Lecture Note (MBC 210) (Osagie Ododo)

ENZYMES
Many chemical reactions can occur spontaneously; others require to be catalyzed to proceed at a significant
rate. Catalysts are molecules that reduce the magnitude of the energy barrier required to be overcame for a
substance to be converted chemically into another. Thermodynamically, the magnitude of this energy barrier
can be conveniently expressed in terms of the free-energy change.

A biochemical reaction is the chemical reactions that comprise the metabolism of all living cells, and need to
be catalyzed to proceed at the pace required to sustain life. Such life catalysts are the enzymes. Each one of
the biochemical reactions of the cell metabolism requires to be catalyzed by one specific enzyme.

Enzymes are protein molecules that have evolved to perform efficiently under the mild conditions required to
preserve the functionality and integrity of the biological systems. Enzymes can be considered then as
catalysts that have been optimized through evolution to perform their physiological task upon which all
forms of life depend. Enzymes are the biological macromolecules which speed up the rate of
biochemical reactions without undergoing any change. They are also called as biological catalysts. An
enzyme is a highly selective catalyst that greatly accelerates both the rate and specificity of metabolic
reactions.
As depicted in figure below, catalysts reduce the magnitude of this barrier by virtue of its interaction with the
substrate to form an activated transition complex that delivers the product and frees the catalyst.

Fig: Mechanism of catalysis. Ea and Ea_ are the energies of activation of the uncatalyzed and catalyzed
reaction. ΔG is the free energy change of the reaction
Most of the characteristics of enzymes as catalysts derive from their molecular structure. Enzymes are
proteins composed by a number of amino acid residues that range from 100 to several hundreds. These
amino acids are covalently bound through the peptide bond that is formed between the carbon atom of the
carboxyl group of one amino acid and the nitrogen atom of the α-amino group of the following. According to
the nature of the R group, amino acids can be non-polar (hydrophobic) or polar (charged or uncharged) and
their distribution along the protein molecule determines its behavior

Scheme of peptide bond formation between two adjacent α-amino acids

Properties of Enzymes
1. Nearly all enzymes are proteins, although a few catalytically active RNA molecules have been
identified.
2. Enzyme catalyzed reactions usually take place under relatively mild conditions (temperatures well
below 100 C, atmospheric pressure and neutral pH) as compared with the corresponding chemical
reactions.
3. Enzymes are catalysts that increase the rate of a chemical reaction without being changed themselves
in the process.
4. Enzymes are highly specific with respect to the substrates on which they act and the products that
they form.
5. Enzyme activity can be regulated, varying in response to the concentration of substrates or other
molecules.
6. They function under strict conditions of temperature and pH in the body

Coenzymes and Prosthetic Groups

Many enzymes require the presence of small, non-protein units or cofactors to carry out their particular
reaction. Cofactors may be either one or more inorganic ions, such as Zn or Fe or a complex organic
molecule called a coenzyme.
A metal or coenzyme that is covalently attached to the enzyme is called a Prosthetic group (heme in
hemoglobin). Some coenzymes, such as NAD , are bound and released by the enzyme during its catalytic
cycle and in effect function as co-substrates. Many coenzymes are derived from vitamin precursors.

Holo Enzymes and Apo Enzymes

A complete catalytically-active enzyme together with its coenzyme or metal ion is called a holoenzyme. The
protein part of the enzyme on its own without its cofactor is termed an apoenzyme.

Active Site of Enzymes

The active site of an enzyme is the region that binds the substrate and converts it into product. It is usually a
relatively small part of the whole enzyme molecule and is a three dimensional entity formed by amino acid
residues that can lie far apart in the linear polypeptide chain.

The active site is often a cleft or crevice on the surface of the enzyme that forms a predominantly nonpolar
environment which enhances the binding of the substrate. The substrate(s) is bound in the active site by
multiple weak forces (electrostatic interactions, hydrogen bonds, van der Waals bonds, hydrophobic
interactions; and in some cases by reversible covalent bonds.

Substrate Specificity of Enzyme

The properties and spatial arrangement of the amino acid residues forming the active site of an enzyme will
determine which molecules can bind and be substrates for that enzyme. Substrate specificity is often
determined by changes in relatively few amino acids in the active site. This is clearly seen in the three
digestive enzymes trypsin, chymotrypsin and elastase.

Mechanism of Action of Enzymes

The substrate(s) is bound in the active site by multiple weak forces which result into the enzyme-substrate
complex. Once bound, active residues within the active site of the enzyme act on the substrate molecule to
transform it first into the transition state complex and then into product, which is released. The enzyme is
now free to bind another molecule of substrate and begin its catalytic cycle again.

