ENZYME ACTION

ENZYME ACTION

The Active Sites of Enzymes And Some Common Features:

The substrates are bound to a specific region of the enzyme called the active site.
Most enzymes are highly selective in the substrates that they bind. Indeed, the catalytic
specificity of enzymes depends in part on the specificity of binding.

The active site of an enzyme is the region that binds the substrates. It also
contains the residues that directly participate in the making and breaking of bonds. These
residues are called the catalytic groups. In essence, the interaction of the enzyme and
substrate at the active site promotes the formation of the transition state. Although
enzymes differ widely in structure, specificity, and mode of catalysis, a number of
generalizations concerning their active sites can be stated:
1. The active site is a three-dimensional cleft formed by groups that come from
different parts of the amino acid sequence indeed, residues far apart ins the sequence may
interact more strongly than adjacent residues in the amino acid sequence.
2. The active site takes up a relatively small part of the total volume of an enzyme.
Most of the amino acid residues in an enzyme are not in contact with the substrate, which
raises the intriguing question of why enzymes are so big. Nearly all enzymes are made up
of more than 100 amino acid residues, which gives them a mass greater than 10 kd and a
diameter of more than 25 Å. The "extra" amino acids serve as a scaffold to create the
three dimensional active site from amino acids that are far apart in the primary structure.
Amino acids near to one another in the primary structure are often sterically constrained
from adopting the structural relations necessary to form the active site. In many proteins,
the remaining amino acids also constitute regulatory sites, sites of interaction with other
proteins, or channels to bring the Substrates to the active sites.
3. Active sites are clefts or crevices. In all enzymes of known structure, substrate
molecules are bound to a cleft or crevice. Water is usually excluded unless it is a reactant.
The nonpolar character of much of the cleft enhances the binding of substrate as well as
catalysis. Nevertheless, the cleft may also contain polar residues. In the nonpolar
microenvironment of the active site, certain of these polar residues acquire special

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properties essential for substrate binding or catalysis. The internal positions of these polar
residues are biologically crucial exceptions to the general rule that polar residues are
exposed to water.
4. Substrates are bound to enzymes by multiple weak attractions. ES complexes
usually have equilibrium constants that range from 10-2 to 10-8 M, corresponding to free
energies of interaction ranging from about -3 to -12 kcal mol-1. The noncovalent
interactions in ES complexes are much weaker than covalent bonds, which have energies
between -50 and -110 kcal mol-1. electrostatic interactions, hydrogen bonds, van der
Waals forces, and hydrophobic interactions mediate reversible interactions of
biomolecules. Van der Waals forces become significant in binding only when numerous
substrate atoms simultaneously come close to many enzyme atoms. Hence, the enzyme
and substrate should have complementary shapes. The directional character of hydrogen
bonds between enzyme and substrate often enforces a high degree of specificity.
5. The specificity of binding depends on the precisely defined arrangement of
atoms in an active site. Because the enzyme and the substrate interact by means of short-
range forces that require close contact, a substrate must have a matching shape to fit into
the site. Emil Fischer's analogy of the lock and key expressed in 1890, has proved to be
highly stimulating and fruitful. However, we now know that enzymes are flexible and
that the shapes of the active sites can be markedly modified by the binding of substrate,
as was postulated by Daniel E. Koshland, Jr., in 1958. The active sites of some enzymes
assume a shape that is complementary to that of the transition state only after the
substrate is bound. This process of dynamic recognition is called induced fit.

Regulatory site of enzymes:

The enzymes having the other than the active site that site is called as regulatory
site of enzyme, these enzymes also referred as Allosteric enzymes. The word ‘allosteric’
means ‘another site’. As the meaning an allosteric enzymes have two sites; namely
regulatory site and a catalytic site or active site.
To the regulatory site certain compounds called modifier or allosteric effectors are
going to bind. The modulator is a normal metabolite which when bind with allosteric site

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of an enzyme modified its kinetic properties, the modulators may be either stimulatory or
inhibitory in nature.
• The allosteric site may be one or more in an enzyme.
• Just like the enzyme’s active site specific for its substrate, The allosteric
site is specific for modulator.
• Allosteric and substrate binding sites may or may not physically adjacent.
• Allosteric enzymes are usually contain multiple sub units and the catalytic
and allosteric site may be located on different subunit.

