BCH 4101 – ADVANCED ENZYMOLOGY

General Principles of Catalysis

A catalyst may be defined as a species which accelerates the rate of a chemical reaction while remaining
unchanged at the end of the catalytic reaction. A reaction in which a catalyst is involved is called a
catalyzed reaction, and the process is called catalysis. In thermodynamic terms, catalysis of a chemical
reaction is achieved by reducing the activation energy for that reaction, the activation energy being the
difference in free energy between the reagent(s) and the transition state for the reaction. This reduction in
activation energy can be achieved either by stabilization (and hence reduction in free energy) of the
transition state by the catalyst, or by the catalyst finding some other lower energy pathway for the
reaction.
The following are the characteristics of catalysis:
1. A catalyst lowers the Gibbs energy of activation by providing a different mechanism for the
reaction. This mechanism enhances the rate and it applies to both the forward and the reverse
directions of the reaction.

Gibbs energy change for (a) an uncatalyzed reaction and (b) a catalyzed reaction. The
catalyzed reaction must involve the formation of at least one intermediate (between the
reactant and thecatalyst). The ∆Go is the same in both cases.

2. A catalyst forms an intermediate with the reactant(s) in the initial step of the mechanism and is
released in the product-forming step. The catalyst does not appear in the overall reaction.
3. Regardless of the mechanism and the energetics of a reaction, a catalyst cannot affect the
enthalpies or Gibbs energies of the reactants and products. Thus, catalysts increase the rate of
approach to equilibrium, but cannot alter the thermodynamic equilibrium constant.

There are three types of catalysis: heterogeneous, homogeneous, and enzymatic. Enzyme catalysis is also
mostly homogeneous in nature. However, because it is of biological origin and is the most complex of the
three types of catalysis, enzyme catalysis is treated as a separate category.

Enzyme Catalysis

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Enzymes accelerate a chemical reaction but in doing so are neither chemically transformed at the
completion of the reaction nor do they alter the equilibrium of the reaction.

Enzymes, in general, provide speed, specificity, and regulatory control to reactions in the body. Enzymes
are usually proteins that act as catalysts, compounds that increase the rate of chemical reactions. Enzyme-
catalyzed reactions have three basic steps:

An enzyme binds the substrates of the reaction it catalyzes and brings them together at the right
orientation to react. The enzyme then participates in the making and breaking of bonds required for
product formation, releases the products, and returns to its original state once the reaction is completed.
Enzymes do not invent new reactions; they simply make reactions occur faster. The catalytic power of an
enzyme (the rate of the catalyzed reaction divided by the rate of the uncatalyzed reaction) is usually in the
range of 106 to 1014. Without the catalytic power of enzymes, reactions such as those involved in nerve
conduction, heart contraction, and digestion of food would occur too slowly for life to exist.
Each enzyme usually catalyzes a specific biochemical reaction. The ability of an enzyme to select just one
substrate and distinguish this substrate from a group of very similar compounds is referred to as
specificity. The enzyme converts this substrate to just one product. The specificity, as well as the speed,
of enzyme-catalyzed reactions result from the unique sequence of specific amino acids that form the
three-dimensional structure of the enzyme.

Some features of enzyme catalysis:

A. The Active Site

To catalyze a chemical reaction, the enzyme forms an enzyme–substrate complex in its active catalytic
site. The active site is usually a cleft or crevice in the enzyme formed by one or more regions of the
polypeptide chain. Within the active site, cofactors and functional groups from the polypeptide chain
participate in transformingthe bound substrate molecules into products.

Initially, the substrate molecules bind to their substrate binding sites, also called the substrate recognition
sites. The three-dimensional arrangement of binding sites in a crevice of the enzyme allows the reacting

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portions of the substrates to approach each other from the appropriate angles. The proximity of the bound
substrate molecules and their precise orientation toward each other contribute to the catalytic power of the
enzyme.
The active site also contains functional groups that directly participate in the reaction. The functional
groups are donated by the polypeptide chain, or by bound cofactors (metals or complex organic molecules
called coenzymes). As the substrate binds, it induces conformational changes in the enzyme that promote
further interactions between the substrate molecules and the enzyme functional groups. (For example, a
coenzyme might form a covalent intermediate with the substrate, or an amino acid side chain might attract
a proton from the reacting substrate). The activated substrates and the enzyme form a transition state
complex, an unstable high-energy complex with a strained electronic configuration that is intermediate
between substrate and product. Additional bonds with the enzyme stabilize the transition state complex
and decrease the energy required for its formation.

