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PAPER : IX Unit – I
Enzyme
Enzymes are proteins that catalyze (i.e., increase the rates of) chemical reactions. Nearly all
known enzymes are proteins. However, certain RNA molecules can be effective biocatalysts
too. These RNA molecules have come to be known as ribozymes. In enzymatic reactions, the
molecules at the beginning of the process are called substrates, and the enzyme converts them
into different molecules, called the products. Almost all processes in a biological cell need
enzymes to occur at significant rates. Since enzymes are selective for their substrates and
speed up only a few reactions from among many possibilities, the set of enzymes made in a
cell determines which metabolic pathways occur in that cell.
Like all catalysts, enzymes work by lowering the activation energy (Ea or ΔG‡) for a reaction,
thus dramatically increasing the rate of the reaction. Most enzyme reaction rates are millions
of times faster than those of comparable un-catalyzed reactions. As with all catalysts,
enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of
these reactions. However, enzymes do differ from most other catalysts by being much more
specific. Enzymes are known to catalyze about 4,000 biochemical reactions. A few RNA
molecules called ribozymes catalyze reactions, with an important example being some parts
of the ribosome. Synthetic molecules called artificial enzymes also display enzyme-like
catalysis.
Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease
enzyme activity; activators are molecules that increase activity. Many drugs and poisons are
enzyme inhibitors. Activity is also affected by temperature, chemical environment (e.g., pH),
and the concentration of substrate. Some enzymes are used commercially, for example, in the
synthesis of antibiotics. In addition, some household products use enzymes to speed up
biochemical reactions (e.g., enzymes in biological washing powders break down protein or
fat stains on clothes; enzymes in meat tenderizers break down proteins, making the meat
easier to chew).
Etymology and history
As early as the late 1700s and early 1800s, the digestion of meat by
stomach secretion and the conversion of starch to sugars by plant extracts
and saliva were known. However, the mechanism by which this occurred
had not been identified
Eduard Buchner
Louis Pasteur
D. D. Khedkar
In the 19th century, when studying the fermentation of sugar to alcohol by
yeast, Louis Pasteur came to the conclusion that this fermentation was
catalyzed by a vital force contained within the yeast cells called
"ferments", which were thought to function only within living organisms.
He wrote that "alcoholic fermentation is an act correlated with the life and
organization of the yeast cells, not with the death or putrefaction of the
cells."
Unit – I : Enzymes
1
In 1878, German physiologist Wilhelm Kühne (1837–1900) first used the
term enzyme, which comes from Greek ενζυμον, "in leaven", to describe
this process. The word enzyme was used later to refer to nonliving
substances such as pepsin, and the word ferment was used to refer to
chemical activity produced by living organisms.
Wilhelm Kühne
In 1897, Eduard Buchner began to study the ability of yeast extracts that lacked any living
yeast cells to ferment sugar. In a series of experiments at the University of Berlin, he found
that the sugar was fermented even when there were no living yeast cells in the mixture. He
named the enzyme that brought about the fermentation of sucrose "zymase". In 1907, he
received the Nobel Prize in Chemistry "for his biochemical research and his discovery of cellfree fermentation". Following Buchner's example, enzymes are usually named according to
the reaction they carry out. Typically, to generate the name of an enzyme, the suffix -ase is
added to the name of its substrate (e.g., lactase is the enzyme that cleaves lactose) or the type
of reaction (e.g., DNA polymerase forms DNA polymers).
Having shown that enzymes could function outside a living cell, the next step was to
determine their biochemical nature. Many early workers noted that enzymatic activity was
associated with proteins, but several scientists (such as Nobel laureate Richard Willstätter)
argued that proteins were merely carriers for the true enzymes and that proteins per se were
incapable of catalysis. However, in 1926, James B. Sumner showed that the enzyme urease
was a pure protein and crystallized it; Sumner did likewise for the enzyme catalase in 1937.
The conclusion that pure proteins can be enzymes was definitively proved by Northrop and
Stanley, who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin.
These three scientists were awarded the 1946 Nobel Prize in Chemistry.
This discovery that enzymes could be crystallized eventually allowed their structures to be
solved by x-ray crystallography. This was first done for lysozyme, an enzyme found in tears,
saliva and egg whites that digests the coating of some bacteria; the structure was solved by a
group led by David Chilton Phillips and published in 1965. This high-resolution structure of
lysozyme marked the beginning of the field of structural biology and the effort to understand
how enzymes work at an atomic level of detail.
Structures and mechanisms
Enzymes are generally globular proteins and range from just 62 amino acid residues in size,
for the monomer of 4-oxalocrotonate tautomerase, to over 2,500 residues in the animal fatty
acid synthase. A small number of RNA-based biological catalysts exist, with the most
common being the ribosome; these are referred to as either RNA-enzymes or ribozymes. The
activities of enzymes are determined by their three-dimensional structure. However, although
structure does determine function, predicting a novel enzyme's activity just from its structure
is a very difficult problem that has not yet been solved.
