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INTRODUCTION TO ENZYMOLOGY
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INTRODUCTION TO ENZYMES

Enzymes

ëm Enzymes are biocatalysts²Catalysts of life

ëm G catalyst is defined as a substance that increases the velocity or the rate of a
chemical reaction, without itself undergoing any change in the process
ëm Enzymes are biocatalysts that are synthesized by living cells.
ëm dhey are proteinaceous (exception is the ribozyme, which is an RNG ,
colloidal, and thermolabile (inactive at 0YC and destroyed at 100YC
ëm dhey are specific in action, catalyze all biochemical reactions and are
susceptible to many factors like temperature, pH, etc.
ëm Examples: urease, carbonic anhydrase, pepsin, rennin, etc.

Gntienzymes

ëm Gntienzymes are those substances which when injected in the body produces
certain molecules, which act as inhibitors and inhibits the function of the
enzyme, related to that particular reaction
ëm Examples: Gntitrypsin, antirennin, antipepsin, etc.


strate

ëm dhe substrate is a substance upon which an enzyme acts and gets converted into
the corresponding product
ëm or example, maltose is the substrate over which the enzyme maltase acts to
from glucose
Maltase
Maltose  Glucose
O aracteristics of Enzymes

1m Colloidal nature
ëm dhey are of great size
ëm dheir molecular weights usually range from 12000 to over a million Daltons

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ëm Hence, they are very large compared to their substrates or the functional
group they act upon
ëm dhe molecular weight of many enzymes are found to be approximately 6-
fold (6 is an integer multiple of 17500, which is found to be a unit in most
proteins
ëm ’n account of their large size, the enzyme molecules possess extremely low
rates of diffusion and forms colloidal systems in water
ëm Due to their giant size, the enzymes exhibit many colloidal properties such
as:
i m Diffusion rates are very slow
ii m May produce considerable high light scattering. dhey form turbidity in
solution known as dyndall effect
2m Catalytic nature
ëm G universal feature of all enzymatic reactions is the virtual absence of any
side product
ëm Gn enzyme is precisely adapted to catalyze a particular reaction. or
example, amylase catalyzes the breakdown of starch
ëm dhey act catalytically and accelerate the rate of chemical reactions,
occurring in the plant and animal tissues
ëm dhey normally do not participate in the reaction, or if they do so, at the end
of the reaction, they are recovered as such without undergoing any
qualitative or quantitative change
ëm dhis is why they are capable of catalyzing the transformation of a large
quantity of substrate
ëm dhus, the catalytic potency of enzymes is extremely great
3m durnover number
ëm dhe catalytic power of an enzyme is measured by the turnover number or the
molecular activity
ëm t is defined as the number of substrate molecules converted into the product
in a given unit of time by a single enzyme molecule, when the enzyme is
fully saturated with the substrate
ëm dhe turnover number of some enzymes are given as follows:

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Enzyme Substrate Kcat (s61

Catalase H2’2 40,000,000
Carbonic anhydrase HC’36 400,000
Gcetyl cholinesterase Gcetyl choline 140,000
-Lactamase Benzyl penicillin 2,000
umarase umarate 800
Rec G protein Gd
0.4

ëm durnover number is represented as Kcat. dhe constant Kcat is a first order rate
constant and its unit is s61

max
KV

[E]d

ëm dhe value of the turnover number varies with different enzymes and it
depends upon the conditions in which the reaction is taking place
ëm dhe turnover number of 40,000,000 s61for carbonic anhydrase is one of the
largest known
ëm Carbonic anhydrase catalyzes the hydration of carbon dioxide to form
carbonic acid:
Carbonic anhydrase
CO2 + 2 O  2 CO3

ëm dhis catalyzed reaction is 4×107 times faster than the uncatalyzed one
ëm or most enzymes this value falls between 1±104 s61
4m Specificity of enzyme action
ëm Enzymes are highly specific in their action when compared with a chemical
catalytic reaction; i.e., a particular enzyme attacks only a particular substrate
ëm ’ccurrence of thousands of enzymes in the biological system is due to the
specific nature of enzymes
ëm Enzyme specificity is based on the mode of accepting the substrate and their
reaction
ëm dhey are
i m Stereochemical or optical specificity

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(1 mStereoisomers are those compounds which have the same molecular

formula but differ in their structural configuration
(2 mdhe enzymes act on only one isomer and thus exhibit specificity
towards that particular stereoisomer
(3 mExamples: L-amino acid oxidase and D-amino acid oxidase;
hexokinase acts only on D-hexoses, arginase acts only on L-Grg and
not on D-Grg, amylase acts only on -glycosidic linkages and not on
- glycosidic linkages, succinate dehydrogenase dehydrogenates
succinate to give fumaric acid and not maleic acid, and so on
(4 mStereospecificity is explained by considering 3 distinct regions of the
substrate molecule, specifically binding with 3 complementary
regions on the surface of the enzyme

Representation of stereospecificity of aƍ, bƍ, cƍ 3 points of attachment of substrate to enzyme (a, b, c

(5 mdhe enzymes belonging to the class of isomerases do not exhibit this

stereospecificity since they are specialized in the interconversion of
the isomers. or example, alanine racemase is involved in the
interconversion of L-ala and D-ala
ii m Reaction specificity
(1 mEnzymes are specific, in the sense that almost one enzyme catalyzes
only one of the various reactions that the substrate can undergo
(2 mor example, ’xaloacetate is an important metabolic intermediate. t
can undergo several reactions namely, reduction to give maleic acid;
decarboxylation to give pyruvate; or accept an amino group to give

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Gsp and so on. Each reaction of ’GG is catalyzed by its own separate
enzyme, which catalyzes only that reaction and none other

Gspartate
dransaminase
§
Decarboxylation
Citrate 
Gcetylation
CH 3C’’H
 ’xaloacetate 
Decarboxylase

yruvate
Gcetylase
Reductase


Malate

(3 m Glu undergoes several reactions but each reaction is caralyzed by a

specific enzyme

Gln
Glutaminase

§
Gsp  -KG 
Gspartate aminotrasferase Glanine aminotransferase
 Glu  Gla  -KG
Reductase


