Class 1.

What are enzymes,

active site,
enzyme unit- sp. Activity,
turn over number,
Enzyme,
cause of tremendous rate acceleration( covalent, acid base catalysis),
enzyme classification
Enzymes : These are biological catalysts that accelerate reactions. Enzymes are generally
highly specific and react with only one substrate to form one product and can enhance reaction
rates by as much as 1017.
With exception of a small group of catalytic RNA molecules, all enzymes are proteins
Enzymes are the largest class of proteins and more than 3000 enzymes known
Some enzymes require cofactors / coenzymes for activity

Coenzymes act as transient carriers of specific functional groups

A cofactor or coenzymes that is very tightly or even covalently bound to the

enzyme protein is called a prosthetic group.
Complete, catalytically active enzyme: holoenzyme
Only protein part (without cofactor and/or coenzyme): apoenzyme or apoprotein
APOENZYME + COFACTOR -------->
HOLOENZYME
 Difference between Enzyme and conventional Chemical Catalyst

Specificity and Selectivity ( Stereospecificity, Chemeoselectivity, regiospecificity or positional

specificity)
Virtually all such selectivities originates from the energy differences in enzyme-substrate transition state complex

Higher reaction rate (an increase in productivity with reduced manufacturing costs due to wages and
overheads)

Milder reaction condition (conditions of temperature, pressure and pH. This decreases the energy
requirements, reduces the capital costs due to corrosion-resistant process equipment and further reduces
unwanted side-reactions)

Capacity for regulation ( Product or substrate inhibition, other regulation)

Disadvantages of Enzyme Catalyzed reactions

The high cost of enzyme isolation and purification
Unstable nature of enzymes, when removed from their natural environment,
Selectivity of enzymes:

A. Streoselectivity: Enzymes can

discriminate between optical isomer ( D,L or
R,S) of a molecule (Stereospecificity

B. Chemeoselectivity: Enzymes
selectively react with on of the different
functional group in a molecule even
when other groups may be more
chemically reactive
( Chemeoselectivity)

C. Regioselectivity: Enzymes
can discriminate between similar
parts ( one of the same or similar Lipase
groups) in different region of
molecule
Active Site of Enzyme :

Small relative to the total volume of the enzyme.

Usually occur in clefts and crevices in the protein. Excluding solvents which would otherwise reduce the
catalytic activity of the enzyme.
Amino acids and cofactors are held in precise arrangement with respect to structure of the substrate.
The specificity of substrate utilization depends on the defined arrangement of the atoms in the enzyme
active site (complements the structure of the substrate molecule).
By binding multiple substrates in a favorable interspatial relationship and/or by altering charge
distribution (resonance) within substrates, Gtransition is much smaller than would be spontaneously
 Binding of a substrate to an enzyme at the active site

Representation of a simple Enzymes affect reaction rates,

enzymatic reaction E + S ES EP E + P not equilibria

Reaction coordinate diagram for a chemical reaction Reaction coordinate diagram comparing enzyme-catalyzed
and uncatalyzed reactions
 Weak interactions between enzyme and substrate are optimized in the transition state

The energy derived from enzyme-substrate

interaction is called binding energy, GB

Enzyme active sites are complementary not

to the substrate per se, but to the transition
state through which substrates pass as they
are converted into products during an
enzymatic reaction

Complementary shapes of a substrate and its binding site on the enzyme

Role of binding energy in catalysis

An imaginary enzyme (sticase) designed to catalyze breakage of a metal stick

 Equilibrium

SP

Reaction

SP
Reaction rate = V = k [S]

If the rate depends only on the

concentration of S (first-order)

From the Transition-State Theory:

k BT G / RT
k e
h
KB Boltzmann constant, h Plancks constant
Whatareinenzymefortremendousrateacceleration?
Theenzymebindsthesubstratemoleculeinsuchawaythatthesusceptiblebondin
closeproximitytothecatalyticgroupontheactivesite(increaseofeffectivemolarity
ofthereactants)andthetransitionstateisreadilyformed

Rateenhancementduetoentropyreductionofsubstrate

Someenzymecombinewithsubstratetoformanunstablecovalentintermediatethat
morereadilyundergoesreactiontoformproducts

Theenzymesmayinducestrainordistortioninthesusceptiblebondofthesubstrate
molecule,makingbondeasiertobreak

Enzymeprovidesfunctionalgroupforacidbase(H+,OH),nucleophili/electrophilic
andcovalentcatalysis

Metaliondependentrateenhancement
Rate enhancements by entropy reduction General acid, base form of amino acid
Covalent catalysis : These are generally two step process: formation and breakdown of covalent
intermediate rather than catalysis of the single reaction directly.

