transaminase Pantothenic Acid Coenzyme A Acyl Group Transfer Thio Ketolase Folic Acid Tetra Hydro Folate (TFH4 ) One Group Transfer- formyl, Methyl Formyl Transferase Biotin Biotin CO2 Pyruvate Carboxylase Cobalamine Methyl Cobalamine Methyl Malonyl Co A Mutase
  • 115. VITAMINS AND COENZYMES Vitamin Coenzyme Reaction type Coenzyme class SOURCE: Compiled from data contained in Horton, H. R., et al. (2002). Principles of Biochemistry , 3rd edition. Upper Saddle River, NJ: Prentice Hall. B 1 (Thiamine) B 2 (Riboflavin) B 3 (Pantothenate) TPP Oxidative decarboxylation Prosthetic group FAD Oxidation/Reduction Prosthetic group CoA - Coenzyme A Acyl group transfer Cosubstrate B 6 (Pyridoxine) PLP Transfer of groups to and from amino acids Prosthetic group Read more: http://www.chemistryexplained.com/Ce- Co/Coenzyme.html#ixzz3oL0qkSi1 B 12 (Cobalamin) 5-deoxyadenosyl cobalamin Intramolecular rearrangements Prosthetic group Niacin Folic acid Biotin NAD + Tetrahydrofolate Biotin Oxidation/Reduction One carbon group transfer Carboxylation Cosubstrate Prosthetic group Prosthetic group
  • 116. Factors affecting enzyme activity: Cofactors (activators ) Cofactor –inorganic ion Enzymes Fe 2 ,Fe3⁺ ⁺ Peroxidase Cu++ Cytochrome oxidase Mg++ Hexokinase Ni++ Urease Mn++ Arginase K+ Pyruvate Kinase Zn++ DNA Polymerase Mo++ Nitrate Reductase Se Glutathione Peroxidase Ca++ Lipase Cl- Salivary Amylase
  • 117. DIAGNOSTIC APPLICATIONS OF ENZYMES • Some diseases can be diagnosed by the estimation of blood level of certain enzymes. • Under normal conditions, the blood levels of these enzymes are low. • But in certain diseases, the blood levels are more. • It is due to release of these enzyme form damaged tissues or organs. • So, estimation of these enzymes in blood helps in the diagnosis of diseases.
  • 118. The following is the list of enzymes whose levels are increased in disease conditions shown Enzyme Disease that can be diagnosed Amylase Acute pancreatitis Alkaline phosphatase Rickets Creatinine phosphokinase Myocardial infarction Glutamic oxaloacetic transaminase(GOT) Myocardial infarction Glutamic pyruvic transaminase (GPT) Liver diseases Isocitrate dehydrogenase(IDH) Hepatitis, Liver metastasis Lactate dehydrogenase(LDL) Myocardial infarction Lipase Acute pancreatitis
  • 119. Amylase • It is an enzyme secreted in the pancreas. • Normally its concentration in blood is very low. • It’s level increases in acute pancreatitis. •So estimation of amylase in blood can provide diagnosis for acute pancreatitis. Alkaline phosphatase • It is formed in the bone. • Rickets and osteomalacia can be diagnosed by an increase in blood level of this enzyme.
  • 120. Acid phosphatase  It is present mainly in the prostate gland.  An increase in it’s blood level helps in the diagnosis of prostatic cancer. Creatinine phosphokinase (CPK)  It is an enzyme present in heart and skeletal muscle.  An increase in it’s blood level helps to diagnose myocardial infarction and muscular dystrophy. Isocitrate dehydrogenase •It is an enzyme of Krebs cycle. •It’s level increases in hepatitis and malignancy of liver.
  • 121. Lactate dehydrogenase(LDL) • LDL level of serum increases in leukemia, generalised cancer and acute hepatitis. • Also the concentration of LDL, (isoenzyme) in serum increases in myocardial infarction. • So estimation of LDL helps in the diagnosis of these diseases. Lipase • Plasma lipase levels are increased in acute pancreatitis and carcinoma of pancreas. • These diseases can be diagnosed by estimating plasma lipase levels.