Most enzymes are much larger than the substrates they act on, and only a small portion of the
enzyme (around 3–4 amino acids) is directly involved in catalysis. The region that contains
these catalytic residues, binds the substrate, and then carries out the reaction is known as the
active site. Enzymes can also contain sites that bind cofactors, which are needed for catalysis.
D. D. Khedkar
Unit – I : Enzymes
2
Some enzymes also have binding sites for small molecules, which are often direct or indirect
products or substrates of the reaction catalyzed. This binding can serve to increase or
decrease the enzyme's activity, providing a means for feedback regulation.
Like all proteins, enzymes are made as long, linear chains of amino acids that fold to produce
a three-dimensional product. Each unique amino acid sequence produces a specific structure,
which has unique properties. Individual protein chains may sometimes group together to form
a protein complex. Most enzymes can be denatured—that is, unfolded and inactivated—by
heating or chemical denaturants, which disrupt the three-dimensional structure of the protein.
Depending on the enzyme, denaturation may be reversible or irreversible.
Specificity
Enzymes are usually very specific as to which reactions they catalyze and the substrates that
are involved in these reactions. Complementary shape, charge and hydrophilic/hydrophobic
characteristics of enzymes and substrates are responsible for this specificity. Enzymes can
also show impressive levels of stereospecificity, regioselectivity and chemoselectivity.
Some of the enzymes showing the highest specificity and accuracy are involved in the
copying and expression of the genome. These enzymes have "proof-reading" mechanisms.
Here, an enzyme such as DNA polymerase catalyzes a reaction in a first step and then checks
that the product is correct in a second step. This two-step process results in average error
rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases.
Similar proofreading mechanisms are also found in RNA polymerase, aminoacyl tRNA
synthetases and ribosomes.
Some enzymes that produce secondary metabolites are described as promiscuous, as they can
act on a relatively broad range of different substrates. It has been suggested that this broad
substrate specificity is important for the evolution of new biosynthetic pathways.
Enzyme Nomenclature
The enzyme nomenclature scheme was developed starting in 1955, when the International
Congress of Biochemistry (Now - International Congress of Biochemistry and molecular
Biology) in Brussels set up an Enzyme Commission. The first version was published in 1961.
The current sixth edition, published by the International Union of Biochemistry and
Molecular Biology in 1992, contains 3196 different enzymes.
The Enzyme Commission number (EC number) is a numerical classification scheme for
enzymes, based on the chemical reactions they catalyze. As a system of enzyme
nomenclature, every EC number is associated with a recommended name for the respective
enzyme.
Strictly speaking, EC numbers do not specify enzymes, but enzyme-catalyzed reactions. If
different enzymes (for instance from different organisms) catalyze the same reaction, then
they receive the same EC number. By contrast, UniProt identifiers uniquely specify a protein
by its amino acid sequence.
Format of number
Every enzyme code consists of the letters "EC" followed by four numbers separated by
periods. Those numbers represent a progressively finer classification of the enzyme.
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Unit – I : Enzymes
3
For example, the tripeptide aminopeptidases have the code "EC 3.4.11.4", whose components
indicate the following groups of enzymes:




EC 3 enzymes are hydrolases (enzymes that use water to break up some other
molecule)
EC 3.4 are hydrolases that act on peptide bonds
EC 3.4.11 are those hydrolases that cleave off the amino-terminal amino acid from a
polypeptide
EC 3.4.11.4 are those that cleave off the amino-terminal end from a tripeptide
Top-level EC numbers
Group
Reaction catalyzed
Typical reaction
To catalyze oxidation/reduction
reactions; transfer of H and O
EC 1
Oxidoreductases atoms or electrons from one
substance to another
Enzyme
example(s) with
trivial name
AH + B → A +
BH
(reduced) Dehydrogenase,
A + O → AO oxidase
(oxidized)
EC 2
Transferases
Transfer of a functional group
from one substance to another.
Transaminase,
AB + C → A + BC
The group may be methyl-, acyl-,
kinase
amino- or phosphate group
EC 3
Hydrolases
Formation of two products from a AB + H2O
substrate by hydrolysis
AOH + BH
EC 4
Lyases
Non-hydrolytic
addition
or
removal
of
groups
from
substrates. C-C, C-N, C-O or C-S
bonds may be cleaved
EC 5
Isomerases
Intramolecule rearrangement, i.e.
isomerization changes within a AB → BA
single molecule
EC 6
Ligases
Join together two molecules by
synthesis of new C-O, C-S, C-N X + Y+ ATP →
Synthetase
or C-C bonds with simultaneous XY + ADP + Pi
breakdown of ATP
D. D. Khedkar
→ Lipase, amylase,
peptidase
RCOCOOH
→
RCOH + CO2 or
Decarboxylase
[x-A-B-Y]
→
[A=B + X-Y]
Unit – I : Enzymes
Isomerase, mutase
4
EC 1 OXIDOREDUCTASE
Oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule (the
reductant, also called the hydrogen or electron donor) to another (the oxidant, also called the
hydrogen or electron acceptor).