The transition state complex decomposes to products, which dissociate from the enzyme. The enzyme
generally returns to its original form. The free enzyme then binds another set of substrates, and repeats the
process.

B. Substrate Binding Sites

Enzyme specificity (the enzyme’s ability to react with just one substrate) results from the three-
dimensional arrangement of specific amino acid residues in the enzyme that form binding sites for the
substrates and activate the substrates during the course of the reaction. The “lock-and-key” and the
“induced-fit” models for substrate binding describe two aspects of the binding interaction between the
enzyme and substrate.

1. LOCK-AND-KEY MODEL FOR SUBSTRATE BINDING

The substrate binding site contains amino acid residues arranged in a complementary three-dimensional
surface that “recognizes” the substrate and binds it through multiple hydrophobic interactions,
electrostatic interactions, or hydrogen bonds. The amino acid residues that bind the substrate can come
from very different parts of the linear amino acid sequence of the enzyme. The binding of compounds
with a structure that differs from the substrate even to a small degree may be prevented by steric
hindrance and charge-repulsion. In the lock-and-key model, the complementarity between the substrate
and its binding site is compared to that of a key fitting into a rigid lock.

2. “INDUCED FIT” MODEL FOR SUBSTRATE BINDING

Complementarity between the substrate and the binding site is only part of the picture. As the substrate
binds, enzymes undergo a conformational change (“induced fit”) that repositions the side chains of the
amino acids in the active site and increases the number of binding interactions. The induced fit model for
substrate binding recognizes that the substrate binding site is not a rigid “lock” but rather a dynamic
surface created by the flexible overall three-dimensional structure of the enzyme.
The function of the conformational change induced by substrate binding, the induced fit, is usually to
reposition functional groups in the active site in a way that promotes the reaction, improves the binding
site of a co-substrate, or activates an adjacent subunit through cooperativity. For example, consider the
large conformational changes that occur in the actin fold of glucokinase when glucose binds. The induced
fit involves changes in the conformation of the whole enzyme that close the cleft of the fold, thereby
improving the binding site for ATP, and excluding water (which might interfere with the reaction) from
the active site. Thus, the multiple interactions between the substrate and the enzyme in the catalytic site
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serve both for substrate recognition and for initiating the next stage of the reaction, formation of the
transition state complex.

C. The Transition State Complex

For a reaction to occur, the substrates undergoing the reaction need to be activated. If the energy levels of
a substrate are plotted as the substrate is progressively converted to product, the curve will show a
maximum energy level that is higher than that of either the substrate or the product. This high energy
level occurs at the transition state. For some enzyme-catalyzed reactions, the transition state is a condition
in which bonds in the substrate are maximally strained. For other enzyme-catalyzed reactions, the
electronic configuration of the substrate becomes very strained and unstable as it enters the transition
state. The highest energy level corresponds to the most unstable substrate configuration, and the condition
in which the changing substrate molecule is most tightly bound to participating functional groups in the
enzyme. The difference in energy between the substrate and the transition state complexis called the
activation energy.

The "Transition state theory" (Henry Eyring, 1935) states: Reactant molecules must overcome an energy
barrier and pass through an activated complex before proceeding on to the product of the reaction.
According to transition state theory, the overall rate of the reaction is determined by the number of

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molecules acquiring the activation energy necessary to form the transition state complex. Enzymes
increase the rate of the reaction by decreasing this activation energy. They use various catalytic strategies,
such as electronic stabilization of the transition state complex or acid-base catalysis, to obtain this
decrease.
Once the transition state complex is formed, it can collapse back to substrates or decompose to form
products. The enzyme does not change the initial energy level of the substrates or the final energy level of
the products.