The Substrate-Enzyme Binding

Originally two models were proposed to explain how an enzyme binds its substrate. The reality is that
different enzymes show features of both models, with some complementarity and some conformational
change

1. The Lock and Key Model

In the lock-and-key model proposed was proposed by Emil Fischer in 1894. According to the model, the
shape of the substrate and the active site of the enzyme are thought to fit together like a key into its lock. The
two shapes are considered as rigid and fixed, and perfectly complement each other when brought together in
the right alignment.

2. The Induced Fit Model

The favourable model of enzyme-substrate interaction is called the induced-fit model. The induced-fit model
was proposed by Daniel E. Koshland, Jr., in 1958. It states that the binding of substrate induces a
conformational change in the active site of the enzyme. In addition, the enzyme may distort the substrate,
forcing it into a conformation similar to that of the transition state. For example, the binding of glucose to
hexokinase induces a conformational change in the structure of the enzyme such that the active site assumes
a shape that is complementary to the substrate (glucose) only after it has bound to the enzyme.

There are four possible major mechanisms of catalysis:

a) Catalysis by Bond Strain
The induced structural rearrangements in this type of catalysis produce strained substrate bonds that attain
transition state more easily. The new conformation forces substrate atoms and catalytic groups like aspartate
into conformations that strain substrate bonds.
b) Covalent Catalysis
The substrate is oriented to active place on the enzymes in such a manner that a covalent intermediate
develops between the enzyme and the substrate, in catalysis that occurs by covalent mechanisms. The best
example of this involves proteolysis by serine proteases that have both digestive enzymes and various
enzymes of the blood clotting cascade. These proteases possess an active site serine whose R group hydroxyl
generates a covalent bond with a carbonyl carbon of a peptide bond and results in the hydrolysis of the
peptide bond.
c) Catalysis Involving Acids and Bases
Other mechanisms add to the completion of catalytic events which are launched by strain mechanisms such
as the usage of glutamate as a general acid catalyst.
d) Catalysis by Orientation and Proximity
Enzyme-substrate interactions induce reactive groups into proximity with one another. Also, groups like
aspartate are chemically reactive, and their proximity towards the substrate favours their involvement in
catalysis.

Isoenzymes
Isoenzymes are different forms of an enzyme which catalyze the same reaction, but which exhibit different
physical or kinetic properties, such as isoelectric point, pH optimum, substrate affinity or effect of inhibitors.

Different isoenzyme forms of a given enzyme are usually derived from different genes and often occur in
different tissues of the body. An example of an enzyme which has different isoenzyme forms is lactate
dehydrogenase (LDH) which catalyzes the reversible conversion of pyruvate into lactate in the presence of
the coenzyme NADH. LDH is a tetramer of two different types of subunits, called H and M, which have
small differences in amino acid sequence. The two subunits can combine randomly with each other, forming
five isoenzymes that have the compositions H , H M, H M , HM and M . The five isoenzymes can be
resolved electrophoretically.

Nomenclature of Enzymes
Many enzymes are named by adding the suffix ‘-ase’ to the name of their substrate. Example. Urease is the
enzyme that catalyzes the hydrolysis of urea, and fructose- 1,6-bisphosphatase hydrolyzes fructose-1,6-
bisphosphate.
However, other enzymes, such as trypsin and chymotrypsin, have names that do not denote their substrate.
Some enzymes have several alternative names. To rationalize enzyme names, a system of enzyme
nomenclature has been internationally agreed. This system places all enzymes into one of six major classes
based on the type of reaction catalyzed. Each enzyme is then uniquely identified with a four-digit
classification number. Example: Trypsin has the Enzyme Commission (EC) number 3.4.21.4, where
1. The first number (3) denotes that it is a hydrolase
2. The second number (4) that it is a protease that hydrolyzes peptide bonds
3. The third number (21) that it is a serine protease with a critical serine residue at the active site, and
4. The fourth number (4) indicates that it was the fourth enzyme to be assigned to this class.
For comparison, chymotrypsin has the EC number 3.4.21.1, and elastase 3.4.21.36.