CATALYTIC TACTICS AND INTERMOLECULAR FORCES

The some more detailed way some of the molecular tactics and intermolecular
forces that enzymes use to implement their catalytic strategies.

VAN DER WAALS INTERACTIONS

Molecules are clouds of electrons in which nuclei are embedded. At any instant in
time, there is a dipole moment, which averages to zero in a nonpolar molecule as
electrons explore the space available to them (defined by the wave function). When two
molecules approach each other, these transient dipole moments become correlated,
resulting in attractive electrostatic interaction. This dipolar interaction is a very short-
range phenomenon, weakening with the sixth power of the distance. However, if two
molecules approach each other too closely, electrostatic repulsion dominates. The two
opposing effects create a preferred separation distance characterizing each atom — the
van der Waals radius — which can be thought of as defining the surface of the molecule.
The energy in van der Waals interactions is relatively low, but can accumulate to become
quite significant. For instance, the van der Waals attractive energy for two methylene
groups is estimated to be 2 kcal mol–1; in enzyme ligandcomplexes, where there is a
large degree of surface contact, this can add up to tens of kcal mol–1.

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HYDROGEN BONDS
“Classical” hydrogen bonds occur when a hydrogen atom that is covalently
bonded to an electronegative atom (oxygen or nitrogen) is only a short distance from an
electronegative atom that has a lone electron pair (again, usually oxygen or nitrogen).
Experimentally determined distances between heavy atoms involved in a hydrogen
bond show that they are slightly closer than the distance expected from their van
der Waals radii, suggesting that the proton s from the partial positive charge that resides
on a hydrogen atom in the polar covalent bond to the electronegative donor atom and the
partial negative charge that resides on the lone pair of electronegative acceptor atom. The
ionic character of hydrogen bonds makes their strength distance dependent but less
sensitive (though not insensitive) to direction. Estimates for the free energy of formation
of typical hydrogen bonds in aqueous

Hydrogen Bonds between an Enzyme and Substrate. The enzyme ribonuclease forms
hydrogen bonds with the uridine component of the substrate.

ACID/BASE CATALYSIS
Acid/base catalysis the participation of an enzyme-bound functional group in
transferring protons is a nearly ubiquitous feature of enzyme reactions. Every ionizable
protein functional group, as well as groups provided from substrates or prosthetic groups,
has been invoked as acid or basic catalysts in enzymology. Two categories of proton
transfer reactions may be considered: those to or from noncarbon atoms (heteroatoms)
and those to or from carbon atoms. The transfer of protons to or from heteroatoms is
generally facile, as evidenced by the high rates of proton.
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ELECTROSTATICS
The interaction of charges can have a strong influence on reaction rates; some
argue that this is the primary origin of the catalytic power of enzymes. Hydrogen bonds
and acid/base catalysis are electrostatic effects that are important enough to be treated in
their own sections. However, beyond the catalytic effects of specifically positioned
protons, constellations of charged amino acid side chains can create regions of positive or
negative electrostatic potential. Similar effects can be produced by the amide groups of
asparagine and glutamine side chains or the dipoles of the peptide groups in the protein
backbone. The combined effects of peptide dipoles can be concentrated by an - helix,
though little is gained by increasing the length of the helix beyond ~2 turns. Electrostatic
fields due either to side-chain charges or dipole moments from the protein structure can
interact with bound molecules, causing changes in pKa values and stabilizing developing
charges in transition states and intermediates. These effects are sensitive to the dielectric
constant that intervenes between the charge/dipole and the substrate/ transition
state/intermediate, and can be amplified by excluding water
.
NUCLEOPHILIC CATALYSIS
Enzymes sometimes use nucleophilic side chains or nucleophilic prosthetic
groups to form an adduct with the substrate or a substrate fragment. The side chains of
serine, threonine, cysteine, histidine, aspartate, glutamate, and tyrosine have been
identified as nucleophiles in a variety of enzymes, including proteases, esterases, kinases,
and lyases, to name just a few. Nucleophilic catalysis is frequently involved in group
transfer reactions, in which a group on one substrate is transferred to an acceptor site on
another substrate. Rather than catalyzing the direct reaction between the two substrates,
the enzyme breaks the reaction into partial reactions in which the group is transferred first
to the enzymatic nucleophile and then to the ultimate acceptor.