Y should be a better leaving group than X.

X is a better attacking group(nucleophile) then Z.
Covalent intermediate should be more reactive than substrate

What kind of groups in proteins are good nucleophiles ?

Cystine thiol-

Serine hydroxyl-

Tyrosine hydroxyl-

Histadine imidazolyl-

Amino acid side chains and the functional groups of some cofactors can serve as nucleophiles in the
formation of covalent bonds with substrates
Acid-base catalysis : A proton (H+) is transferred in the transition state.

Specific acid-base catalysis:

Protons from hydronium ion (H3O+ ) and hydroxide ions (OH - ) act directly
as the acid and base group.

General acid-base catalysis:

Catalytic group participates in protein transfer stabilize the transition state of

the chemical reaction.
Protons from amino acid side chains, cofactors, organic substrates act as
Bronsted-Lowry acid and base group.

Histidine pKa is around 7. It is the most effective general acid or base.

acid group (A-H) acting

as a partial proton donor basic group (B:) acting
for carbonyl oxygen of as proton acceptor
the ester

For even greater catalysis, enzyme can utilize acid and base simultaneously
Example: RNase A: His 12
General Base
Abstracts a proton from 2 hydroxyl of 3 nucleotide.
His 119
General acid
Donates a proton to 5 hydroxyl of nucleoside.
 How a catalyst circumvents unfavorable charge
development during cleavage of a amide

Specific
General
acid-base
acid-base
catalysis
catalysis

Hydrolysis of an amide bond - same reaction as

that catalyzed by chymotrypsin and other proteases
 Metal ions based enzyme catalysis:

Electrostatically stabilizing or shielding negative charges.

Act much like a proton but can be present in high concentration at neutral pH and can have
multiple positive charges
Act to bridge a substrate and nucleophilic group.
Bind to substrates to ensure proper orientation.
Participate in oxidation/reduction mechanisms through change of oxidation state.

Can stabilize developing negative charge on a leaving

Can stabilize developing negative group, making it a better leaving group.
charge on a leaving group, making it
a better leaving group. Can shield negative charges on substrate group that will
otherwise repel attack of nucleophile.

Can increase the rate of a hydrolysis reaction

by forming a complex with water, thereby
increasing waters acidity.
 Enzyme Units
The activities of enzyme are usually measured in terms of the activity unit (U) which is defined as the
amount which will catalyse the transformation of 1 micromole of the substrate per minute under
standard conditions (Typically, this represents 10-6 - 10-11 Kg for pure enzymes and 10-4 - 10-7 Kg for
industrial enzyme preparations)
katal (kat) which is defined as the amount which will catalyse the transformation of one mole of
substance per second (1 kat = 60 000 000 U) [ It is an impracticable unit and has not yet received
widespread acceptance]
Sometimes non-standard activity units are used, such as Soxhet, Anson and Kilo Novo units, which are
based on physical changes such as lowering viscosity and supposedly better understood by industry.
The activity is a measure of enzyme content that is clearly of major interest when the enzyme is to be
used in a process. For this reason, enzymes are usually marketed in terms of activity rather than weight.

Specific Activity of Enzyme

The specific activity (e.g. U Kg-1) is a parameter of interest ( an index of purity ).

