Types of Enzyme catalysis

The enzymatic catalysis begins with substrate binding.

The binding energy is the free energy released in the formation of a large
number of weak interactions between the enzyme and the substrate.
We can envision this binding energy as serving two purposes: it establishes
substrate specificity and increases catalytic efficiency.
Only the correct substrate can participate in most or all of the interactions
with the enzyme and thus maximize binding energy, accounting for the
exquisite substrate specificity exhibited by many enzymes.
Furthermore, the full complement of such interactions is formed only
when the substrate is in the transition state.
Thus, interactions between the enzyme and the substrate not only favor
substrate binding but stabilize the transition state, thereby lowering the
activation energy.

The binding energy can also promote structural changes in both the
enzyme and the substrate that facilitate catalysis, a process referred to as
induced fit.
In most enzymes, the binding energy used to form the ES complex is
just one of several contributors to the overall catalytic mechanism.

Once a substrate is bound to an enzyme, properly positioned

catalytic functional groups aid in the cleavage and formation of
bonds by a variety of mechanisms, including general acid-base
catalysis, covalent catalysis, and metal ion catalysis.

These are distinct from mechanisms based on binding energy,

because they generally involve transient covalent interaction with a
substrate or group transfer to or from a substrate.

(i). General Acid-Base Catalysis:

Many biochemical reactions involve the formation of unstable

charged intermediates that tend to break down rapidly to their
constituent reactant species, thus impeding the reaction.
Acids and bases can accelerate chemical reactions by donating and
accepting a proton, respectively.
Bases may also support a reaction by increasing the nucleophilic
property of the attacking group.
Irrespective of the mode of reaction enhancement, charged
transition state is formed that is highly unstable.

An uncatalyzed attack by water during hydrolytic reactions, for

instance leads to the formation of a transition state that is highly
unstable because of the development of positive and negative
charges, as shown:
The transition state can be stabilised by diluting the +δ charge on
water by transferring one of its proton to a basic group (Lys, His, Ser,
etc) of the enzyme (general base catalysis).

Similarly the – δ charge in the transition state can be stabilised by

transfer of a proton (general acid catalysis) from an acidic group
(Asp, Glu, etc) of the enzyme.

Likewise, these +δ and –δ charges can also be stabilised during

electrostatic catalysis by carboxylate groups of the enzyme or its
bound metal ions (Mg++/ Mn++).

These stabilising effects of the enzymes thus promote the formation

of transition state, and hence, the enhancement of the rate of the
reactions.
Charged intermediates can often be stabilized by the transfer of
protons to or from the substrate or intermediate to form a species
that breaks down more readily to products.

For non-enzymatic reactions, the proton transfers can involve either

the constituents of water alone or other weak proton donors or
acceptors.
Catalysis of this type that uses only the H+ (H3O+) or OH- ions present
in water is referred to as specific acid-base catalysis.

If protons are transferred between the intermediate and water faster

than the intermediate breaks down to reactants, the intermediate is
effectively stabilized every time it forms.

No additional catalysis mediated by other proton acceptors or

donors will occur.
In many cases, however, water is not enough.

The term general acid-base catalysis refers to proton transfers

mediated by other classes of molecules.

For nonenzymatic reactions in aqueous solutions, this occurs only

when the unstable reaction intermediate breaks down to reactants
faster than protons can be transferred to or from water.

Many weak organic acids can supplement water as proton donors in

this situation, or weak organic bases can serve as proton acceptors.

In the active site of an enzyme, a number of amino acid side chains

can similarly act as proton donors and acceptors.

These groups can be precisely positioned in an enzyme active site to

allow proton transfers, providing rate enhancements of the order of
102 to 105.
This type of catalysis occurs on the vast majority of enzymes.

In fact, proton transfers are the most common biochemical

reactions.

The active sites of some enzymes contain amino acid functional groups, such as those
shown here, that can participate in the catalytic process as proton donors or proton
acceptors.
2. Covalent Catalysis
In covalent catalysis, a transient covalent bond is formed between
the enzyme and the substrate.
Consider the hydrolysis of a bond between groups A and B:

In the presence of a covalent catalyst (an enzyme with a nucleophilic group X:)
the reaction becomes

This alters the pathway of the reaction, and it results in catalysis only when
the new pathway has a lower activation energy than the uncatalyzed
pathway.
Both of the new steps must be faster than the uncatalyzed reaction.
A number of amino acid side chains (Glu, Asp, Lys, Arg, Cys, His, Ser, Tyr),
and the functional groups of some enzyme cofactors can serve as
nucleophiles in the formation of covalent bonds with substrates.
These covalent complexes always undergo further reaction to
regenerate the free enzyme.