  • 122. Transaminases These are enzymes involved in transamination reactions. The two important transaminases present in serum are: 1. Serum glutamic oxaloacetic transaminase (SGOT). 2. Serum glutamic pyruvic transaminase (SGPT). SGOT •The level of SGOT increases in myocardial infarction. • So, myocardial infarction can be diagnosed by estimating SGOT. SGPT • It’s level increases in hepatic damage. • So, estimation of SGPT helps in the diagnosis of liver diseases like infective hepatitis and obstructive jaundice.
  • 123. The following are the applications of enzymes in the fields of medicine and pharmacy: •Drugs acting through enzymes •Therapeutic uses of enzymes •Use of enzymes in the manufacture of bulk drugs Drugs acting through enzymes: •A number of drugs act by inhibiting enzymes. •Enzymes act on drugs and convert them into inactive metabolites. Drug + Enzyme  Inactive metabolite Drug + Enzyme inhibitor---- No inactive metabolite. So the duration of action of the drug is increased. Medicinal significance of Enzymes
  • 124. • In presence of compounds which inhibit the enzyme (i.e. enzyme inhibitor), the formation of inactive metabolite is prevented. So the drug remains inactive form. So the duration of action of the drug is prolonged. • For example, acetylcholine is inactivated by the enzyme acetyl cholinesterase. This enzyme is inhibited by anticholinesterase like physostigmine. So in presence of physostigmine, the action of acetylcholine is increased. • Similarly, Allopurinol used for the treatment of gout acts by inhibiting the enzyme xanthine oxidase. This enzyme is required for the conversation of Xanthine to uric acid.
  • 125. Therapeutic uses of Enzymes Enzymes are used for the treatment of number of diseases. The following are the few examples: •Enzymes like pepsin, papain and amylase are administered for improving digestion. •The enzyme hyaluronidase is used for diffusion of a number of drugs. •The enzymes streptokinase and urokinase are used for dissolving blood clot. •The enzymes trypsin is used for liquefying the lens. So it is used in the treatment of cataract. •The enzyme asparaginase is used for the treatment of cancer.
  • 126. Use of enzymes in the manufacture of Bulk drugs Many enzymes are used in pharmaceutical industry for the manufacture of bulk drugs. The following are a few examples: •The enzyme penicillin acylase is used for the production of 6-amino penicillanic acid from penicillin G. 6-amino penicillanic acid is needed for the synthesis of several Beta lactam antibiotics. •The enzyme glucose oxidase is needed for the production of fructose syrup. •Amylase is needed for the production of dextrin. •The enzyme papain is used in the production of protein hydrolysate.

Notas do Editor

  1. Kinases are specialized transferases that regulate metabolism by transferring phosphate from ATP to other molecules.AST,ALT,hexokinase Kinases are specialized transferases that regulate metabolism by transferring phosphate from ATP to other molecules.AST,ALT,hexokinase
  2. Isomerases are a general class of enzymes which convert a molecule from one isomer to another.. The general form of such a reaction is as follows: A–B → B–A
  3. facilitate intramolecular rearrangements in which bonds are broken and formed or they can catalyze conformational changes.
  4. The 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.
  5. Activation energy is the push needed to start a reaction
  6. Within the active site of the ES complex, the reaction occurs to convert substrate to product (P):
  7. The products are then released, allowing another substrate molecule to bind the enzyme - this cycle can be repeated millions (or even more) times per minute The overall reaction for the conversion of substrate to product can be written as follows: E + S  ES  E + P
  8. The temp at which enzymatic reaction occur fastest is called Optimum temperature
  9. pepsin (a stomach enzyme) functions best at a low (acidic) pH. At pH 1, pepsin is in it’s functional shape; it would be able to bind to its substate. At pH 5, the enzyme’s shape is different and it no longer has an active site able to bind the substrate. The change in enzyme activity is observed as a difference in reaction rate.