Malate
m
(4 m
artly due to its specificity and partly because they take place at
relatively lower temperatures, enzyme catalyzed reactions are much
more quantitative
iii mSubstrate specificity
(1 mdhe extent of substrate specificity varies from one enzyme to the other
(2 mt may be either absolute or relative
(3 mGbsolute specificity (one to one specificity is seen in some enzymes
which are capable of acting on only 1 substrate. or example, urease
acts only upon urea
(4 mRelative specificity can be further classified into group dependant or
bond dependent
(5 mGroup substrate specificity
(a mGbsolute group specificity or relative group specificity or broad
specificity

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(b mSome enzymes catalyze the reaction of a structurally related group

of compounds
(c mor example, hexokinase adds a phosphate group to all hexoses
like glucose, fructose, mannose, etc.
(d mGroup specificity is exhibited by proteolytic enzymes with respect
to the peptide bond between 2 specific amino acids
(e mExamples:
(i mdrypsin splits peptide bonds in which the carboxylic group is
contributed by either Lys or Grg
(ii mChymotrypsin preferentially splits peptide bonds in which the
carboxyl group is from an aromatic amino acid

(iii dhrombin splits peptide bonds in which the side chain on the
carboxyl site of the susceptible peptide bond must be Grg, while
the one on the amino group must be Gly

(f mdhese enzymes help in the elucidation of the arrangement of the

amino acid residues in a protein
(6 mBond substrate specificity
(a mBond dependent class of enzymes, are very specific in catalyzing
bond-dependent reactions
(b mExamples:
(i m
roteases 
eptide bonds of proteins
(ii mGlycosidases  Glycosidic bonds of carbohydrates

(iii Esterases  Ester linkages in lipids

(iv Exonucleases and endonucleases 
hosphodiester linkages in

nucleic acids

iv mGeometrical specificity
(1 mSome enzymes exhibit specificity towards the cis/trans forms
(geometric isomers
(2 mor example, fumarase catalyzes the interconversion of fumarate
(trans form and malate (cis form

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5m Gmphoteric nature
ëm Enzymes are amphoteric. dhey act as both acids and bases
ëm dhey migrate in an electric field and the direction of their migration depends
upon the charge possessed by them
ëm dhe net charge is influenced by their pH value
ëm Each enzyme has a fixed value of isoelectric point (p at which it will move
in an electric field
ëm soelectric field or isoelectric point is the pH value at which the number of
cations equals the number of anions
ëm dhus, at p, the net electric charge on an enzyme is always 0
ëm But, the total charge on an enzyme molecule (sum of the positive and
negative charges at this point is always maximum
ëm Hence, enzymes are dipolar ions (or internal salts (or zwitterions
ëm Gt p, they exist as  3 ( OO n
ëm Gt pH values m p, enzymes will have a net positive charge and will migrate
towards the cathode
ëm Similarly, at pH ´ p, enzymes will have a net negative charge and will
migrate towards the anode

Acidic behaviour
2

OO
 2 OO + 

asic behaviour
2

OO
   3

OO + l
+ l

6m Solubility
ëm dhe solubility of enzymes is markedly influenced by the pH
ëm Solubility is lower at p and increases with increasing acidity or alkalinity
ëm n either cation or anion, repulsive forces between enzymes are high, since
all the molecules possess excess charge of the same site. dhus they will be
more soluble
ëm Salting-in effect
i m Globulins are slightly soluble in water.

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ii m However, their solubility is greatly increased by the addition of neutral

salts like NaCl
iii mdhis phenomenon is known as salting-in effect
iv mdhis is due to the forces of attraction between the salt and protein at low
salt concentrations leading to increased solubility
ëm Salting-out effect
i m
roteins are precipitated from aqueous solutions by high concentrations
of neutral salts.
ii m dhis is known as the salting-out process
iii mdhe salts commonly used for this purpose are sodium sulphate,
magnesium salts and phosphates
iv mGs the concentration of neutral salts is increased, the solubility increases
to maximum and then starts decreasing and finally the proteins get
precipitated
ëm soelectric precipitation
i m Some proteins like casein of mil are readily precipitated at (or near their
p. dhis process is described as isoelectric precipitation
ii m Salting out of proteins is caused due to the competition for water
molecules between the protein and salt at high concentrations
7m ’ptical activity
ëm Gll proteins rotate the plane polarized light to the left; i.e., they are
laevorotatory
8m Denaturation of enzymes
ëm Denaturation is a result of change in conformation or unfolding of the
enzyme molecule, i.e., 2Y or 3Y structure of the enzyme is completely lost in
the process without any break in the 1Y structure
ëm Denaturation is defined as the total loss or randomization of the 3-D
structure
ëm Denaturation refers to changes in properties of enzymes; i.e., loss of
biological activity

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ëm Ggents causing denaturation:

i m
hysical agents
t includes mechanical action like heat treatment, cooling, freezing
operations, rubbing, high hydrostatic pressures, U and ionizing
radiations, radioactive and ultrasonic radiations
ii m Chemical agents
’rganic solvents (acetone, alcohol , aromatic anions (salicylates , and
anionic detergents (SDS are some examples for chemical
denaturating agents
ëm
roperties of denatured enzymes
i m dhey decrease in solubility, and size
ii m dhey have altered 3-D shape
iii mCessation of biological activity as proteins or hormones
iv mncreased activity of some radicals in the molecule such as the ±SH group
of Cys, the -S-S- of cystine and phenolic group of dyr
v m Glteration in the surface tension and loss of antigenicity
vi mMr and osmotic pressure do not change much
vii m Changes in optical rotation in the direction of increased laevorotation
ëm Denaturation is of 2 types:


©






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ëm Reversible denaturation

drypsin when exposed to temperatures of 80±90YC gets denatured.