ELECTROPHILIC CATALYSIS
The 20 genetically encoded amino acids provide an abundance of nucleophilic
groups but no electrophilic groups (with the exception of a proton donated by an acid, but

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this is in a category of its own). Nonetheless, many biochemical reactions require

catalysis by an electrophile, necessitating the recruitment of electrophilic prosthetic
groups. These can be either small organic molecules, such as pyridoxal phosphate, or
metal ions. Electrophilic catalysts speed reactions by forming an adduct with a substrate
and activating them for the loss of an electrophilic-leaving group such as CO2 or H+.
When an electron-deficient group leaves a molecule for instance, in a deprotonation
reaction it will leave behind an electron pair. It is the job of the electrophilic catalyst to
stabilize this new lone pair, usually by delocalization in the case of organic prosthetic
groups, or by direct charge neutralization in the case of metals.

REDOX CATALYSIS
A large number of biochemical reactions are net redox reactions. In addition to
these, there are many reactions that do not involve a net redox change but do require a
reactive intermediate to be generated by redox chemistry. Although some redox reactions
occur directly between two substrates, in a large number of cases a substrate oxidizes or
reduces the enzyme directly, which subsequently reacts with the next substrate.
Consequently, a large number of enzymes do redox chemistry.

THEORES OF MECHANISM OF ENZYME ACTION.

Lock-and-Key Model of Enzyme-Substrate Binding.
In 1890, Emil Fisher proposed a model to explain the great specificity of
enzymes. He explained the interaction between substrate and enzymes in terms of “Lock
and Key”. According to this concept, the calytic site of the enzyme by itself is
complementory in shape to that of the substrate.

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Lock-and-Key Model of Enzyme-Substrate Binding. In this model, the active site of

the unbound enzyme is complementary in shape to the substrate.

The enzymic reaction is possible if the substrate matches the active center as the
key fits the lock. If the substrate [“Key”] become slightly modified, it no longer fits the
active center [“Lock”] and no reaction takes place. Also, the presence of a substrate at the
active site may exclude water molecules and thus make the region more non-polar. Both
of these factors could be responsible for some degree of change in the tertiary structure.
Therefore, in the lock and key mechanism, the active site is always structurally intact,
with the catalytic sites aligned and freely accessible. Thus, a suitable reacting group,
whether part of an appropriately bound substrate or not, can come into contact with the
region of catalytic activity and some degree of reaction take place.

Induced-Fit Model of Enzyme-Substrate Binding.

In order to account for the above observation, Koshland (1958) proposed the
induced fit hypothesis. According to this hypothesis, the structure of the substrate may be
complementary to that of the active site in the enzyme substrate complex. But not in the
free enzyme during the binding of the substrate, which results in the required matching of
structures.

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Induced-Fit Model of Enzyme-Substrate Binding. In this model, the enzyme changes

shape on substrate binding. The active site forms a shape complementary to the substrate
only after the substrate has been bound.

The induced fit hypothesis requires that the active site be flexible and the
substrate be rigid, allowing the enzyme to wrap itself around the substrate, thereby
bringing together the corresponding catalytic sites and reacting groups. Also, in this
mechanism, different catalytic components might be separated by a considerable margin
in the free enzyme, minimizing the risk of a chance collision of a reactive group with
both of them. It is also possible that access to the catalytic groups of free enzyme might
be blocked. Only when the binding group of the substrate is recognized by the
corresponding site of the enzyme and the binding process proceeds, does conformational
change take place, which results in all the relevant groups in the substrate and enzyme
coming together.

FACTORS AFFECTING ENZYME ACTIVITY.