Classification of Enzymes
Earlier, enzymes were assigned names based on the one who discovered it. With further researches,
classification became more comprehensive.
According to the International Union of Biochemists (I U B), enzymes are divided into six functional classes
and are classified based on the type of reaction in which they are used to catalyze. The six kinds of enzymes
are hydrolases, oxidoreductases, lyases, transferases, ligases and isomerases

1. Oxidoreductases
Catalyze oxidation-reduction reactions where electrons are transferred. These electrons are usually in the
form of hydride ions or hydrogen atoms. The most common name used is a dehydrogenase and sometimes
reductase is used. An oxidase is referred to when the oxygen atom is the acceptor.
 2. Transferases
Catalyze group transfer reactions. The transfer occurs from one molecule that will be the donor to another
molecule that will be the acceptor. Most of the time, the donor is a cofactor that is charged with the group
about to be transferred.Example: Hexokinase used in glycolysis.

3. Hydrolases
Catalyze reactions that involve hydrolysis. It usually involves the transfer of functional groups to water.
When the hydrolase acts on amide, glycosyl, peptide, ester, or other bonds, they not only catalyze the
hydrolytic removal of a group from the substrate but also a transfer of the group to an acceptor compound
For example: Chymotrypsin.

4. Lyases
Catalyze reactions where functional groups are added to break double bonds in molecules or the reverse
where double bonds are formed by the removal of functional groups. For example: Fructose bisphosphate
aldolase used in converting fructose 1,6- bisphospate to G3P and DHAP by cutting C-C bond.

5. Isomerases
Catalyze reactions that transfer functional groups within a molecule so that isomeric forms are produced.
These enzymes allow for structural or geometric changes within a compound. For example: phosphoglucose
isomerase for converting glucose 6-phosphate to fructose 6-phosphate. Moving chemical group inside same
substrate.

6. Ligases
They are involved in catalysis where two substrates are ligated and the formation of carbon-carbon, carbon-
sulfide, carbon-nitrogen, and carbon-oxygen bonds due to condensation reactions. These reactions are
coupled to the cleavage of ATP.

Significance of Enzymes
1. In the absence of an enzyme, biochemical reactions hardly proceed at all, whereas in its presence the
rate can be increased up to 10 -fold. Thus, they are crucial for normal metabolism of living systems.

2. Besides in the body, extracted and purified enzymes have many applications.

Medical applications of enzymes include:

1. To treat enzyme related disorders.
2. To assist in metabolism
3. To assist in drug delivery.
4. To diagnose & detect diseases.
5. In manufacture of medicines.
Industrial applications of enzymes include:
1. Amylase, lactases, cellulases are enzymes used to break complex sugars into simple sugars.
2. Pectinase like enzymes which act on hard pectin is used in fruit juice manufacture.
3. Lipase enzymes act on lipids to break them in fatty acids and glycerol. Lipases are used to remove
stains of grease, oils, butter.
4. Enzymes are used in detergents and washing soaps.
5. Protease enzymes are used to remove stains of protein nature like blood, sweat etc.

Factors Affecting Enzyme Activity

There are several factors that affect the speed of an enzyme’s action, such as the concentration of the
enzyme, the concentration of the substrate, temperature, hydrogen ion concentration (pH), and the presence
of inhibitors.

1. Concentration of Enzyme
As the concentration of the enzyme is increased, the velocity of the reaction proportionately increases. This
property is used for determining the activities of serum enzymes during the diagnosis of diseases.

2. Concentration of Substrate
In the presence of a given amount of enzyme, the rate of enzymatic reaction increases as the substrate
concentration increases until a limiting rate is reached, after which further increase in the substrate
concentration produces no significant change in the reaction rate. At this point, so much substrate is present
that essentially all of the enzyme active sites have substrate bound to them. In other words, the enzyme
molecules are saturated with substrate. The excess substrate molecules cannot react until the substrate
already bound to the enzymes has reacted and been released (or been released without reacting).

3. Effect of Temperature
The protein nature of the enzymes makes them extremely sensitive to thermal changes. Enzyme activity
occurs within a narrow range of temperatures compared to ordinary chemical reactions. As you have seen,
each enzyme has a certain temperature at which it is more active. This point is called the optimal
temperature, which ranges between 37 to 40C°.

The enzyme activity gradually lowers as the temperature rises more than the optimal temperature until it
reaches a certain temperature at which the enzyme activity stops completely due to the change of its natural
composition.

On the other hand, if the temperature lowers below the optimal temperature, the enzyme activity lowers until
the enzyme reaches a minimum temperature at which the enzyme activity is the least. The enzyme activity
stops completely at 0C°, but if the temperature rises again, then the enzyme gets reactivated once more.