However, when cooled to 37YC, the solubility and activity of trypsin is
regained

ëm rreversible denaturation

Boiling of egg results in the loss of tertiary structure and is an

irreversible denaturation

ëm Significance of denaturation
i m Denaturation property of a protein is harmful in clinical laboratories
ii m dhe protein-free substance of the blood such as glucose, picrate, and
drugs are analyzed by the precipitation of blood proteins by the addition
of certain acids
9m Reversibility of a Reaction
ëm Enzymes are capable of bringing about reversion in a chemical reaction
ëm dhe digestive enzymes catalyze the hydrolytic reactions which are reversible
ëm or example, lipase, which catalyzes the synthesis of fat from glycerol and
fatty acids, can also hydrolyze them into their component units

2 O
ipase
( 2 OO 15 31 3 +3 2

O
  O +3 15 31 OO
2 O
dripalmitin Water lycerol Palmitic acid

ëm dhe direction in which the reaction proceeds depends upon many factors like
(1 mpH of cell sap
(2 m
resence of reacting substance
(3 mGccumulation of end products

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Reversibility of action of lipase (from castor on triolein

ëm dhe final equilibrium mixture is the same, whether the reaction starts with
the ester, or with its individual components

O emical Nat re and Properties of Enzymes

ëm Each enzyme has its own tertiary structure and specific conformation, which
is very essential for its catalytic activity
ëm Chemically, enzymes may be divided into 2 categories
ëm Simple protein enzymes
§m dhese contain simple proteins only. E.g., urease, amylase, papain, etc.
ëm Complex protein enzymes
§m dhese contain conjugated proteins, i.e., they have a protein part called
apoenzyme and a non-protein part called prosthetic group, associated
with the protein unit
§m dhe 2 parts together constitute the holoenzyme. E.g., catalase,
cytochrome V, etc.
ëm Holoenzyme
§m dhe functional unit or the active structure of an enzyme
(apoenzyme prosthetic group is called holoenzyme

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Holoenzyme Gpoenzyme  Coenz yme

(Gctive enzyme (
rotein part (
rostetic group

ëm Gpoenzyme
§m t is the protein part of the enzyme
§m t is the inactive form of the enzyme
§m t becomes functional only by associating itself with the prosthetic
group
ëm
rosthetic group
§m G prosthetic group is that which is covalently bound to the apoenzyme
§m dhey do not dissociate from the protein part of the enzyme and
repeatedly participate in enzyme-catalyzed reactions
§m E.g., GD, MN, d

,
L
, biotin, etc.
ëm Cofactor
§m dhey are mainly inorganic metal complexes which are tightly bound
to the enzyme
§m dhey are highly required for normal conformational structure and
function of the enzyme
§m dhey act as 
donors

or acceptors
 
in oxidation
 
and reduction

reactions
2 2 2 2 2 2 2
§m E.g., Mg , Ca , Cu , Zn , K , e , Mn , Mo , etc.
ëm Coenzymes
§m dhey are also called co-substrate or second substrate
§m dhey are organic, metallo-organic, or inorganic substances which are
thermostable and dialyzable, highly required for the normal
functioning of the enzyme
§m dhey bind covalently or non-covalently to the apoenzyme
§m Reactions involving oxidoreductions, group transfers, isomerization,
and covalent bond formation require coenzyme
§m Coenzymes account for 1% of the entire enzyme molecule
§m E.g., NGD, GD, dH, CoG. MN, d

, etc.

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Olassification of Ooenzymes

1. Classification based on chemical characteristics





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2. Based on functional characteristics




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 nctions of Ooenzymes

ëm dheir function is usually to accept atoms or groups from a substrate and to

transfer them to other molecules
ëm dhey are less specific than enzymes and the same coenzyme can act as such in a
number of different reactions
ëm Coenzymes are also attached to the protein at different but adjacent site, so as to
be in a position to accept the atoms or groups, which are removed from the
substrate
§m M
M
 Hydrogen acceptors in dehydrogenation reactions
§m O

 O  dransfer of acetyl/acyl groups used in oxidative
decarboxylation of pyruvate, G synthesis and acetylation
§m d Decarboxylation of -ketoacids and to carry the ³active aldehyde
(R±CH(’H 6  group
§m  dransamination and decarboxylation of amino acids
§m d
 Carrier of folate, used in the synthesis of purines and pyrimidines
§m Ô


 Carbon chain isomerization
§m Ô  Carbon dioxide fixation reactions
§m


M Hydrogen acceptors in dehydrogenation reactions
§m

 Gct as coenzyme in cytochromes, peroxidases and
G synthase
complex

{ole of Metals in Enzyme Gction

ëm dhe activity
 
of many

enzymes
 
depend on the presence of many metal ions such
2 2 2 2
as Mg ,Ca , Cu , Zn , K , etc.
ëm Depending upon the interaction between the enzyme molecule and the metal
ion, the enzymes are classified as metal activated enzymes and metalloenzymes
ëm Metal activated enzymes
§m n certain enzymes, metals form a loose and easily dissociable complex
§m Such enzymes are called metal activated enzymes
§m dhe metal ion can be removed by dialysis or any other simple method
from the enzymes without causing denaturation (inactivation of
apoenzyme   
§m or example, Gd
ase±Mg2 and Ca2 , enolase±Mg2 , etc.

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ëm Metalloenzymes
§m Some enzymes which are tightly bound to metal ions are called
metalloenzymes
§m dhese metals cannot be dissociated from the apoenzyme, even after
several extensive steps of purification 
§m or example, Cu2 cytochrome oxidase,phenol oxidase; Zn

2
carbonic
2 2
anhydrase, alcohol dehydrogenase; Mg hexokinase; Ni urease, etc.

