Enzymes

• Biological Catalyst- increase the rate of chemical reactions

• Most of them are proteins
• They are high molecular weight ( 12000 to 1 million)
compounds made up of chains of amino acids linked together
by peptide bonds.
• Substrate is a Substance in which enzyme act and produce a
product .
• Each enzyme is specific to certain substance or substances
and produce a particular product or products.
• Enzymes increase the rate of chemical reactions by lowering
the free energy barrier that separates the reactants and
products
• The basic characteristics of enzymes includes
 Almost all the enzymes are proteins and they follow the
physical and chemical reactions of proteins
 Enzymes are sensitive and labile to heat
 Enzymes are water soluble
 Enzymes could be precipitated by protein precipitating
agents such as ammonium sulfate and trichloroacetic acid.
• Enzymes differ from ordinary chemical catalysts by:
– Higher reaction rates, 106-1012
– Milder reaction conditions (temp, pH, …)
– Greater reaction specificity (no side products)
– Capacity for regulation
 Classification & Nomenclature of Enzyme
• According to the International union Of Biochemistry an
enzyme name has two parts:
‐First part is the name of the substrates for the enzyme.
‐Second part is the type of reaction catalyzed by the
enzyme. This part ends with the suffix “ase”.
Example: Lactate dehydrogenase
• The Enzyme Commission (EC) numbers divide enzyme into six
main groups – according to the type of rxn catalyzed.
• The first Enzyme Commission (1961) devised a system for
classification of enzymes. These code numbers, prefixed by EC,
which are now widely in use, contain four elements separated by
points, with the following meaning:
 the first number shows to which of the six main divisions (classes) the
enzyme belongs
 the second figure indicates the subclass,
 the third figure gives the sub-subclass,
 the fourth figure is the serial number of the enzyme in its sub-
subclass.
• The main divisions and subclasses are:
The Six Classes

 EC 1. Oxidoreductases
 EC 2. Transferases
 EC 3. Hydrolases
 EC 4. Lyases
 EC 5. Isomerases
 EC 6. Ligases
EC 1 : Oxidoreductases :
oxidation – reduction reaction
H2/ O2 /e-1 transfered between molecule
eg: dehyogenase (hydride transfer)
Oxidases (e transfer to molecular O2 )
Oxygenases (O2 transfer from molecular O2 )
Peroxidases (e transfer to peroxide)
Ist digit – Main class
IInd digit - H2/ e-1 donor
IIIrd digit - H2/ e-1 acceptor
EC 2 : Transferases

o transfer of whole functional groups (e.g. NH2 group)

o excluding oxidoreductases
o A‐X + B ↔ BX + A

IInd digit – Group transferred (carbon/ aldehyde/ ketone grp etc)

IIIrd digit - Group transferred ( methyl / hydroxy methyl etc )
EC 3 : Hydrolases

 Hydrolysis reactions involving various functional groups

 Includes esterases, glycosidases, lipases, proteases
 A‐X + H2O ↔ X‐OH + HA

IInd digit – type of bond hydrolysed ( ester , glycosidic etc)

IIIrd digit - type of bond hydrolysed (carboxylic ester, thiol ester etc)
( further describes)
EC 4 : Lyases

 catalyze non‐hydrolytic removal of functional groups from

substrates, often creating a double bond in the product
 A‐B → A=B + X‐Y
XY
IInd digit - Bond Broken (C- C, C-O, C-N, C-S)
IIInd digit - Type of grp removed (Carboxylic, aldehyde etc)
EC 5. Isomerases

catalyzes isomerization reactions, including racemizations

and cis‐tran isomerizations.

IInd digit – type of reaction

IIIrd digit – Substrate (amino acids, hydroxyl acids etc)
EC 6. Ligases

catalyzes the synthesis of various (mostly C‐X) bonds,

coupled with the breakdown of energy‐containing
substrates, usually ATP
 X + Y + ATP = X- Y + ADP + Pi

IInd digit – type of bond synthesized (C- O, C-S etc)

IIIrd digit – bond being formed
 Enzyme Structure
Active Site – a special pocket or cleft
It contains 3D amino acid structure complementary to
the substrate.
Binds the substrate and form ES complex and is
converted to EP and dissociate and form E and P
Enzymes may be simple prtns or complex enzymes.
Cofactor – Non protein component of Enzyme.
Metal ion (Zn2+, Fe2+)or organic molecule.
If the cofactor is organic molecule – Coenzyme
eg : derivative vitamins, NAD+, etc
 Heat stable & low molecular wt organic compd
Holoenzyme - Active enzyme with cofactor
Apoenzyme – protein portion of the holoenzyme
Heat liable and unstable part
It gives 3D structures for enzymatic chemical rXn
When a cofactor is tightly bound, it is very difficult to remove
without damaging the Enzyme. - Prosthetic Group
• Turn Over Number (Kcal)
is the no : of substrate molecules metabolized per enzyme
molecule per unit time with units of min-1 or s-1
Kcal = Vmax / [E] T
Specificity of the Enzyme :
It is determined by