Reactions
For example, an enzyme that catalyzed this reaction would be an oxidoreductase:
A– + B → A + B–
OR
AH2 → A + BH2
In this example, A is the reductant (electron donor) and B is the oxidant (electron acceptor).
In biochemical reactions, the redox reactions are sometimes more difficult to see, such as this
reaction from glycolysis:
Alcohol:NAD oxidoreductase (1:1:1:1)
Alcohol + NAD ⇒
Aldehyde + NADH + H
In this reaction, NAD+ is the oxidant (electron acceptor), and glyceraldehyde-3-phosphate is
the reductant (electron donor).
Nomenclature
Proper names of oxidoreductases are formed as "donor:acceptor oxidoreductase"; however,
other names are much more common. The common name is "donor dehydrogenase" when
possible, such as glyceraldehyde-3-phosphate dehydrogenase for the second reaction above.
Common names are also sometimes formed as "acceptor reductase", such as NAD+
reductase. "Donor oxidase" is a special case where O2 is the acceptor.
Classification
Oxidoreductases are classified as EC 1 in the EC number classification of enzymes.
Oxidoreductases can be further classified into 22 subclasses and 3 subsubclasses.
Examples: Alcohol dehydrogenase, Oxygenase, Peroxidase, Catalase, etc.
EC 2 TRANSFERASE
A transferase is an enzyme that catalyzes the transfer of a functional group (e.g. a methyl or
phosphate group) from one molecule (called the donor) to another (called the acceptor). For
example, an enzyme that catalyzed this reaction would be a transferase:
A–X + B → A + B–X
In this example, A would be the donor, and B would be the acceptor. The donor is often a
coenzyme.
Choline acetyltransferase (2:3:1:6)
Acetyl − CoA + Choline ⇒
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Unit – I : Enzymes
CoA + O − Acetyl choline
5
Nomenclature
Proper names of transferases are formed as "donor:acceptor grouptransferase." However,
other names are much more common. The common names of transferases are often formed as
"acceptor grouptransferase" or "donor grouptransferase." For example, a DNA
methyltransferase is a transferase that catalyzes the transfer of a methyl group to a DNA
acceptor.
Classification
Transferases are classified as EC 2 in the EC number classification. Transferases can be
further classified into nine subclasses.
Examples: Transaminase, Glycosyl transferase, Transketolase, etc.
EC 3 HYDROLASE
A hydrolase is an enzyme that catalyzes the hydrolysis of a chemical bond. For example, an
enzyme that catalyzed the following reaction is a hydrolase:
A–B + H2O → A–OH + B–H
L−Argenine Ureohydrolase (3:5:3:1)
L − Argenine + H20 ⇒
L − Ornithine + Urea
Nomenclature
Systematic names of hydrolases are formed as "substrate hydrolase." However, common
names are typically in the form "substratease." For example, a nuclease is a hydrolase that
cleaves nucleic acids with the help of water. It catalyze reactions by hydrolytic cleavage. In
the reaction donor group is transferred to water molecule.
Classification
Hydrolases are classified as EC 3 in the EC number classification of enzymes. Hydrolases
can be further classified into 13 subclasses and 4 subsubclasses, based upon the bonds they
act upon.
Examples: Lipase, Urease, Glycosidase, Peptidase, Amidase, etc.
EC 4 LYASE (DESMOLASE)
A lyase is an enzyme that catalyzes the breaking of various chemical bonds by means other
than hydrolysis and oxidation i.e. electronic rearrangements , often forming a new double
bond or a new ring structure. For example, an enzyme that catalyzed this reaction would be a
lyase:
ATP → cAMP + PPi
Lyases differ from other enzymes in that they only require one substrate for the reaction in
one direction, but two substrates for the reverse reaction.
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Unit – I : Enzymes
6
A-B + X-Y → A-X + B-Y
L−Malate hydrolyase (4:2:1:2)
L − Malate + H20 ⇒
Fumarate + H2O
Nomenclature
Systematic names are formed as "substrate group lyase." When the reverse reaction is more
important, synthase may be used in the name.
Classification
Lyases are classified as EC 4 in the EC number classification of enzymes. Lyases can be
further classified into seven subclasses.
Examples: Fumarase, Histidase, Decarboxylase, Dehydratase, Aldolase, etc.
EC 5 ISOMERASES
The names of isomerases are formed as "substrate isomerase" (for example, enoyl CoA
isomerase), or as "substrate type of isomerase" These are enzymes which can transform
substrates in their isomers by any one of the isomerization reactions.