Enzyme Catalytic Mechanisms

Enzymes achieve their enormous rate of accelerations via the same catalytic mechanisms used by
chemical catalysts. The types of catalytic mechanisms enzymes employ have been largely classified as
1 Acid-base catalysis
2 Covalent catalysis
3 Metal ion catalysis
4 Proximity and orientation catalysis
5 Preferential binding of the transition state complex

1. Acid-Base Catalysis

Enzymatic processes utilizing acid and base catalysis is involved with proton transfer; in practice there are
very few enzymes that do not have acidic or basic catalytic groups at their active sites. However, unlike
organic reactions which can be carried out under a very wide range of pH conditions to suit the reaction,
enzymes have a strict limitation in that they operate at physiological pH, in the range 5–9. Given this
restriction, and the fairly small range of amino acid side chains available for participation in acid/base
chemistry, a remarkably diverse range of acid/base chemistry is achieved.

Amino acid side chains used for acid/base catalysis.

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General acid catalysis takes place when the substrate is protonated by a catalytic residue which in turn
gives up a proton. The active site acidic group must, therefore, be protonated at physiological pH but its
pKa must be just above (i.e. in the range 7–10). If the pKa of a side chain was in excess of 10 then it
would become thermodynamically unfavorable to transfer a proton.

General acid catalysis

General base catalysis takes place either when the substrate is deprotonated, or when water is
deprotonated prior to attack on the substrate.

General base catalysis

Enzyme active site bases must therefore be deprotonated at physiological pH but have pKa values just
below. In some cases, the pKa values of active site acidic and basic groups can be strongly influenced by
their micro-environment. Thus, for example, the enzyme acetoacetate decarboxylase contains an active
site lysine residue which forms an imine linkage with its substrate – its pKa value was found to be 5.9,
much less than the expected value of about 9. When an active site peptide was obtained containing the
catalytic lysine, it was found to be adjacent to another lysine residue. So it is likely that the proximity of
another positively charged residue would make the protonated form thermodynamically less favourable,
and hence reduce the pKa. The same effect can be observed in the pKa values of ethylenediamine, where
the pKa for the monoprotonated form is 10.7 as usual, but the pKa for the doubly protonated form is 7.5.

Abnormally low lysine pKa in acetoacetate decarboxylase

Similarly, if a charged group was involved in a salt bridge with an oppositely charged residue, its pKa
would be altered, or if it was in a hydrophobic region of the active site, which would destabilize the
charged form of the group. Finally, it is worth noting that histidine, whose side chain contains an
imidazole ring of pKa 6–8, can act either as an acidic or a basic residue, depending on its particular local
pKa, making it a versatile reagent for enzymatic acid-base chemistry.
Acid-base catalysis by enzymes is made effective by the optimal positioning of the active site acid/base
groups in close proximity to the substrate, generating a high effective concentration of the enzyme
reagent. This can be illustrated in the case of glycoside hydrolysis. The mechanism of the non- enzymatic
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reaction involves protonation of the glycosidic group by external acid to form a good leaving group,
followed by formation of an oxonium intermediate. Glycoside hydrolysis can be accelerated dramatically
by positioning an acidic group in close proximity to the glycosidic leaving group. Enzymes which
catalyze glycoside hydrolysis also employ an acidic catalytic group to protonate the glycosidic leaving
group – but the enzymatic reaction is some 30 000-fold faster even than the intramolecular reaction,
suggesting that the enzyme is able to further stabilize the transition state for this reaction.
Enzymes also have the ability to carry out bifunctional catalysis: protonation of the substrate at the same
time as deprotonation in another part of the molecule. An example of bifunctional catalysis is the enzyme
ketosteroid isomerase, whose active site contains two catalytic residues: Asp-38 which acts as a catalytic
base; and Tyr-14 which acts as an acidic group. The mechanism involves the formation of a dienol
intermediate via a concerted step involving simultaneous deprotonation of the substrate by Asp-14 and
protonation of the substrate carbonyl by Tyr-14. In the second step the tyrosinate group acts as a base, and
the substrate is re- protonated by the protonated Asp-38.

Mechanism for ketosteroid isomerase

Bifunctional catalysis is thought to make possible the deprotonation of substrates with apparently high
pKa values. Thus, in the given example, deprotonation adjacent to a ketone in solution to form an enolate
species would involve removal of a proton of pKa 18–20, which would be impractical at pH 7. However,
simultaneous protonation to form an enol intermediate makes the reaction thermodynamically much more
favourable.
Also, enzymes which bind metal cofactors such as Zn2+and Mg2+can utilize their properties as Lewis
acids, i.e. electron pair acceptors. An example is the enzyme thermolysin. In this enzyme, Glu-143 acts as
an active site base to deprotonate water for attack on the amide carbonyl, which is at the same time
polarized by co-ordination by an active site Zn 2+ ion. The protonated glutamic acid probably then acts as
an acidic group for the protonation of the departing amine.