Standard Conditions
These are meant to refer to optimal conditions, especially with regard to pH, ionic strength, temperature,
substrate concentration and the presence and concentration of cofactors and coenzymes.
Conditions for maximum initial activity are not necessarily those for maximum stability. Great care has to
be taken over the consideration of these factors when the most efficient catalyst for a particular purpose is
to be chosen
 Enzyme Nomenclature
Enzymes are classified according the report of a Nomenclature Committee appointed by
the International Union of Biochemistry (1984).
This enzyme commission assigned each enzyme a recommended name and a 4-part
distinguishing number. It should be appreciated that some alternative names remain in
such common usage that they will be used, where appropriate, in this text. The enzyme
commission (EC) numbers divide enzymes into six main groups according to the type of
reaction catalysed:
(1) Oxidoreductases which involve redox reactions in which hydrogen or oxygen atoms or
electrons are transferred between molecules. This extensive class includes the
dehydrogenases (hydride transfer), oxidases (electron transfer to molecular oxygen),
oxygenases (oxygen transfer from molecular oxygen) and peroxidases (electron transfer to
peroxide). For example: glucose oxidase (EC 1.1.3.4, systematic name, b-D-
glucose:oxygen 1-oxidoreductase).

-D-glucose + oxygen D-glucono-1,5-lactone + hydrogen peroxide

(2) Transferases which catalyse the transfer of an atom or group of atoms (e.g. acyl-,
alkyl- and glycosyl-), between two molecules, but excluding such transfers as are
classified in the other groups (e.g. oxidoreductases and hydrolases). For example:
aspartate aminotransferase (EC 2.6.1.1, systematic name, L-aspartate:2-oxoglutarate
aminotransferase; also called glutamic-oxaloacetic transaminase or simply GOT).

L-aspartate + 2-oxoglutarate oxaloacetate + L-glutamate

(3) Hydrolases which involve hydrolytic reactions and their reversal. This is presently
the most commonly encountered class of enzymes within the field of enzyme technology
and includes the esterases, glycosidases, lipases and proteases. For example:
chymosin (EC 3.4.23.4, no systematic name declared; also called rennin).

-casein + water para--casein + caseino macropeptide

(4) Lyases which involve elimination reactions in which a group of atoms is removed from
the substrate. This includes the aldolases, decarboxylases, dehydratases and some
pectinases but does not include hydrolases. For example: histidine ammonia-lyase (EC
4.3.1.3, systematic name, L-histidine ammonia-lyase; also called histidase).

L-histidine urocanate + ammonia

(5) Isomerases which catalyse molecular isomerisations and includes the epimerases,
racemases and intramolecular transferases. For example: xylose isomerase (EC 5.3.1.5,
systematic name, D-xylose ketol-isomerase; commonly called glucose isomerase).

-D-glucopyranose -D-fructofuranose
(6) Ligases, also known as synthetases, form a relatively small group of enzymes which
involve the formation of a covalent bond joining two molecules together, coupled with the
hydrolysis of a nucleoside triphosphate. For example: glutathione synthase (EC 6.3.2.3,
systematic name, -L-glutamyl-L-cysteine:glycine ligase (ADP-forming); also called
glutathione synthetase).

ATP + -L-glutamyl-L-cysteine + glycine ADP + phosphate + glutathione

 Scheme of Classification and Numbering of Enzyme-Catalysed Reactions
The first Enzyme Commission, in its report in 1961, devised a system for classification
of enzymes that also serves as a basis for assigning code numbers to them.
These code numbers, prefixed by EC, which are now widely in use, contain four
elements separated by points, with the following meaning:
(i) the first number shows to which of the six main divisions (classes) the enzyme
belongs,
(ii) the second figure indicates the subclass,
(iii) the third figure gives the sub-subclass,
(iv) the fourth figure is the serial number of the enzyme in its sub-subclass .
Enzyme Commssion Class. Subclass. Sub-subclass. Serial number in sub-subclass
EC 1. 1. 1. 1
With NAD or NADP as acceptor
Oxidoreductases
alcohol dehydrogenase
Acting on the CH-OH group of donors

Systematic name: ATP:glucose phosphotransferase

Classification number: 2.7.1.1.
2. Transferase (class)
7. phosphotransferase (subclass)
ATP+DglucoseADP+Dglucose6phospate 1. phosphotransferase with a hydroxyl group as acceptor
( Sub-Subclass)
Hexokinase 1. D-glucose as the phosphoryl group acceptor
(Sl.no. of sub-sub class)
 Class 1. Oxidoreductases.