Isomers are compounds with the same molecular formula but different structural formulas.
Isomers do not necessarily share similar properties unless they also have the same functional
groups. This should not be confused with a nuclear isomer, which involves a nucleus at
different states of excitement. There are many different classes of isomers, like stereoisomers,
enantiomers, geometrical isomers, etc.
Classification
Isomerases have their own EC classification of enzymes: EC 5. Isomerases can be further
classified into six subclasses and five subsubclasses depending in isomerization type viz.
racemization, isomerization of geometric, intramolecular oxidoreductases, intramolecular
transferases, intramolecular lyases and other isomerases.
A–B–C
║
A–B–C
H−
H−
C−
C−
⇒
Isomerase
COOH
COOH
⇒
Isomerase
A–B–C
║
C–B–A
H−
C − COOH
HOOC − C −
H
Racemase (5:1:1:1)
L − Alanine ⇒
D − Alanine
Examples : Triose phosphate isomerase, Glucose phosphate isomerase, Epimerase, Mutase
D. D. Khedkar
Unit – I : Enzymes
7
EC 6 LIGASES / SYNTHETASES
A ligase (from the Latin verb ligāre — "to bind" or "to glue together") is an enzyme that can
catalyse the joining of two large molecules by forming a new chemical bond, usually with
accompanying hydrolysis of a small chemical group pendant to one of the larger molecules.
Due to its biosynthetic role it is referred as SYNTHETASES. In the enzymic reaction
catalyzed by ligases needs energy provided by energy rich molecule like ATP. In general,
ligase catalyses the following reaction:
Ab + C ⇒
A–C + b
ATP ⇝ADP+iP
or sometimes
Ab + cD ⇒
ATP ⇝ADP+iP
A–D + b + c
where the lowercase letters signify the small, pendant groups.
Acetate:CoA Ligase (6:2:1:1)
Acetate + CoA + ATP ⇒
Acetyl CoA + AMP + 2iP
The ligase enzyme works more efficiently at room temperature and at the PH of 7.
Nomenclature
The common names of ligase enzymes often include the word "ligase," such as DNA ligase,
an enzyme commonly used in molecular biology laboratories to join together DNA
fragments. Other common names for ligases include synthetases, because they are used to
synthesize new molecules, or carboxylases when they are used to add carbon dioxide to a
molecule.
Note that "synthetases" should not be confused with synthases, as synthases do not use
energy from nucleoside triphosphates (such as ATP, GTP, CTP, TTP, and UTP) and belong
to the lyase group, whereas ligases do use nucleoside triphosphates.
Classification
Ligases are classified as EC 6 in the EC number classification of enzymes. Ligases can be
further classified into six subclasses and 5 subsubclasses.
ENZYME STRUCTURE
Enzymes can have molecular weights ranging from about
10,000 to over 1 million. A small number of enzymes are not
proteins, but consist of small catalytic RNA molecules. Often,
enzymes are multiprotein complexes made up of a number of
individual protein subunits.
Many enzymes catalyze reactions without help, but some
require an additional non-protein component called a co-factor.
Co-factors may be inorganic ions such as Fe2+, Mg2+, Mn2+, or
D. D. Khedkar
Unit – I : Enzymes
8
Zn2+, or consist of organic or metalloorganic molecules knowns as co-enzymes.
The complete functional enzyme is called as Holoenzyme which possesses Apoenzyme
(Protein Part) and Cofactor/ Coenzyme (Prosthetic Group or Non Protein Part)
APOENZYME
It’s a huge part of complete enzyme occupies 80 – 90 % of the entire space. The apoenzyme
has a specific confirmation and molecular dimension always found in tertiary structure. It has
affinities towards the substrates, coenzyme, cofactor and product. Importantly, it establishes a
specialized area with a specific morphology called as “active site”. The apoenzyme is a
multimeric structure having many subunits to form a typical shape.
ACTIVE SITE
This is a special area of an enzyme surface that offers a site of binding to the substrate
molecule and catalyse a reaction. The surface is lined with amino acid residues whose
substituent groups bind the substrate and catalyse the chemical transformation. Following are
some of the properties of an active site:
1. Existence of an active site is due to the tertiary structure of apoenzyme resulting in 3 D
native confirmation.
2. Denaturation of the enzyme results in derangement of active site.
3. They are made up of amino acids (catalytic residues) which are far from each other in a
linear sequence (e.g. Lysozyme – 129 amino acid long protein form active site by
assembling 35, 52, 62, 63 and 101 number amino acids)
4. Active sites are regarded as clefts, crevises or pockets occupying a small region in a big
enzyme molecule.
5. The active site is flexible in nature and promote the specific substrate binding and adust
with it by reorientation.