• Functional group of the Substrate

• Functional group of the Enzyme

• Physical proximity of these functional groups

1. Relative, low or bond Specificity
enzymes act on substrate having similar structure or bond.
eg: Amylase – act on starch, dextrin etc
2. Moderate, structural or group specificity
enzyme specific not only to the bond but also to the
structure surrounding it
Eg : pepsin – hydrolyse central peptide bond as well as
aminoacids like phenyl alanine, tyrosine & tryptophan
3. Absolute, high or substrate specificity
Act on only one substrate
Eg: lactase – act only lactose
4. Optical or stereo specificity
Enzyme is specific not only to substrate but also to optical
configuration
Eg : L aminoacid oxidase – act on L aminoacid
5. Dual specificity
2types
Enzyme act on two substrate by one reaction type
Enzyme act on one substrate by two diff reactions
 Two theories are there to explain specificity of Enzyme Reaction

LOCK AND KEY THEORY:-

 Enzyme active site is
complementary to
substrate.
 Two theories are there to explain specificity of Enzyme Reaction
INDUCED FIT MODEL
• enzyme structure is flexible, not rigid
• The active site adjusts to fit the shape
of the substrate.
• At the same time the substrate adjusts
its shape to better adapt to the
geometry of the active site.
• As a result, the reacting section of the
substrate becomes aligned exactly with
the groups in the active site that
catalyze the reaction.
Methods of enzyme
purification
• Enzymes are manufactured in bioreactors and
is in crude form.

• Is purified before further use

• Extraction is followed by purification

• Purification depends on
– Techniques

– Purity of enzyme
• 3 major purification methods – based on
– Ionic property of enzyme

– Ability to get absorbed

– Difference in size of molecule

I. Ironic property of enzyme
1. Salting out
 By varying pH of the soln or by adding chemical
agent which carryout pptn
 At isoelectric pH enzymes have min solubility so
ppt out as pure crystals
 Also by addn of Ammonium Sulphate & Acetone
 Normaly used ions based on decrease order of
ability to ppt is given below - Hofmeister series
Anions - Citrate, Tartarate, Sulphate, Acetate etc
Cations – Th4+, Al3+,Ba2+ etc
I. Ironic property of enzyme

Chemical agent dissolved in

aquous phase – salting in –
prtns get soluble

Once attained a critical level ,

prtn – prtn interaction
overcomes prtn – water
interaction – agent displays
water molecule

Result salting out

 I. Ironic property of enzyme
2. Electrophoresis
• Separation of proteins by the differential
migration through a gel according to the size and
ionic charge of the molecules in an electrical
field.
• Examples of gels used are
• starch,
• acrylamide,
• agarose or
• mixtures of acrylamide and agarose.
I. Ironic property of enzyme
I. Ironic property of enzyme
3. SDS PAGE
• SDS polyacrylamide
gel electrophoresis
• Separates by size
• Proteins are
complexed with SDS
to give the same
charge density
I. Ironic property of enzyme

• After a time, the current is turned off and the

proteins stained by 1% coomassie blue to
make them visible
• The separated proteins appear as distinct
bands .
I. Ironic property of enzyme
4. Ion exchange chromatography
I. Ironic property of enzyme

• Selective adsorption and exchange of ions takes

place in adsorption site ion exchange column

• Anion exchanger eg :- DEAE Cellulose

( Di Ethyl Amino Ethyl)
• Cation Exchanger eg:- DM Cellulose
(Di Methyl)
II. Absorbing property of enzyme
1. Affinity chromatography
• Separate by specificity = enzy – substrate interaction
• Elution: Bound proteins eluted by adding high
concentration of ligand
II. Absorbing property of enzyme

2. Adsorption Chromatography
• based on adsorption
• Enzyme adsorbed on particular site of carrier
molecule in column
• Depends on effective distribution coefficient
• Eg : starch, diatomaceous earth
• Recovery of extra cellular enzymes
III. Size of enzyme

1. Size exclusion chromatography

• Gel filtration
• Support material - Gel
• Separate by size
• As wash with buffer:
• Small molecules enter the beads
• Large molecules move between the beads
• Larger particle separate first and Smaller one
later
III. Size of enzyme
III. Size of enzyme

2. Dialysis

• Passage of solute from higher concen to lower

concn thru semi permeable membrane
• Used in enzyme concentration
• A dialysis bag is submerged in the buffer
• Buffer#1 diffuses out the bag while the
buffer#2 diffuses into the bag and tiny
undesirable molecules diffuse out (salt or
other small molecule).
III. Size of enzyme