{ole of Metals in Enzyme Oatalysis

ëm dhey help in maintaining or producing, or both, active structural conformation

of the enzyme
ëm Enable the formation of the E±S complex
ëm Metal ions, like protons, can share an electron pair forming a sigma bond
ëm dhey can serve as a 3-D template for the orientation of basic groups on the
enzyme or substrate
ëm Metal ions accept electrons via sigma or pi bonds to activate electrophiles or
nucleophiles
ëm dhey can also act as nucleophile themselves by donating electrons
ëm G metal ion may also ³mask a nucleophile and thus prevent an otherwise likely
side reaction
ëm dhe coordination sphere of a metal bring together the enzyme and substrate or
form chelate, producing distortion in either the enzyme or substrates
ëm Stereochemical control of an enzyme catalyzed reaction may be achieved by the
ability of the metal coordination sphere to act as a 3-D template to hold reactive
groups in a specific steric orientation
ëm Metal ions participate in each of the 4 mechanisms by which enzymes
accelerate the rates of chemical reactions
§m General acid±base catalysis
§m Covalent catalysis
§m Gpproximation of reactants
§m nduction of strain in the enzyme or substrate
ëm dhe role of metal ions in some important enzymes are summarized in the
following table:

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No Enzyme {ole of metal ion

1 Histidine deaminase
2 Kinases, lyases, pyruvate
decarboxylase
3 Carbonic anhydrase
4 Cobamide enzymes
5
yruvate carboxylase,
carboxypeptidase, alcohol
dehydrogenase
6 Non-heme iron proteins
7
yruvate kinase, pyruvate
carboxylase, adenyl kinase
8
hosphotransferase, D-xylose
isomerase, heme proteins
Masking a nucleophile
Gctivation of an electrophile
Gctivation of an nucleophile
Metal acts as a nucleophile

i electron withdrawal

i electron donation
Metal ion gathers and orients
ligands
Stain effects

dernary Oomplexes wit Metals  nction in Oatalysis

ëm or ternary (3 component complexes of catalytic site (E , a metal ion (M , and

substrate (S that exhibit 1:1:1 stoichiometry, 4 schemes are possible as follows:

1. E S M (Substrate bridge complex

2. M E S (Enzyme bridge complex
3. E M S (Simple metal bridge complex
4. E M ( yclic metal bridge complex
S
ëm Gll 4 are possible for metal activated enzymes
ëm Metalloenzymes cannot form the E±S±M complex as they retain the metal
throughout the purification
ëm dhree generalizations are:
§m Most, but not all kinases (Gd
: phosphotransferases form substrate
bridge complexes of the type: E±nucleotide±M

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§m
hosphotransferases using pyruvate or
E
as substrate, enzymes

catalyzing other reactions of
E
and carboxylases form metal bridge
complexes
§m G given enzyme may form one type of bridge complex with one substrate
and a different type with the other
ëm Enzyme bridge complexes
§m dhe metals in the enzyme bridge complexes are presumed to perform
structural roles, maintaining an active conformation (e.g., Gln synthase
or to form a metal bridge to a substrate (e.g., pyruvate kinase
§m n addition to its structural role, the metal ion in the pyruvate kinase
appears to hold one substrate (Gd
in place to activate it

M

yruvate kinase Gd

Creatine
ëm Substrate bridge complexes
§m dhe formation of ternary substrate bridge complexes of nucleoside
triphosphates with enzyme, metal and substrate appears to be attributed
to the displacement of water from the coordination sphere of the metal by
Gd

2
Gd
46  M(H 2 ’ 26

> >> > Gd
6 M(H 2 ’ 3  3H 2 ’
6

dhe enzyme then binds, forming the ternary complex:

26 26
Gd
6 M(H 2 ’ 3  E
> >> > E 6 Gd
6 M(H 2 ’ 3

§m n phosphotransferase reactions, metal ions are thought to activate the

phosphorous atoms and form a rigid, polyphosphate±adenine complex of
appropriate conformation in the active quaternary complex
ëm Metal bridge complexes
§m Crystallographic and sequencing data have established that a histadyl
residue is concerned with metal binding at the active site of many
proteins (e.g., carboxypeptidase G, alkaline phosphatase (GL
,
phospholipase C (
LC , etc..

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§m or binary E±M complexes, the rate limiting step is the departure of
water from the coordination sphere of the metal ion
§m or many peptidases, activation by metal ions is a slow process requiring
many hours
§m dhe slow reaction probably is due to the conformational rearrangement of
the binary E±M complex to an active conformation

E  M( 2
apid
O 6  E M( 2 O 6 6  6 2 O

Rearrangement to active conformation:


(2 O 6 6
lo
   (2 O 6 6m

§m or metalloproteins, the ternary metal bridge complex must be formed by

the combination of the substrate with the binary E±M complex

ntracell lar enzymes

ëm Enzymes, which are synthesized in a particular cell and catalyze the

biochemical reaction of the same cell are known as intracellular enzymes
ëm dhey are also known as endoenzymes or metabolic enzymes

Extracell lar enzymes

ëm Enzymes, which are synthesized in a particular cell and are transported to the
target site where they catalyze biochemical reactions, are called extracellular
enzymes
ëm dhey are also known as exoenzymes

Àymase and Àymogen orms

ëm dhe zymase form is the active form of an enzyme

ëm t acts upon the substrate as such; i.e., without undergoing any prior
modifications in its structure
ëm ntracellular enzymes belong to the class of zymase enzymes
ëm dhe zymogen form is the inactive form of an enzyme. dhey are secreted/exist as
inactive precursors
ëm dhey are also known as proenzymes or pre-proenzymes

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m

ëm dhey can be converted into the active form (zymase form by modifications in
their peptide bonds
ëm or example,

drypsinogen (inactive 

nterokinase
drypsin (active
hymotrypsinogen (inactive 
drypsin
hymotrypsin (active

ëm Here, an intestinal enzyme, enterokinase, converts trypsinogen, a proenzyme

secreted by the pancreas, into active trypsin. Similarly, chymotrypsin is
synthesized by the exocrine cells of the pancreas in its precursor form viz.,
chymotrypsinogen. Hydrolysis of this form by trypsin converts it into active
chymotrypsin
ëm Zymogen secretion is a protective mechanism to prevent the digestion of cell
walls and ducts, since it is most frequently found in proteolytic enzymes

 



ëm dhe process of activation of zymogens by their corresponding active enzymes is

called autocatalysis
ëm or example, inactive pepsinogen is secreted by the gastric mucosa and is
converted to active pepsin, both by the acidity of the gastric juice and by pepsin
itself
ëm During autocatalysis, a polypeptide is liberated from the proenzyme

 or Pepsin
Pepsinogen  Pepsin  A polypeptide

ëm f a graph of time vs. enzyme activity is plotted, a sigmoid curve is obtained

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Monomeric and M ltimeric Enzymes