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 Mechanism for thermolysin. R, peptide chain

2. Covalent (Nucleophilic) Catalysis

Nucleophilic (or covalent) catalysis is a type of catalysis not often seen in organic reactions, but quite
often utilized by enzymes. It involves nucleophilic attack of an active site group on the substrate, forming
a covalent bond between the enzyme and the substrate, and hence a covalent intermediate in the reaction
mechanism. This is a particularly effective strategy for enzyme-catalyzed reactions for two reasons.
 An enzyme is able to position an active site nucleophile in close proximity and correctly aligned
to attack its substrate, generating a very high effective concentration of nucleophile.
 Since enzyme active sites are often largely excluded from water molecules, an enzyme active site
nucleophile is likely to be ‘desolvated’.
o A charged nucleophile in aqueous solution would be surrounded by several layers of water
molecules, which greatly reduce the polarity and effectiveness of the nucleophile.
However, a desolvated nucleophile at a water-excluded active site will be a much more
potent nucleophile than its counterpart in solution.

Enzymes have a range of potential nucleophiles available to them.

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 Amino acid side chains used for nucleophilic catalysis in enzymatic reactions

Probably the best nucleophile available to enzymes is the thiol side chain of cysteine, which is seen
operating in proteases and acyl transfer enzymes. The Ɛ-amino group of lysine is used in a number of
cases to form imine linkages with ketone groups in substrates, as in the example of acetoacetate
decarboxylase. This enzyme catalyzes the decarboxylation of acetoacetate to acetone.

Mechanism for acetoacetate decarboxylase

The other nitrogen nucleophile available to enzymes is the versatile imidazole ring of histidine. This
group is more often used for acid/base chemistry, but is occasionally used as a nucleophile in, for
example, phosphotransfer reactions. Finally, enzymes have oxygen nucleophiles available in the form of
the hydroxyl groups of serine, threonine and tyrosine, and the carboxylate groups of aspartate and
glutamate.

3. Metal Ion catalysis

Close to 1/3rd of all known enzymes require the presence of metal ions for catalytic activity. This group of
enzymes include the metalloenzymes, which contain tightly bound metal ion cofactors, most commonly
transition metal ions such as Fe2+, Fe3+, Cu2+, Mn2+, or Co2+. These catalytically essential metal ions are
distinct from ions such asNa+, K+, or Ca2+, which often play a structural rather than a catalytical role in
enzymes. Although ions such as Mg2+ and Zn2+ may be either structural or catalytic.
Metal ions participate in the catalytic process in three major ways:

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  By binding to substrates to orient them properly for reaction
 By mediating oxidation-reduction reactions through reversible changes in the metal ion’s
oxidation state
 By electrostatically stabilizing or shielding negative charges.
In many metal ion-catalyzed reactions, the metal ion acts in much the same way as a proton to neutralize
negative charge. Yet metal ions are often much more effective catalysts than protons because metal ions
can be present in high concentrations at neutral pH and may have charges greater than +1.
A metal ion’s charge also makes its bound water molecules more acidic than free H 2O and therefore a
source of nucleophilic OH- ions even below neutral pH. An example of this phenomenon occurs in the
catalytic mechanism of carbonic anhydrase.

CO2 + H2O HCO3- + H+

Carbonic anhydrase contains an essential Zn2+ ion that is implicated in the enzyme’s catalytic mechanism
as follows:
1. Zn2+ is tetrahedrally coordinated by three invariant His side chains an H 2O molecule. This Zn2+-
polarized H2O ionizes through base catalysis that is facilitated by a fourth His residue.
2. The resulting Zn2+-bound OH- nucleophilically attacks the nearby enzymatically bound CO2,
thereby converting it to HCO-3.
3. The catalytic site is then regenerated by the binding and ionization of another H2O at the Zn2+.