The covalent bond formed between the enzyme and the substrate
can activate a substrate for further reaction in a manner that is
usually specific to the particular group or coenzyme.
Example: Chymotrypsin Action Proceeds in Two Steps Linked by a Covalently
Bound Intermediate
•A number of proteolytic enzymes participate in the breakdown of proteins in the digestive
systems of mammals and other organisms.
•One such enzyme, chymotrypsin, cleaves peptide bonds selectively on the carboxyl
terminal side of the large hydrophobic amino acids such as tryptophan, tyrosine,
phenylalanine, and methionine.
•Chymotrypsin is a good example of the use of covalent modification as a catalytic
strategy.
•The enzyme employs a powerful nucleophile to attack the unreactive carbonyl group of
the substrate.
•This nucleophile becomes covalently attached to the substrate briefly in the course of
catalysis.
The catalytic process begins with the nucleophilic attack of the
hydroxyl gp of Ser195 on the carbonyl carbon of the sessile peptide
bond forming a tetrahedral intermediate.
The proton of the Ser195 hydroxyl is transferred to the imidazole of
His57, which functions as a general base.
The negatively charged triad partner, Asp102 stabilizes the positive
charge on His57 which it acquires on accepting the proton from
Ser195.
This arrangement prevents the development of potentially
destabilizing positive charge on the serine hydroxyl and also makes
it better nucleophile.
The acylation cycle of the enzymatic reaction comes to an end after
the His57 uses the acquired proton to protonate the amino group in
the portion of the substrate being displaced.
This results in the expulsion of the leaving group with consequential
conversion of the tetrahedral intermediate into an acylenzyme.
An activated water molecule then binds to His57 and performs
nucleophilic attack on the carbonyl carbon of the acylenzyme
intermediate exactly the same way as described for the first cycle of
the reaction except that the proton is extracted from water this
time.

The peptide involves the formation and decomposition of the

tetrahedral intermediate, wherein the whole process is facilitated
by general acid-base catalysis mediated by histidine side chain.
3. Metal Ion Catalysis
Metals, whether tightly bound to the enzyme or taken up from
solution along with the substrate, can participate in catalysis in several
ways.
Ionic interactions between an enzyme-bound metal and a substrate
can help orient the substrate for reaction or stabilize charged reaction
transition states.
This use of weak bonding interactions between metal and substrate is
similar to some of the uses of enzyme-substrate binding energy.
Metals can also mediate oxidation-reduction reactions by reversible
changes in the metal ion’s oxidation state.
Nearly a third of all known enzymes require one or more metal ions
for catalytic activity.
Metal ions can function catalytically in several ways.

A metal ion may serve as an electrophilic catalyst, stabilizing a

negative charge on a reaction intermediate.

The metal ion may generate a nucleophile by increasing the acidity

of a nearby molecule, such as water in the hydration of CO2 by
carbonic anhydrase.

The metal ion may bind to substrate, increasing the number of

interactions with the enzyme and thus the binding energy.

Most enzymes employ a combination of several catalytic strategies

to bring about a rate enhancement.
4. Catalysis by approximation.
Many reactions include two distinct substrates.

In such cases, the reaction rate may be considerably enhanced by

bringing the two substrates together along a single binding surface
on an enzyme.

NMP kinases bring two nucleotides together to facilitate the

transfer of a phosphoryl group from one nucleotide to the other
The initial binding of the substrate to the enzyme site is of utmost
importance in enzymatic catalysis.

This brings the reacting groups closer to the catalytic groups on the
enzyme active side on one hand and compensates, at least in part,
for the binding associated entropy loss by releasing the binding
energy on the other.
The binding of the substrate molecules in close proximity to each other
increases its effective concentration, leading to further reduction in the
entropy loss due to enzyme-substrate complex formation.
This proximity improvement is vividly called proximation, approximation
or propinquity effect.
Another possible way in which the crossing of the activation energy
barrier may be facilitated is the introduction of strain into the reactants
due to compulsive binding of the substrate at the enzyme active site and
thereby allowing more binding energy to be available for the transition
state.
This is in line with the induced fit model of the active site, which
presumes that the affinity of the enzyme to the transition state is greater
than to substrate itself.
Binding of the substrate to the active site, therefore, induces structural
rearrangements in the substrate, taking it closer to the conformation of
the transition state and hence reducing the energy of activation and
increasing the rate of the reaction.
Binding of the substrate to the enzyme repositions the catalytic groups
and the substrate.

The newly acquired orientation of the enzymatic catalytic groups enables

them to become more effective in the catalytic process.

Indeed as shown in the figure, when the rate of the hydrolysis of the ester
bond in aspirin was measured with its own carboxylate group acting as
catalytic group, it was found to be some 100 folds higher as compared to
the uncatalyzed hydrolysis of similar compounds.
As much as 13 M solution of an external base is required as catalyst
to give the same rate as the intra molecular acid-base catalysis
exhibited by the carboxylase group during hydrolysis of aspirin.

This means that the effective concentration of carboxylate in

intracellular catalysis is 13 times higher as compared to what it
actually is.

The effective increase in the concentration is achieved by entropy

factor because enzyme-substrate complex behaves like a single
molecule with adequate compensation for any decrease in the
entropy coming from the decrease in energy due to interactions
leading to complex formation.

The intermolecular reactions, on the other hand, involve two or

more molecules associating to form one complex, leading to an
increase in order with the consequential loss in entropy, making the
reaction less favourable.
 Strain Distortion
Certain structures such as three-membered and four-membered
ring structures, such as epoxides are highly reactive due to the
strain distortion inherent to the unfavored bond angles inherent to
the ring.

Enzyme active sites can also utilize strain distortion within a bound
substrate to increase the reactivity of the molecule and favor the
formation of the transition state.

Many enzymes that function by the induced fit model also utilize
strain distortion within their catalytic mechanism.
Within the unbound state they remain in a low catalytic state,
however the interaction with the substrate induces the
destabilization of the enzyme active site or may induce strain within
the substrate causing the initiation of the catalytic activity of the
enzyme.