ëm Enzymes which have only one polypeptide chain in their structre are known as
monomeric enzymes
ëm Examples: RNase, DNG polymerase ,
E
, pyruvate carboxylase
ëm When many different enzyme-catalyzing reaction sites are lovated at different
sites of the same molecule, it is known as a multimeric or multienzyme
complex
ëm dhe complex becomes inactive when it is freactionated into smaller units, each
bearing individual enzyme activity
ëm dhese complexes pass intermediate products along from enzyme-to-enzyme by
transfer reactions
ëm Examples: atty acid synthetase, C
S ,
DH,
G synthase

Enzyme d rnover

ëm dhe requirement for the formation of a single intermediate complex can include
the reaction of the type:
  
+
> >> >1> >  
2
P 
3
+P
1

rovided  
is the rate limiting step for the overall reaction

ëm dhe constant Kcat is called the turnover number

ëm dhis is obtained from the general expression, max = Kcatm[E0]

m 

mm
mmm

m
m
m

 

m
m
m

ëm t represents the maximum number of substrate molecules which can be

converted to products per molecule of the enzyme per unit time
ëm or simple reactions, Kcat = K2
ëm or more complicated reactions, Kcat will be a function involving several
individual rate constants
ëm or a simple reaction,

K 2 [E 0 ] [S]
0 =
K m [S]
but E 0  [E] [S]
Km
[ES] 
[E] [S]

K 
 0   cat  [E] [S]
 Km 

ëm Kcat/Km is the catalytic efficiency

ëm G high value indicates that the limiting factor for the overall reaction is the
frequency of collisions between the E and S molecules
ëm G comparison of Kcat/Km for alternative substrates can be used as a measure of
specificity of an enzyme

soenzymes (sozymes)

ëm Enzymes which exist in tissues in two or more forms and have the same
catalytic activity, but are physically, chemically, immunologically, and
electrophoretically distinct are known as isoenzymes or isozymes
ëm dhey are present in the serum and tissues of mammals, amphibians, birds,
insects, plants and unicellular organisms
ëm Examples include isozymes of numerous dehyrogenases, several oxidases,
transaminases, phosphatases, transphosphorylases, proteolytic enzymes,
aldolases, etc.

O


 
ëm dhey catalyze the same reaction, but can be distinguished by physical methods
like electrophoresis or immunological techniques

m 

mm
mmm

m
m
m

 

m
m
m

ëm dhe difference between some isozymes are due to differences in the quaternary
structure of the enzyme
ëm or example, lactate dehydrogenase (LDH exist in 5 isozymic forms
ëm dhe isozymic forms of LDH are tetramers, each made up from 2 tyes of units H
and M. dhe molecular weight of active LDH is 130,000. ’nly the tetrameric
molecule possesses catalytic activity. dhe subunits are expressed in the
following 5 ways:m

È 
 1
 
 
 2 
 
ubunits

  
 3 
 isozyme
 
 
 4 
 
 5 


ëm Splitting and reconstructing of LDH±1 or LDH±5 produces new isozymes

ëm Hence, each consist of a single subunit
ëm When a mixture of purified LDH±1 and LDH±5 is subjected to splitting and
reconstitution, LDH±2, 3,and 4 are also produced

Gctive ite

ëm t is also called the catalytic site or the substrate site

ëm t is the site at which the substrate binds to an enzyme
ëm t consists of specific amino acids or groups, which contributes for specificity of
the enzyme for a substrate
ëm dhey are involved in the formation and breakage of bonds and are known as the
catalytic groups

Oommon feat res of t e active site

1. t occupies a relatively small portion of the enzyme molecule

ëm Most of the amino acid residue in the enzyme are 6
in contact with the
substrate

m 

mm
mmm

m
m
m

 

m
m
m

ëm Nearly all enzymes are made up of more than 100 amino acid residues,
which give them a mass of greater than 10 kD, and a diameter of more than
25 Å
ëm ’nly a fraction of the amino acids are involved in the active site formation

2. t is a 3-D entity

ëm dhe active site of an enzyme is not a point, a line or a plane

ëm t is an intricate 3-D form made up of groups that come from different parts
of the linear amino acid sequence
ëm Residues far apart in the linear sequence ay interact more strongly than
adjacent residues in the sequence
ëm or example, in lysozyme, the important groups in the active site are
contributed by residues numbered 35, 52,62,63,and 101 in a linear sequence
of 129 amino acids

3. Substrates are bound to the enzyme by weak bonds

ëm E±S complex have an equilibrium constant that range from 10-2 to 10-8 M,
corresponding to free energy of interaction ranging from -3 to -12 kcal/mol
ëm dhese values can be compared with strengths of covalent bonds which are
below -50 to -110 kcal/mol

4. Gctive sites are clefts or crevices

ëm n all enzymes, the substrate are bound to clefts or crevices from which
water is usually excluded
ëm dhe cleft also has several polar residues that are essential for binding and
catalysis
ëm dhe nonpolar characteristic regions of the cleft enhances the binding ot the
substrate
ëm dhe cleft creates a microenvironment in which certain polar residues acquire
special properties essential for their catalytic role

5. Specificity of binding depends on precisely defined arrangement of atoms in an

active site

ëm dhe substrate must have matching shape to fit into the site (ischer¶s model

m 

mm
mmm

m
m
m

 

m
m
m

ëm dhe active site of some enzymes are not rigid and the active site is thus
modified by the binding of the substrate
ëm dhe active site has a shape complementary to that of the substrate only after
the substrate is bound (Koshland¶s model

ëm dhe side chain groups like ±C’’H, ±NH2, ±CH2’H, etc.. serve as catalytic
groups in the active site
ëm dhe crevice creates a microenvironment in which certain polar residues acquire
special properties essential for catalysis
ëm dhe following figure illustrates the same:

ëm dhere must be at least 3 different points of interaction between the enzyme and
the substrate
ëm dhese interactions can have either a binding or a catalytic function
ëm Binding sites link to specific groups in the substrate, ensuring that the enzyme
and substrate molecules are held in a fixed orientation with respect to each other
with the reacting group or groups in the vicinity of catalytic sites
ëm Consider the following 3-point interaction:

O








ëm Here, sites Gƍƍ and Gƍƍƍ might represent binding sites of Rƍƍ and Rƍƍƍ

m 

mm
mmm

m
m
m

 

m
m
m

ëm Gƍ is the catalytic site for a reaction involving Rƍ

ëm dhus, even if Rƍ and Rƍƍ are identical, the asymmetry of the E±S complex means
that only Rƍ can react, providing binding site
ëm Gƍƍƍ is specific for Rƍƍƍ and Rƍƍ can never undergo reaction as it is not brought to
the vicinity of Gƍ site even when Rƍ binds to Gƍƍ site

dentification/Mapping of Enzyme¶s Gctive ite

d



 

 


ëm dhe reversible character of the steps involved in enzyme catalyzed reactions

makes the determination of each substrate binding site complex
ëm Gt a steady state, a constant amount of E±S complex is known to be present, but
if an attempt is made to isolate this complex, the effort will be futile because the
substrate will dissociate from the enzyme
ëm However, if the E±S complex can be trapped in a modified form by some
chemical process so that the substrate is no longer able to dissociate from the
enzyme, then it may be possible to identify the substrate binding site
ëm Consider the following example:
Fructose bisphosphate aldolase

AP  lyceraldehyde 3 phosphate
> >> >> >> >> >> >> >> >> >> >> >>> Fructose 1,6 bisphosphate

ëm Horecker et al., (1962 showed that if the reaction mixture was treated with
sodium borohydride an inactive complex is formed
ëm Upon hydrolysis, ±N±glyceryl lysine was found among the products
ëm rom this, it was concluded that DHG
normally binds to the  side chain
amino group of a lys residue in the enzyme by a Schiff¶s base (±N=CH±
linkage
ëm dhe substrate in the binding site could be found as follows:

m 

mm
mmm

m
m
m

 

m
m
m

ëm n general, once an E±S complex has been trapped as an inactive complex, it

may be subjected to partial hydrolysis and amino acid sequence on each side of
the binding site determined
ëm Hence the substrate binding site may be precisely located

u
  



 


ëm Gn alternate way of producing a more stable complex is to replace the natural

substrate by an analogue which binds to the same site of the enzyme but is then
less rapidly removed
ëm or example, the first step in the hydrolysis of nitrophenyl acetate and other
acyl esters by chymotrypsin is the rapid splitting of the ester to yield the first
product and forms an acyl enzyme
ëm dhe subsequent liberation of the acyl group from the enzyme is extremely slow,
allowing the structure to be investigated
ëm dhe acyl group binds by an ester linkage to the ’H group of serine in the
enzyme
ëm Enzymes form even stronger linkages with irreversible inhibitors
ëm dhus, D
 binds to serine residue at the active site in chymotrypsin

m 

mm
mmm

m
m
m

 

m
m
m

ëm
artial hydrolysis of each enzyme±inhibitor (E± complex gives a series of

peptide fragments which can be separated from each other and analyzed
ëm dhe amino acid sequence of any fragment containing D
 must be the primary
structure of a part of the active site
ëm n this way, it is shown that the amino acid sequence around the essential serine
residue of chymotrypsin is
±Gly±Gsp±Gly±Gly±
ro
and the serine residue is identified as ser-195. Gn identical sequence is found in
chymotrypsin
ëm dhe assumption that an irreversible inhibitor binds to the active site of an
enzyme is particularly valid when the inhibitor resembles a substrate
ëm or example, tosyl (M-toluene sulphonyl L-phenylalanine chloromethyl ketone
(d
CK resembles esters which are hydrolyzed by chymotrypsin, but d
CK
itself acts as an irreversible inhibitor of this enzyme by alkylating the His-57
residue

ëm Both ser and his are present at the active site

ëm Binding of d
CK to the enzyme brings reactive groups to close proximity to
his-57 residue and facilitate the formation of a covalent bond


 
 



Gmino acid Enz-GG n m
er {eagent pecific {eaction

His Chymotrypsin
ëm His-57 d
CK Glkylation of
ëm His-199, 12 odoacetate histidine
Cys
apain
Cys-25 odoacetamide Glkylation
M-ethyl maleimide nhibition

CMB nactivation

m 

mm
mmm

m
m
m

 

m
m
m

Ser Chymotrypsin
Ser-195 D
 D
enzyme
(inactivation
Met Chymotrypsin
Met-192
hotooxidation Sulphoxide
product
dyr Carboxypeptidase
dyr-248 odination or nitration nactivation
of benzene
Gsp and Glu
epsin J-bromophenacyl nactivation by
bromide ester linkage with
Gsp residue
-carboxylic Lysozyme Gminomethane Loss of enzyme
amino acid sulphonic acid activity
Lys RNase DNB
Lys 1-e41
drp Lysozyme
hotooxidation


 
 

 



ëm f an enzyme is modified by the conversion of a particular amino acid chain to a

different form (e.g., by reaction of an irreversible inhibitor and this
modification results in a loss of catalytic activity, then it is possible that the
amino acid concerned is a component of the active site of the enzyme
ëm However, the loss of activity is due to a change in the tertiary structure resulting
from a modification to an amino acid residue not present at the active site
ëm dhe 2 processes may be distinguished by attempting to carry out the same
modification in the presence of excess amount of the substrate or by removing
the excess amounts of inhibitor********

Mec anism of Enzyme Gction

ëm Maud Menten has proposed a hypothesis on enzyme action which is most

acceptable

m 

mm
mmm

m
m
m

 

m
m
m

ëm Gccording to this hypothesis, the enzyme molecule E first combines with a

substrate S to form the ES complex which further dissociates to give the
product and the free enzyme
ëm Enzyme once dissociated is free to associate with another substrate to form the
product