EFFECT OF pH:
Enzymes are affected by changing in pH. Enzyme action is greatest within
a narrow range of pH because all the enzymes are active. Changing the pH changes the
H bonds, and thus the shape of the active site. Therefore, the substrate can no longer bind
to the active site and so enzyme action decreases. The most favorable pH value the point
where the enzyme is most active is known as the optimum pH. Enzymes have an
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optimum pH at which their activity decreases. This is because the amino acid side chains
in the active site may act as weak acid and basses with critical functions that depend on
their maintaining a certain state of ionization.

The pH effect is mainly based on two factors:

1.Ionization of the enzyme, particularly at the active site Like all proteins, enzyme
molecules possess numerous ionizable groups whose state of ionizability depends on pH.
For an enzyme molecule to be active as a catalyst, certain of these groups must be ionized
while others must remain unionized. This state of affairs would obviously prevail only
within a limited pH range, which would depend on the pka values of the groups
concerned.

2. Ionization of the substrate, or the coenzyme, In some cases, substrate, like the
enzyme, is also capable of being ionized. Thus in such cases also it would be responsible
to assume that only one ionic form of the substrate molecules might be capable of
undergoing the reaction. This specific ionic substrate would also exist over a limited pH
range.

EFFECT OF TEMPERATURE.
Like most chemical reactions, the rate of an enzyme catalysed reaction increases
as the temperature is raised. However, this happens only up to certain temperature
commonly known as optimum temperature. Above which enzymes become denatured
and lose activity which in turn is due to loss of the secondary and tertiary structure of the
protein. As the enzyme inactivated, the reaction which it catalyses slow down and
ultimately stops. At low temperature enzyme action is low because the movement of
molecule is low. Some enzymes lose their activity when frozen. Thus optimum
temperature of an enzyme may be defined as the temperature at which its activity is
maximum.

EFFECT OF SUBSTRATE CONCENTRATION.

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Substrate concentration is one of the most important factor, which determines the
velocity of enzyme reactions. In nearly all cases, when initial velocity is plotted against
substrate concentration, a section of a rectangular hyperbola is obtained.

The Chymotrypsin Mechanism.

Bovine pancreatic chymotrypsin (Mr 25,191) is a protease, an enzyme that

catalyzes the hydrolytic cleavage of peptide bonds. This protease is specific for peptide
bonds adjacent to aromatic amino acid residues (Trp, Phe, Tyr). The three-dimensional
structure of chymotrypsin with functional groups in the active site emphasized. The
reaction catalyzed by this enzyme illustrates the principle of transition-state stabilization
and also provides a classic example of general acid-base catalysis and covalent catalysis.
Chymotrypsin enhances the rate of peptide bond hydrolysis by a factor of at least 109. It
does not catalyze a direct attack of water on the peptide bond; instead, a transient
covalent acyl-enzyme intermediate is formed. The reaction thus has two distinct phases.
In the acylation phase, the peptide bond is cleaved and an ester linkage is formed between
the peptide carbonyl carbon and the enzyme. In the deacylation phase, the ester linkage is
hydrolyzed and the nonacylated enzyme regenerated.

In chymotrypsin, Ser195 is linked to His57 and Asp102 in a hydrogen-bonding

network referred to as the catalytic triad. When a peptide substrate binds to
chymotrypsin, a subtle change in conformation compresses the hydrogen bond between
His57 and Asp102, resulting in a stronger interaction, called a low-barrier hydrogen
bond. This enhanced interaction increases the pKa of His57 from ~7 (for free histidine) to
12, allowing the His residue to act as an enhanced general base that can remove the
proton from the Ser195 hydroxyl group. Deprotonation prevents development of a very
unstable
positive charge on the Ser195 hydroxyl and makes the Ser side chain a stronger
nucleophile. At later reaction stages, His57 also acts as a proton donor, protonating the
amino group in the displaced portion of the substrate (the leaving group). As the Ser195
oxygen attacks the carbonyl group of the substrate, a very short-lived tetrahedral
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intermediate is formed in which the carbonyl oxygen acquires a negative charge. This
charge, forming within a pocket on the enzyme called the oxyanion hole, is stabilized
by hydrogen bonds contributed by the amide groups of two peptide bonds in the
chymotrypsin backbone. One of these hydrogen bonds (contributed by Gly193) is present
only in this intermediate and in the transition states for its formation and breakdown; it
reduces the energy required to reach these states. This is an example of the use of binding
energy in catalysis.