6. The active site possesses “S” – Substrate binding and “C” catalytic Site
7. The substrate binds to active site by means of covalent or weak non covalent interaction.
8. The enzyme specificity is due to the specific shape and size of the active site.
9. The coenzyme or cofactors are associated with the active site.
10. The active site predominantly possesses the “serine” amino acid.
The active site plays cricial role in the completion of catalytic reaction by forming a Enzyme
– Substrate and Enzyme – Product Complex.
COENZYME
Its a nonproteinaceous organic substance with very low molecular weight, that usually
contains a vitamin or mineral and combines with a specific protein, the apoenzyme, to form
an active enzyme system.
An organic prosthetic group (nonprotein portion of the enzyme) whose presence is required
for the activity of many enzymes. The prosthetic groups attached to the protein of the enzyme
(the apoenzyme) may be regarded as dissociable portions of conjugated proteins. Neither the
apoenzyme nor the coenzyme moieties can function singly. In general, the coenzymes
function as acceptors of electrons or functional groupings, such as the carboxyl groups in αketo acids, which are removed from the substrate. Coenzymes are having affinities towards
both substrate and the apoenzyme and soometimes considered as a second substrate or cosubstrate. On particpation in the catalysis it irreversiblly alters its structure and hence could
D. D. Khedkar
Unit – I : Enzymes
9
not be recyled. It requires freshly in every catalysis. It plays very important role in the
activation and deactivation of the reactions.
Most of the coenzymes are derived from the water soluble vitamins (B – Complex). Wellknown coenzymes include the pyridine nucleotides, nicotinamide adenine dinucleotide
(NAD) and nicotinamide adenine dinucleotide phosphate (NADP); thiamine pyrophosphate
(TPP); flavin mononucleotide (FMN) and flavinadenine dinucleotide (FAD); iron
protoporphyrin (hemin); uridine diphosphate (UDP) and UDP-glucose; and adenosine
triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP).
Coenzyme A (CoA), a coenzyme in certain condensing enzymes, acts in acetyl or other acyl
group transfer and in fatty acid synthesis and oxidation. Folic acid coenzymes are involved in
the metabolism of one carbon unit. Biotin is the coenzyme in a number of carboxylation
reactions, where it functions as the actual carrier of carbon dioxide. See also Adenosine
triphosphate (ATP); Enzyme; Nicotinamide adenine dinucleotide phosphate (NADP).
The coenzymes does not decides the enzyme specificity. They may participate in various
reactions. The coenzymes provides stereospecificity to the enzyme. They are classified
according to type of reaction they participate in –
Biosynthesis
Coenzyme A is synthesized in a five-step process from pantothenate:
1. Pantothenate (Vitamin B5) is phosphorylated to 4'-phosphopantothenate by the
enzyme pantothenate kinase (PanK; CoaA; CoaX)
2. A cysteine is added to 4'-phosphopantothenate by the enzyme
phosphopantothenoylcysteine synthetase (PPC-DC; CoaB) to form 4'-phospho-Npantothenoylcysteine (PPC)
3. PPC is decarboxylated to 4'-phosphopantetheine by phosphopantothenoylcysteine
decarboxylase (CoaC)
4. 4'-phosphopantetheine is adenylylated to form dephospho-CoA by the enzyme
phosphopantetheine adenylyl transferase (CoaD)
5. Finally, dephospho-CoA is phosphorylated using ATP to coenzyme A by the enzyme
dephosphocoenzyme A kinase (CoaE).
Function
Since coenzyme A is chemically a thiol, it can react with carboxylic acids to form thioesters,
thus functioning as an acyl group carrier. It assists in transferring fatty acids from the
cytoplasm to mitochondria. A molecule of coenzyme A carrying an acetyl group is also
referred to as acetyl-CoA. When it is not attached to an acyl group it is usually referred to as
'CoASH' or 'HSCoA'.
List of coenzyme A activated acyl groups





Acetyl-CoA
Propionyl-CoA
Acetoacetyl-CoA
Coumaroyl-CoA (used in flavonoid and stilbenoid biosynthesis)
Acyl derived from dicarboxylic acids
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Unit – I : Enzymes
10
Coenzymes are classified on the basis of type of reaction they are participating in –
I.
II.
III.
Hydrogen Transfering : NAD, FAD, FMN, Cytochrome, etc.
Group Transfering : ATP, Biotin, CoA, Thymine Pyrohosphate, etc.
Isomerases & Lyases : B12 Coenzymes, Glutathion, etc.
COFACTOR
A cofactor is a non-protein chemical compound that is bound (either tightly or loosely) to a
protein and is required for the protein's biological activity. These proteins are commonly
enzymes and cofactors can be considered "helper molecules/ions" that assist in biochemical
transformations. With enzymes, cofactors are also often further classified depending on how
tightly they bind to the protein, with loosely-bound cofactors termed coenzymes and tightlybound cofactors termed prosthetic groups. Some sources also limit the use of the term
"cofactor" to inorganic substances. An inactive enzyme, without the cofactor is called an
apoenzyme, while the complete enzyme with cofactor is the holoenzyme.
