Enzyme nomenclature and IUB classification

Mr. Santhosh.N M.Sc,

PG Biochemistry dept, JSSCACS college,
Mysuru
BRIEF HISTORY OF ENZYMES

Until the 19th century,

• Souring of milk

• Fermentation of sugar to alcohol

In 1833, active preparations

• Diastase (amylase),

• Pepsin.

This were given the general name ferments.

• Justus von Liebig recognized that these ferments could be
non-living materials obtained from living cells, but Louis Pasteur and others
still maintained that ferments must contain living material.
• Appropriately, it was in yeast that a factor was discovered which settled the
argument in favour of the inanimate theory of catalysis: brothers Eduard
and Hans Buchner showed, in 1897, that sugar fermentation could take
place when a yeast cell extract was added even though non living cells were
present.
• In 1926, James Sumner crystallized urease from jack-bean extracts and, in
the next few years, many other enzymes were purified and crystallized.
Necessity for the classification of enzyme

• Trypsin, protein hydrolysis,

• Lactase, hydrolysis,
• Fumarase, hydrates, and
• Catalase, decomposition.

IUBMB(IUB) and EC in nomenclature

Enzyme commission’s system of classification

Enzyme with a code and each code consists of 4

element.
Each elements denotes the digits.
Nomenclature of enzyme by EC
Class 1: Oxidoreductase
Oxidation/reduction (H,O or e-)
Sub class: Donor group
Sub-subclass: acceptor group

Ex:
Class 2: Transferases
Transfer of functional groups;
AX+B BX+A
EX:
Class 3: Hydrolases
Hydrolysis of molecule by water molecule.
AX+H2O XOH+HA
Class 4: lyases
Non hydrolytic removal of groups from substrate
Class 5 : Isomerase
Interconversion of isomers
Class 6: Ligases
Bond formation mediated by NTPs
X + Y + ATP XY + ADP + Pi

EX:
Localization of enzyme
Synthesised protein in
ribosome is further
localization to different
compartments in the
cell.
Isolation of enzyme
Cell disruption
which can done via a number of number of different processes of
choice e.g Detergents lysis, Osmolysis, freeze- thaw cycles, enzymatic
lysis, ultra sonication, Homogenisation

Centrifugation
At a specific speed depending on the organ, tissue, organelle or
fluid.
Subsequent deferential
centrifugation technique
is helpful in the isolation
of desired enzymes.
Passive
diffusion of
molecule
Ion exchange chromatography
Cation exchanger-
Negative charge
surface
Anion exchanger -
Positive charge surface
Size exclusion chromatography

Polyacrylamide-Sephacryl
Dextran -Sephadex
Agarose –Sepharose

Gel permeation – Organic

Gel filtration - Aqueous
Affinity chromatography
Protein Characterization
Characterization of proteins and peptides involves three different processes:

1. Determining the Amino Acid Composition

• Involves finding out the amino acids that make up the protein and their number.
2. Determining the Amino Acid Sequence
• Involves finding out the sequence of amino acids of the proteins in their order.
3. Determining the Molecular mass of the Protein
Determination of Amino Acid Composition

The peptide is first hydrolyzed into its constituent amino acids by heating it in 6M
HCl at 110oC for 24‐72 hrs.

•The R groups remain intact, except for:

• Trp – indole ring damaged

• Asn, Gln – converted to Asp, Glu

• Gly does not react

Amino acid analysis
• The amino acids can be derivatized with ninhydrin or o‐phthalaldehyde to make
fluorescent derivatives that are easy to detect.
• These are chromatographed by reverse‐phase HPLC (high‐ pressure liquid
chromatography).
– The characteristic retention times are used to identify the amino acids.

– The fluorescence level can be quantified to determine the amount of that amino
acid.
Amino acid analysis.
Specific activity
The number of enzyme unites per milligram of protein.
Specific activity= enzyme unit(IU) /protein in mg.
Criteria of purity of enzyme
Enzyme units
Enzyme unit (IU)
The amount of enzyme causing transformation of 1 μmole of
substrate per minute at 25°C under optimal conditions of
measurement.
1 enzyme unit = 1 μmol/min.
Katal (kat)
The katal is the accepted SI unit of enzyme activity. One katal is
that amount of enzyme that catalyzes the transformation of 1 mole of
substrate per second.
1 katal = 1 mol/sec.
**1IU = 16.67 nanokat
Lineweaver–Burk or double reciprocal plot
Competitive inhibition
Uncompetitive inhibition
Non-competitive inhibition
Bisubstrate enzyme catalysed reaction
60% of known biochemical reactions are bisubstrate reactions

• Transferase reactions

• oxidation-reduction reactions
W.W. Cleland
• Substrates designated A, B, C, and D.
• Products designated P, Q, R, and S.
• Stable enzyme designated E, F, and G
*E being the free enzyme
The numbers of reactants and products in a given reaction are
specified, in order, by the terms Uni (one), Bi (two), Ter (three), and
Quad (four).

Reaction requiring one substrate and yielding three products is

designated a Uni Ter reaction.

Reaction requiring two substrate and yielding two products is

designated a Bi Bi reaction.
Sequential Reactions
Ordered Bi Bi reaction

Many NAD+ and NADP+ requiring dehydrogenases follow an Ordered Bi Bi

mechanism in which the coenzyme is the leading reactant.
 Random Bi Bi Reaction

Some dehydrogenases and kinases operate through Random Bi Bi

mechanisms.
Ping Pong Reactions
Mechanisms in which one or more products are released before all
substrates have been added are known as Ping Pong reactions.

Chymotrypsin ,transaminase, and some flavoenzymes, react with Ping

Pong mechanisms
lock-and-key hypothesis:
Emil Fischer’s discover, that glycolytic
enzymes can distinguish between
stereoisomeric sugars. He made the
hypothesis like;
“specificity of an enzyme (the lock) for its
substrate (the key) arise from their
geometrically complementary shapes”
Induced-fit hypothesis

“Structure of a substrate may be

complementary to that of the active site
in the enzyme-substrate complex, but not
in the free enzyme: a conformational
change takes place in the enzyme during
the binding of substrate which results in
the required matching of structures”
Thank you