To this class belong all enzymes catalysing oxidoreduction reactions. The substrate that is oxidized is
regarded as hydrogen donor. The systematic name is based on donor:acceptor oxidoreductase.

The common name will be dehydrogenase, wherever this is possible; as an alternative, reductase can be
used. Oxidase is only used in cases where O2 is the acceptor.

The second figure in the code number of the oxidoreductases, unless it is 11, 13, 14 or 15, indicates the
group in the hydrogen (or electron) donor that undergoes oxidation: 1 denotes a -CHOH- group, 2 a -CHO
or -CO-COOH group or carbon monoxide, and so on, as listed in the key.

The third figure, except in subclasses EC 1.11, EC 1.13, EC 1.14 and EC 1.15, indicates the type of
acceptor involved: 1 denotes NAD(P)+, 2 a cytochrome, 3 molecular oxygen, 4 a disulfide, 5 a quinone or
similar compound, 6 a nitrogenous group, 7 an iron-sulfur protein and 8 a flavin.

In subclasses EC 1.13 and EC 1.14 a different classification scheme is used and sub-subclasses are
numbered from 11 onwards.
It should be noted that in reactions with a nicotinamide coenzyme this is always regarded as acceptor,
even if this direction of the reaction is not readily demonstrated. The only exception is the subclass EC 1.6,
in which NAD(P)H is the donor; some other redox catalyst is the acceptor.

Although not used as a criterion for classification, the two hydrogen atoms at carbon-4 of the
dihydropyridine ring of nicotinamide nucleotides are not equivalent in that the hydrogen is transferred
stereospecifically.
EC 1Oxidoreductases
EC 1.1 Acting on the CH-OH group of donors sub-subclassesup to 50
EC 1.1.1 With NAD or NADP as acceptor
EC 1.1.2 With a cytochrome as acceptor
EC 1.1.3 With oxygen as acceptor
EC 1.1.4 With a disulfide as acceptor
EC 1.1.5 With a quinone or similar compound as acceptor
EC 1.1.99 With other acceptors

EC 1.2 Acting on the aldehyde or oxo group of donors sub-subclassesup to 50

EC 1.2.1 With NAD or NADP as acceptor

EC 1.2.2 With a cytochrome as acceptor
EC 1.2.3 With oxygen as acceptor
EC 1.2.4 With a disulfide as acceptor
EC 1.2.7 With an iron-sulfur protein as acceptor
EC 1.2.99 With other acceptors

EC 1.97Other oxidoreductases
 Factors That Influence Enzyme
Activity
Temperature
pH and ionic strength
Organic solvents
Proteases
Inhibitors/effectors
Metals, Oxygen, other small molecules
Pressure, radiation, and shear
 Enzyme Control Mechanisms

Substrate level (reversible inhibition)

Feedback (pathway level)
Allostery
Compartmentalization (cellular level)
 Examples of Enzymatic Reactions
Chymotrypsin is a protease specific for peptide bonds adjacent to aromatic amino acids
(Trp, Tyr, Phe)

Key active-site amino acid

residues Ser195, His57, and
Asp102

Aromatic amino
Pocket in which the amino acid side chain
acid side chain of the
substrate is bound

Three polypeptide chains linked

by disulfide bonds
The chymotrypsin mechanism
involves acylation and
deacylation of a Ser residue
 Regulatory Enzymes
Allosteric enzymes undergo conformational changes in response to modulator binding

Conformational change

Subunit interactions in an
allosteric enzyme, and
interactions with inhibitors
and activators
 Feedback inhibition

Buildup of the end product ultimately

slows the entire pathway

In many pathways a regulatory Example of heterotropic

step is catalyzed by an allosteric inhibition

allosteric enzyme
 Enzyme Control Mechanisms

Substrate level (reversible inhibition)

Feedback (pathway level)
Allostery
Compartmentalization (cellular level)