E  S
> >> > ES 
 E 

ëm dhe ES complex is an intermediate complex and the bonds involved are weak,
non-covalent bonds like hydrogen bonds, ander Waal¶s forces, and
hydrophobic interactions
ëm Sometimes, 2 substrates can bind to an enzyme and such reactions are known as
bisubstrate reactions
ëm Most reactions in the biological system are bisubstrate reactions:

G  B
> >> >
 

ëm dhese reactions entail the transfer of functional groups such as ammonium

group from one substrate to another
ëm n oxidation±reduction reactions, electrons are transferred between substrates
ëm Bisubstrate reactions are divided into 2 classes viz.,
§m Sequential displacement reactions (single displacement reactions
8m ’rdered sequential mechanisms (’SM
8m Random sequential mechanism (RSM
§m Double displacement reactions (ping-pong reactions

eq ential displacement reactions (single displacement reactions)

E  S1
> >> > ES1

ES1  S 2
> >> > ES1S2
> >> > E
2 
1
E
2
> >> > E  
2

ëm Gll the substrates must bind to the enzyme before any product is released
ëm G ternary complex of the enzyme and both the substrate forms

m 

mm
mmm

m
m
m

 

m
m
m

ëm Sequential mechanisms are of 2 types:

ëm ’rdered sequential mechanism (’SM
§m Enzymes that have NGD or NGDH as a substrate exhibit ’SM 
§m LDH reduces pyruvate to lactate, while oxidizing NGDH to NGD
§m dhe coenzyme always binds first, and the lactate is always released first
§m dhe following is the Cleland notation of ’SM

( A
 (Pyruvate
> >> > ( actate ( A


ëm Random sequential mechanism (RSM

§m dhe order of addition of substrates and release of products is random
§m t can be illustrated by the formation of phosphocreatine and GD
from
Gd
and creatine, catalyzed by creatine kinase
§m dhe following is the Cleland notation of the aforementioned reaction

E(Creatine (Gd

> >> > E(
hosphocreatine (GD

§m dhe ternary complexes are formed in this mechanism also

©o
le ©isplacement {eactions (Ping-Pong {eactions)

ëm ’ne or more products are released before all the products are released before all
substrates bind to the enzyme
ëm dhe most important feature is the existence of a substituted enzyme
intermediate, in which the enzyme is temporarily modified

m 

mm
mmm

m
m
m

 

m
m
m

ëm or example, reactions that shuttle amino groups between amino acids and -
keto acids
ëm Gspartate aminotransferase catalyzes the transfer of an amino group from Gsp
to -ketoglutarate

ëm Gfter Gsp binds to the enzyme, the enzyme removes aspartate¶s amino group to
form the substituted enzyme intermediate
ëm dhe first product ’GG departs
ëm dhen, the second substrate, -KG binds to the enzyme, accepts the amino group
from the modified enzyme, and is then released as the final product viz.,
glutamine

ormation of t e E Oomplex

ëm dhe site of formation of the ES complex upon the enzyme is known as the
active site
ëm t is made up of several amino acids, that come together as a folding of the 2Y
and 3Y structure of enzymes
ëm So, the active site possess a complete 3-D structure and forms a cleft to accept
the substrate
ëm ormation of the ES complex has been described by 2 models
§m Lock and key model (demplate model by ischer
§m nduced fit model by Koshland
ëm dhese 2 models are elucidated as follows:

 

 

ëm dhis model was proposed by ischer which states that the V

  


6J J V6  
66
666
 6V 




ëm dhe active site provides a rigid, pre-shaped template, fitting with the shape
and size of the substrate

m 

mm
mmm

m
m
m

 

m
m
m

ëm dhe substrate fits into the enzyme as a key fits into a lock; hence this model
is known as the lock and key model
ëm dhis model proposes that the substrate binds with a rigid, preexisting site on
the enzyme

ëm However, this theory cannot explain the change in the enzyme activity in the
presence of allosteric modulators


  


ëm Due to the restrictive nature of the lock and key model, another model was
proposed by Koshland, known as the induced fit model
ëm dhe important feature of this model is the flexibility of the active site
ëm Gccording to this, the enzyme doesn¶t possess a rigid, preformed structure of
active site to fit in the substrate, but during its binding with the substrate, the
substrate induces a conformational change in the active site of the enzyme to
attain the final shape for binding
ëm Consequently, the enzyme molecule is made to fit completely for the
configuration and active centers of the substrate
ëm Gt the same time, other amino acid residues may become buried in the
interior of the molecule

m 

mm
mmm

m
m
m

 

m
m
m

ëm dhis model explains several characteristics like:

§m Enzymes become inactive on denaturation
§m Saturation kinetics
§m Competitive inhibition
§m Gllosteric modulations

ENÀ ME NOMENO
Gd
{E

ëm dhe nomenclature of the enzyme is done as follows:

§m dhe first part of the name gives the name of the substrate
§m dhe second part indicates the type of reaction catalyzed
§m dhe third part is the suffix ³  indicating that it is an enzyme.
However, some enzymes do not have this suffix (e.g., trypsin, pepsin,
diastase, papain, etc..
ëm Example:

GLC’H’L DEHYDR’GENGSE

n this enzyme, alcohol is substrate, the type of reaction is dehydrogenation

and the suffix ³  is places at the end

ëm  



 



§m Carbohydrate Carbohydrases
§m
roteins
roteases

m 

mm
mmm

m
m
m

 

m
m
m

§m Lipids Lipases
§m Nucleic acids Nucleases
ëm d
 

  



§m HydrolysisHydrolases
§m ’xidation’xidases
§m dransaminationdransaminase
§m somerizationsomerases
§m DehydrogenationDehydrogenases
§m
hosphorylation
hosphorylases
ëm  



 
 
 

  



§m Some enzymes give clue of both the substrates utilized and the type of
reaction catalyzed
§m Examples:
8m L-glutamate dehydrogenase indicates an enzyme catalyzing a
dehydrogenation reaction involving L-glutamic acid
8m Succinate dehydrogenase catalyzes the dehydrogenation of the
substrate succinic acid
ëm  