4. Catalysis by Proximity and Orientation/Entropy Reduction

Reactions that are intramolecular abound in organic reactions i.e., they involve two or more functional
groups within the same molecule, rather than functional groups in different molecules. Intramolecular
reactions generally proceed much more rapidly and under much milder reaction conditions than their
intermolecular counterparts, which makes sense since the two reacting groups are already ‘in close
proximity’ to one another.
A useful concept in quantitating proximity effects is that of effective concentration. In order to define the
effective concentration of a participating group (nucleophile, base, etc.), there is a need to compare the
rate of the intramolecular reaction with the rate of the corresponding intermolecular reaction where the
reagent and the participating group are present in separate molecules. The effective concentration of the
participating group is defined as the concentration of reagent present in the intermolecular reaction
required to give the same rate as the intramolecular reaction.
The same effect operates in enzyme active sites, and is a major factor in enzyme catalysis. The binding of
substrates and cofactors at an enzyme active site of defined three-dimensional structure brings the
reactants into close proximity to one another and to the enzyme active site functional groups. This
increases the probability of correct positioning for reaction to take place, so it speeds up the reaction. An
important factor in this analysis is that the enzyme structure is already held rigidly (or fairly rigidly at
least) in the correct conformation for binding and catalysis. The catalytic power of proximity effects, or
‘pre-organization’, has been demonstrated by the synthesis of host– guest systems which mimic enzymes
by binding substrates non-covalently.

Reactants must come together with the proper spatial relationship for a reaction to occur.
By simply binding their substrates, enzymes facilitate their catalyzed reactions in four ways:

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  Enzymes bring substrates into contact with their catalytic groups and in reactions involving more
than one substrate, with each other. Although studies have shown that this alone can only enhance
reaction rates by a factor of 5.
 Enzymes bind their substrates in the proper orientations for reactions. Molecules are not equally
reactive in all directions. Rather, they react more readily if they have the proper relative
orientation. Enzymes thus, align their substrates and their catalytic groups in order to optimize
reactivity.
 Charge groups may help stabilize the transition state of the reaction, a phenomenon known as
electrostatic catalysis. The charge distribution around the active sites may also guide polar
substrates toward their binding site.
 Enzymes prevent the relative translational and rotational motions of their substrates and their
catalytic groups. This effect can promote rate enhancements of up to approx. 107.

Bringing substrates and catalytic groups together in a reactive orientation orders them and therefore
has a substantial entropic penalty. The free energy required to overcome this entropy loss is supplied
by the binding energy of the substrate(s) to the enzyme and contribute to the decreased activation
energy (∆Gǂ).

5. Transition State Stabilization

Like all catalysts, enzymes reduce the activation energy of the reaction by stabilizing the transition state
for the reaction. But in the case of enzymes, there are usually several transition states involved in an
enzymatic reaction since an enzyme binds its substrate reversibly at the enzyme active site.
In order to achieve optimal catalysis, enzymes selectively bind the transition state, rather than the
substrate. Hence, it is not advantageous for enzymes to bind their substrates too tightly. This is evident
when one looks at substrate binding constants. Typical KM values for enzymes are in the mm–µm range
(10-3 - 10-6M), whereas dissociation constants for binding proteins and antibodies whose function is to
bind small molecules tightly are in the range nm–pm (10-9 - 10-12M).

6. Involvement of protein dynamics in enzyme catalysis

Our understanding of such high rates of catalysis in enzymes is still incomplete. Chemical models for
enzyme catalysts, and catalytic antibodies which function via transition state stabilization, are orders of
magnitude less active catalysts than in situ. Other factors are therefore likely used by the enzymes to
achieve the high rates of catalysis.

Examination of protein structure in solution by nuclear magnetic resonance (NMR) spectroscopy has
revealed that there is a significant amount of internal motion in a protein, on a timescale of 1–10 ns. Such
internal motion could transmit kinetic energy from a distant part of the protein to the active site in order to
assist in catalysis. It has been proposed that dynamic fluctuations in the protein structure are used by
enzymes to organize the enzyme–substrate complex into a reactive conformation.
The role of protein dynamics in enzyme catalysis is therefore a topic of considerable interest. Studies of
some selected enzymes have revealed, in a number of cases, that amino acid residues distant from active
sites can have a dramatic effect on enzyme activity. Replacement of Gly-120 to valine in dihydrofolate
reductase disrupts the internal motion of a protein loop, and so interferes with the conversion of the
ternary complex to the reactive conformation, hence this step becomes partially rate-limiting, reducing the

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rate of hydride transfer 500-fold. Thus, protein dynamics may have an important role to play in enzyme
catalysis.

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