Some enzymes or enzyme complexes require several cofactors. A good example is the
multienzyme complex pyruvate dehydrogenase. This enzyme complex at the junction of
glycolysis and the citric acid cycle requires five organic cofactors and one metal ion : loosely
bound thiamine diphosphate (ThDP), covalently bound lipoamide and flavin adenine
dinucleotide (FAD), and the cosubstrates nicotinamide adenine dinucleotide (NAD+) and
coenzyme A (CoA) and a metal ion (Mg2+).
Organic cofactors are often vitamins or are made from vitamins. Many contain the nucleotide
adenosine monophosphate (AMP) as part of their structures, such as ATP, coenzyme A, FAD
and NAD+. This common structure may reflect a common evolutionary origin as part of
ribozymes in an ancient RNA world. It has been suggested that the AMP part of the molecule
can be considered a kind a "handle" by which the enzyme can "grasp" the coenzyme to switch
it between different catalytic centers.
The succinate dehydrogenase complex showing several cofactors,
including flavin, iron-sulfur centers and heme.
Classification
Cofactors can be divided into two broad groups: organic cofactors, such as flavin or heme,
and inorganic cofactors: such as the metal ions Mg2+, Cu+, Mn2+ or iron-sulfur clusters.
Organic cofactors are sometimes further divided into coenzymes and prosthetic groups. The
term coenzyme refers specifically to enzymes and as such to the functional properties of a
protein. On the other hand "prosthetic group", emphasizes the nature of the binding of a
cofactor to a protein (tight or covalent) and thus refers to a structural property. Different
sources give slightly different definitions of coenzymes, cofactors and prosthetic groups.
Some consider tightly-bound organic molecules as prosthetic groups and not as coenzymes,
while others define all non-protein organic molecules needed for enzyme activity as
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Unit – I : Enzymes
11
coenzymes, and classify those that are tightly bound as coenzyme prosthetic groups.
Unsurprisingly, these terms are often used loosely.
A 1979 letter in Trends in Biochemical Sciences noted the confusion in the literature and the
essentially arbitrary distinction made between prosthetic groups and coenzymes and proposed
the following scheme. Here, cofactors were defined as an additional substance apart from
protein and substrate that is required for enzyme activity and a prosthetic group as a
substance that undergoes its whole catalytic cycle attached to a single enzyme molecule.
However, the author could not arrive at a single all-encompassing definition of a "coenzyme"
and proposed that this term be dropped from use in the literature.
MECHANISM OF ENZYME ACTION
Enzymes are generally globular proteins and range from just 62 amino acid residues in size,
for the monomer of 4-oxalocrotonate tautomerase, to over 2,500 residues in the animal fatty
acid synthase. A small number of RNA-based biological catalysts exist, with the most
common being the ribosome; these are referred to as either RNA-enzymes or ribozymes. The
activities of enzymes are determined by their three-dimensional structure. However, although
structure does determine function, predicting a novel enzyme's activity just from its structure
is a very difficult problem that has not yet been solved.
Most enzymes are much larger than the substrates they act on, and only a small portion of the
enzyme (around 3–4 amino acids) is directly involved in catalysis. The region that contains
these catalytic residues, binds the substrate, and then carries out the reaction is known as the
active site. Enzymes can also contain sites that bind cofactors, which are needed for catalysis.
Some enzymes also have binding sites for small molecules, which are often direct or indirect
products or substrates of the reaction catalyzed. This binding can serve to increase or
decrease the enzyme's activity, providing a means for feedback regulation.
Like all proteins, enzymes are made as long, linear chains of amino acids that fold to produce
a three-dimensional product. Each unique amino acid sequence produces a specific structure,
which has unique properties. Individual protein chains may sometimes group together to form
a protein complex. Most enzymes can be denatured—that is, unfolded and inactivated—by
heating or chemical denaturants, which disrupt the three-dimensional structure of the protein.
Depending on the enzyme, denaturation may be reversible or irreversible.
D. D. Khedkar
Unit – I : Enzymes
12
Specificity
Enzymes are usually very specific as to which reactions they catalyze and the substrates that
are involved in these reactions. Complementary shape, charge and hydrophilic/hydrophobic
characteristics of enzymes and substrates are responsible for this specificity. Enzymes can
also show impressive levels of stereospecificity, regioselectivity and chemoselectivity.
Some of the enzymes showing the highest specificity and accuracy are involved in the
copying and expression of the genome. These enzymes have "proof-reading" mechanisms.
Here, an enzyme such as DNA polymerase catalyzes a reaction in a first step and then checks
that the product is correct in a second step. This two-step process results in average error
rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases.