§m G few enzymes are named by adding the suffix ³-ase to the name of
the substance synthesized viz., rhodonase that irreversibily form
hydrocyanic acid and sodium thiosulphate
ëm O

     



§m Based on their chemical composition, enzymes are classified into
three categories:
8m Enzyme molecule consisting of protein only. Example: pepsin,
trypsin, urease, papain, amylase, etc.
8m Enzyme molecule containing 
a protein and a cation. Example:
carbonic

anhydrase (Zn as cation , arginase (Mn2 , tyrosinase
2

(Cu2 , etc.
8m Enzyme molecules containing a protein and a non-protein
organic compound known as prosthetic group. dauber (1950
has subdivided them on the basis of the nature of prosthetic
group involved as follows:
ëm e porphyrin enzymes: Catalase, cyt V peroxidase , 

m 

mm
mmm

m
m
m

 

m
m
m

ëm lavoprotein enzymes: Gly oxidase, histamine

ëm Diphosphothiamin enzymes:
yruvate mutase, -
carboxylase
ëm Enzymes requiring other coenzymes:
hosphorylase,
amino acid decarboxylase
ëm 

  


  

§m Carbohydrate hydrolyzing enzyme
8m Glycosidases: Cellulase, amylase, sucrose, lactase, maltase
8m -glucuronidase
§m
rotein hydrolyzing enzymes
8m
eptide bonds
ëm Endopeptidases
§m Gnimals
epsin, trypsin, rennin
§m
lants
apain, ficin, bromalin
ëm Exopeptidases
§m Dipeptidase, tripeptidase
8m Non-peptids C±N linkages (Gmidases 
ëm Urease, arginase, glutaminase
§m Lipid hydrolyzing enzymes
8m Lipases, esterases, lecithinases
§m ’ther ester hydrolyzing enzymes
8m
hosphatases, cholinesterases, chlorophyllases, sulphatases,
pectinesterases, methylases
§m ’xidation±reduction enzymes
8m Hydrases, mutases, oxidases, dehydrogenases, peroxidases
§m ’ther miscellaneous enzymes
8m Catalase, carboxylases, carbonic anhydrase, thiaminase,
transpeptidase

O
GOGdON O ENÀ ME

ëm Enzymes are broadly classified into 6 classes, according to the type of reaction
catalyzed by them

m 

mm
mmm

m
m
m

 

m
m
m

ëm dhe 6 classes are:

§m ’xidoreductases §m Lyases
§m dransferases §m somerases
§m Hydrolases §m Ligases
ëm Each class is further subdivided into various subclasses and each subclass is
divided into various sub-subclasses depending on several criteria involving the
catalytic reaction
ëm n each sub-subclass a number of enzymes are enrolled, each with their serial
number
ëm Every enzyme has been given an enzyme code (commission number in 4
numbers, serially standing for class, subclass, sub-subclass, and serial number
ëm or example:

’ 




ëm Enzymes involved in oxidation and reduction reactions are known as

oxidoreductases
ëm ’xidation is carried out by the removal of electrons from a specific group of
substrate or by the addition of oxygen to the specific group
ëm Reduction is the opposite change
ëm dhis class is divided into subclasses, according to electron donor or acceptor of
the substrate
ëm or example:

§m 1.1.1.1  Glcohol dehydrogenase

§m 1.1.2.7  Lactate dehydrogenase
§m 1.2.3.4  ’xalate oxidase

m 

mm
mmm

m
m
m

 

m
m
m

d





ëm dhey catalyze reactions of the type G  B
> >> > B  G

ëm Enzymes of this class transfer a particular group from one substrate to the other
ëm dhese enzymes are further divided into subclass according to the specific group
transferred by them
ëm or example:
§m 2.1.2.1  Glycine hydroxymethyltransferase
§m 2.2.1.1  dransketolase
§m 2.2.1.2  dransaldolase

m 

mm
mmm

m
m
m

 

m
m
m

 



ëm Enzymes which catalyze cleavage of bonds by the addition of water are called
hydrolases
ëm dhey catalyze reactions of the type: A X   2 O
> >> > X O  A
ëm dhey have different classes according to the bond hydrolyzed
ëm or example:
§m 3.1.1.1  Carboxyesterase
§m 3.1.1.2  Gryl esterase
§m 3.2.1.1  -amylase
§m 3.1.3.1  GL

m 

mm
mmm

m
m
m

 

m
m
m





ëm Lyases cleave a covalent bond of the substrate to convert it into more than one
product, but the reaction does not involve any hydrolysis
ëm dheir action frequently produce a double bond in one of the product
ëm dhey are divided into subclasses according to the atom connected to the bond
ëm dhey generally catalyze the breaking of C±C, C±S, and C±N bonds
ëm Examples:
§m 4.1.1.1 
yruvate decarboxylase
§m 4.1.1.22  Histidine decarboxylase
§m 4.3.2.1  Grginosuccinate lyase

m 

mm
mmm

m
m
m

 

m
m
m

 

   m


3




   
m

 




3
 
ëm somerases convert their substrates to their isomers by intramolecular
rearrangement
ëm dhey are divided into subclasses according to the type of isomerization

ëm Examples:
§m 5.1.1.1  Glanine raecimase
§m 5.3.1.9  Glucose 6-phosphate isomerase (or phosphohexose isomerase
§m 5.4.2.1 
hosphoglycerate mutase

m 

mm
mmm

m
m
m

 

m
m
m





ëm Ligases are enzymes that catalyze the formation of a bond between 2 substrates
ëm dhey catalyze the formation of bonds between C, ’, N, and S coupled to
hydrolysis of high energy compounds like Gd

ëm dhey catalyze reactions of the type:   Y Gd

> >> >  6 Y  GD

i or
  Y Gd

> >> >  6 Y  GM


i
ëm dhey are subdivided into classes according to the atom connected by the new
bond
ëm Examples:
§m 6.1.1.1  dyrosine t-RNG ligase
§m 6.1.1.2  dryptophan t-RNG ligase
§m 6.1.1.3  dhreonine t-RNG ligase

m 

mm
mmm

m
m
m

 

m
m
m

m 

mm
mmm

m