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THE LYSOZYME MECHANISM.

Lysozyme is a natural antibacterial agent found in tears and egg

whites. The hen egg white lysozyme (Mr 14,296) is a monomer with
129 amino acid residues. This was the first enzyme to have its three-
dimensional structure determined, by David Phillips and colleagues in
1965. The structure revealed four stabilizing disulfide bonds and a cleft
containing the active site. More than five decades of lysozyme
investigations have provided a detailed picture of the structure and
activity of the enzyme, and an interesting
story of how biochemical science progresses.

The substrate of lysozyme is peptidoglycan, a carbohydrate found in

many bacterial cell walls. Lysozyme cleaves the (_1n4) glycosidic COO
bond between the two types of sugar residue in the molecule, N-
acetylmuramic acid (Mur2Ac) and N-acetylglucosamine (GlcNAc), often
referred to as NAM and NAG, respectively. Six residues of the
alternating Mur2Ac and GlcNAc in peptidoglycan bind in the active site,
in binding sites labeled A through F. Model building has shown that the
lactyl side chain of Mur2Ac cannot be accommodated in sites C and E,
restricting Mur2Ac binding to sites B, D, and F. Only one of the bound
glycosidic bonds is cleaved, that between a Mur2Ac residue in site D
and a GlcNAc residue in site E. The key catalytic amino acid residues in
the active site are Glu35 and Asp52. The reaction is a nucleophilic
substitution, with OOH from water replacing the GlcNAc at C-1 of
Mur2Ac. With the active site residues identified and a detailed
structure of the enzyme available, the path to understanding the
reaction mechanism seemed open in the 1960s. However, definitive
evidence for a particular mechanism eluded investigators for nearly

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four decades. There are two chemically reasonable mechanisms that

could generate the observed product of lysozymemediated cleavage of
the glycosidic bond. Phillips and colleagues proposed a dissociative
(SN1 type) mechanism in which the GlcNAc initially dissociates in step
1 to leave behind a glycosyl cation (a carbocation) intermediate. In this
mechanism, the departing GlcNAc is protonated by general acid
catalysis by Glu35, located in a hydrophobic pocket that gives its
carboxyl group an unusually high pKa. The carbocation is stabilized by
resonance involving the adjacent ring oxygen, as well as by
electrostatic interaction with the negative charge on the nearby Asp52.
In step 2 ,water attacks at C-1 of Mur2Ac to yield the product. The
alternative mechanism involves two consecutive direct-displacement
(SN2-type) steps. In step 1 , Asp52 attacks C-1 of Mur2Ac to displace
the GlcNAc. As in the first mechanism, Glu35 acts as a general acid to
protonate the departing GlcNAc. In step 2 , water attacks at C-1 of
Mur2Ac to displace the Asp52 and generate product.

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THE DNA METHYL TRANSFERASE ACTIVITY.

According to this mechanism, DNA methylation is initiated by a
nucleophilic attack of an SH group from a catalytic cysteine residue
located in the conserved amino acids sequence motif IV (GPPC) (Kumar
et al. 1994) on the C6 position of the target cytosine, yielding a
covalent intermediate between the base and the enzyme. Thereby, the
C5 position of the cytosine gets activated and becomes capable of
performing a nucleophilic attack on the methyl group bound to the
cofactor
substrate AdoMet. The enzyme facilitates the nucleolytic attack on the
C6 atom by a transient protonation of the cytosine ring at the
endocyclic nitrogen atom N3, which is stabilized by the glutamate
residue from a highly conserved motif VI (ENV). The covalent complex
between the methylated base and the DNA is resolved by
deprotonation at the C5 position, which leads to the elimination of the
cysteinyl group and the reestablishment of aromaticity. Then, the
methylated base together with the cofactor product, S-adenosyl-L-
homocysteine, is released. In addition to the residues already
mentioned, the second arginine residue in motif VIII (RXR) plays an
important role in the catalytic mechanism of DNA m5C
methyltransferases.

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FIGURE: Schematic picture of the catalytic mechanisms proposed for

DNA MTases and RNA MTases.

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