Similar proofreading mechanisms are also found in RNA polymerase, aminoacyl tRNA
synthetases and ribosomes.
Some enzymes that produce secondary metabolites are described as promiscuous, as they can
act on a relatively broad range of different substrates. It has been suggested that this broad
substrate specificity is important for the evolution of new biosynthetic pathways.
MODELS OF MODES OF ENZYME ACTION
I.
"Lock and key" model (Template Theory/Fisher Theory, Concept of
Intermolecular Fit, Reciprocal Fit Approach )
Enzymes are very specific, and it was suggested by Emil Fischer in 1894 that this was
because both the enzyme and the substrate possess specific complementary geometric shapes
that fit exactly into one another. This is often referred to as "the lock and key" model.
However, while this model explains enzyme specificity, it fails to explain the stabilization of
the transition state that enzymes achieve. The "lock and key" model has proven inaccurate,
and the induced fit model is the most currently accepted enzyme-substrate-coenzyme figure.
II.
Induced fit
In 1958, Daniel Koshland suggested a modification to the lock and key model: since
enzymes are rather flexible structures, the active site is continually reshaped by interactions
with the substrate as the substrate interacts with the enzyme. The favored model for the
enzyme-substrate interaction is the induced fit model. This model proposes that the initial
interaction between enzyme and substrate is relatively weak, but that these weak interactions
rapidly induce conformational changes in the enzyme that strengthen binding.
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Unit – I : Enzymes
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Catalysis by induced fit
The advantages of the induced fit mechanism arise due to the stabilising effect of strong
enzyme binding. There are two different mechanisms of substrate binding: uniform binding,
which has strong substrate binding, and differential binding, which has strong transition state
binding. The stabilizing effect of uniform binding increases both substrate and transition state
binding affinity, while differential binding increases only transition state binding affinity.
Both are used by enzymes and have been evolutionarily chosen to minimize the ΔG of the
reaction. Enzymes which are saturated, that is, have a high affinity substrate binding, require
differential binding to reduce the ΔG, whereas small substrate unbound enzymes may use
either differential or uniform binding.
These effects have led to most proteins using the differential binding mechanism to reduce
the ΔG, so most proteins have high affinity of the enzyme to the transition state. Differential
binding is carried out by the induced fit mechanism - the substrate first binds weakly, then the
enzyme changes conformation increasing the affinity to the transition state and stabilizing it,
so reducing the activation energy to reach it.
It is important to clarify, however, that the induced fit concept cannot be used to rationalize
catalysis. That is, the chemical catalysis is defined as the reduction of ΔG‡ (when the system
is already in the ES‡) relative to ΔG‡ in the uncatalyzed reaction in water (without the
enzyme). The induced fit only suggests that the barrier is lower in the closed form of the
enzyme but does not tell us what the reason for the barrier reduction is.
III.
Substrate Strain Theory
This is the principal effect of induced fit binding, where the affinity of the enzyme to the
transition state is greater than to the substrate itself. This induces structural rearrangements
which strain substrate bonds into a position closer to the conformation of the transition state,
so lowering the energy difference between the substrate and transition state and helping
catalyze the reaction.
However, the strain effect is, in fact, a ground state destabilization effect, rather than
transition state stabilization effect. Furthermore, enzymes are very flexible and they cannot
apply large strain effect.
In addition to bond strain in the substrate, bond strain may also be induced within the enzyme
itself to activate residues in the active site.
D. D. Khedkar
Unit – I : Enzymes
14
ENZYME REGULATION
Homeostasis is the mechanism by which an organism maintains its body in dynamic
equilibrium. A slight change in a concentration of a fluid within the organism may cause
major changes within its body. In living cells, there are different kinds of enzymes working
together. Living cells synthesis or break down molecules for normal metabolism and growth.
Enzyme regulation is one example. Enzymes are used to catalyze (speed up) reactions within
the body. The regulation of enzymes help maintain the body's equilibrium. An enzyme can be
in either one of two modes: on or off. That is controlling the synthesis of enzymes and
controlling the activity of enzymes (feedback inhibition). Basically enzyme regulation takes
advantage of these two modes. When a concentration of one product is too high, a negative
feedback loop can occur and stop the enzyme that catalyzes that specific product. Enabling a
lowering of reaction rate and lowering the concentration over time.
Enzymes activities are regulated by five basic techniques.
1. Allosteric control. Allosteric proteins have different regulatory and catalytic binding sites.
Allosteric proteins are cooperative proteins, where binding of a substrate in one active site
affects the activity of the rest of the binding sites. Some substrate binding will favor the
protein to be in the inactive T (tense) state, while other substrate binding will favor the
protein to be in the active R (relaxed) state, depending on the biological needs. Allosterically
regulated enzymes do not however obey Michaelis-Menten kinetics but instead follow
sigmoidal kinetics.
2. Isoenzymes. Isoenzymes have different animo acid sequences but catalyze the same
reaction as enzymes. They usually have different Km and Vmax values, and different
regulatory techniques. The advantages of isoenzymes is that it can catalyze the same reaction
under the different environments within the different organelles. Isozymes are an important
entity in metabolism for servicing a specific tissue or developmental sequence. For example
lactate dehydrogenase (LDH) has two isozymes that have an amino acid sequence that is 75%
similar. The H isozyme is present in the heart muscle and the M isozyme is expressed in the
skeletal muscle.
3. Reversible covalent modification. An enzyme's activity can be altered by covalently
attaching a different group to its active site. It blocks the natural substrate from binding to the
active site. The most common forms of covalent modification are phosphorylation and
dephosphorylation as well as aceylation and deacylation. Not all forms of covalent
modification are readily reversible. For example, an attachment of a lipid group will inhibit
the signal-transduction pathway in some proteins.
4. Proteolytic Activation. Many enzymes are present in the body in their inactive forms call
zymogen or prownzyme. They are not activated until a digestive enzyme cleaves it. The
cleavage alters the three dimension shape of the enzyme, forming the active site in the right
orientation. The zymogens become active enzymes in an irreversible reaction, typically the
hydrolysis of bonds in the zymogen.
5. Control by Limiting Amount of Enzyme. The amount of enzymes gets produced can be
controlled at the transcription level.
D. D. Khedkar
Unit – I : Enzymes
15
FACTORS REGULATING ENZYME ACTION
1.
2.
3.
4.
5.
6.
Enzyme Concentration
Substrate Concentration
pH
Temperature
Inhibitors
Activators
D. D. Khedkar
Unit – I : Enzymes
16
ENZYME KINETICS AS AN APPROACHTO UNDERSTANDING MECHANISM
Biochemists commonly use several approaches to study the mechanism of action of purified
enzymes. A knowledge of the three-dimensional structure of the protein provides important
information, and the value of structural information is greatly enhanced by classical protein
chemistry and modern methods of site-directed mutagenesis (changing the amino acid
sequence of a protein by genetic engineering; see Fig. 9–12). These technologies permit
enzymologists to examine the role of individual amino acids in enzyme structure and action.
However, the central approach to studying the mechanism of an enzyme-catalyzed reaction is
to determine the rate of the reaction and how it changes in response to changes in
experimental parameters, a discipline known as enzyme kinetics. This is the oldest approach
to understanding enzyme mechanisms and remains the most important. We provide here a
basic introduction to the kinetics of enzyme-catalyzed reactions. More advanced treatments
are available in the sources cited at the end of the chapter.
Substrate Concentration Affects the Rate of Enzyme-Catalyzed Reactions
A key factor affecting the rate of a reaction catalyzed by an enzyme is the concentration of
substrate, [S]. However, studying the effects of substrate concentrationis complicated by the
fact that [S] changes during the course of an in vitro reaction as substrate is converted to
product. One simplifying approach in kinetics experiments is to measure the initial rate (or
initial velocity), designated V0, when [S] is much greater than the concentration of enzyme,
[E]. In a typical reaction, the enzyme may be present in nanomolar quantities, whereas [S]
may be five or six orders of magnitude higher. If only the beginning of the reaction is
monitored (often the first 60 seconds or less), changes in [S] can be limited to a few percent,
and [S] can be regarded as constant. V0 can then be explored as a function of [S], which is
adjusted by the investigator. The effect on V0 of varying [S] when the enzyme concentration
is held constant is shown in Figure 6–11. At relatively low concentrations of substrate, V0
increases almost linearly with an increase in [S]. At higher substrate concentrations, V0
D. D. Khedkar
Unit – I : Enzymes
17
increases by smaller and smaller amounts
in response to increases in [S]. Finally, a
point is reached beyond which increases in
V0 are vanishingly small as [S] increases.
This plateau-like V0 region is close to the
maximum velocity, Vmax.
The ES complex is the key to
understanding this kinetic behavior, just as
it was a starting point for our discussion of
catalysis. The kinetic pattern in Figure 6–
11 led Victor Henri, following the lead of
Wurtz, to propose in 1903 that the
combination of an enzyme with its
substrate molecule to form an ES complex
is a necessary step in enzymatic catalysis.
This idea was expanded into a general
theory of enzyme action, particularly by
Leonor Michaelis and Maud Menten in
1913. They postulated that the enzyme first combines reversibly with its substrate to form an
enzyme-substrate complex in a relatively fast reversible step:
The ES complex then breaks down in a slower second step to yield the free enzyme and the
reaction product P: Because the slower second reaction (Eqn 6–8) must limit the rate of the
overall reaction, the overall rate must be proportional to the concentration of the species that
reacts in the second step, that is, ES.
D. D. Khedkar
Unit – I : Enzymes
18