4. Effect of pH
The potential of hydrogen (pH) is the best measurement for determining the concentration of hydrogen ion
(H) in a solution. It also determines whether the liquid is acidic, basic or neutral. Generally, all liquids
with a pH below 7 are called acids, whereas liquids with a pH above 7 are called bases or alkalines.
Liquids with pH 7 are neutral and equal the acidity of pure water at 25 C°. You can determine pH of any
solution using the pH indicators

Enzymes are protein substances that contain acidic carboxylic groups (COOH) and basic amino groups (NH
So, the enzymes are affected by changing the pH value. Each enzyme has a pH value that it works at with
maximum efficiency called the optimal pH. If the pH is lower or higher than the optimal pH, the enzyme
activity decreases until it stops working. For example, pepsin works at a low pH, i.e, it is highly acidic, while
trypsin works at a high pH, i.e, it is basic. Most enzymes work at neutral pH 7.4.

Enzyme PH activity

5. Effect of Activators
Some of the enzymes require certain inorganic metallic cations, like Mg, Mn,Zn, Ca, Co, Cu, Na, K etc., for
their optimum activity. Rarely, anions are also needed for enzyme activity, e.g. a chloride ion (CI) for
amylase

Enzyme inhibition
An enzyme inhibitor is a molecule that binds to an enzyme and decreases its activity. By binding to enzymes'
active sites, inhibitors reduce the compatibility of substrate and enzyme and this leads to the inhibition of
Enzyme-Substrate complexes' formation, preventing the catalysis of reactions and decreasing (at times to
zero) the amount of product produced by a reaction. It can be said that as the concentration of enzyme
inhibitors increases, the rate of enzyme activity decreases, and thus, the amount of product produced is
inversely proportional to the concentration of inhibitor molecules. Not all molecules that bind to enzymes are
inhibitors; enzyme activators bind to enzymes and increase their enzymatic activity, while enzyme substrates
bind and are converted to products in the normal catalytic cycle of the enzyme.

The binding of an inhibitor can stop a substrate from entering the enzyme's active site and/or hinder the
enzyme from catalyzing its reaction. Inhibitor binding is either reversible or irreversible. Irreversible
inhibitors usually react with the enzyme and change it chemically (e.g. via covalent bond formation). These
inhibitors modify key amino acid residues needed for enzymatic activity. In contrast, reversible inhibitors
bind non-covalently and different types of inhibition are produced depending on whether these inhibitors
bind to the enzyme, the enzyme-substrate complex, or both.
Enzyme inhibitors also occur naturally and are involved in the regulation of metabolism. For example,
enzymes in a metabolic pathway can be inhibited by downstream products. This type of negative feedback
slows the production line when products begin to build up and is an important way to maintain homeostasis
in a cell. Other cellular enzyme inhibitors are proteins that specifically bind to and inhibit an enzyme target.
This can help control enzymes that may be damaging to a cell, like proteases or nucleases. A well-
characterized example of this is the ribonuclease inhibitor, which binds to ribonucleases in one of the tightest
known protein–protein interactions. Natural enzyme inhibitors can also be poisons and are used as defenses
against predators or as ways of killing prey.

Reversible inhibitors
Types of reversible inhibitors
Reversible inhibitors attach to enzymes with non-covalent interactions such as hydrogen bonds, hydrophobic
interactions and ionic bonds. Multiple weak bonds between the inhibitor and the active site combine to
produce strong and specific binding. In contrast to substrates and irreversible inhibitors, reversible inhibitors
generally do not undergo chemical reactions when bound to the enzyme and can be easily removed by
dilution or dialysis.

There are four kinds of reversible enzyme inhibitors. They are classified according to the effect of varying
the concentration of the enzyme's substrate on the inhibitor

1. Competitive inhibition
In competitive inhibition, the substrate and inhibitor cannot bind to the enzyme at the same time, as shown in
the figure on the right. This usually results from the inhibitor having an affinity for the active site of an
enzyme where the substrate also binds; the substrate and inhibitor compete for access to the enzyme's active
site. This type of inhibition can be overcome by sufficiently high concentrations of substrate (Vmax remains
constant), i.e., by out-competing the inhibitor. However, the apparent Km will increase as it takes a higher
concentration of the substrate to reach the Km point, or half the Vmax. Competitive inhibitors are often
similar in structure to the real substrate (see examples below).

2. Uncompetitive inhibition
In uncompetitive inhibition, the inhibitor binds only to the substrate-enzyme complex. This type of inhibition
causes Vmax to decrease (maximum velocity decreases as a result of removing activated complex) and Km to
decrease (due to better binding efficiency as a result of Le Chatelier's principle and the effective elimination
of the ES complex thus decreasing the Km which indicates a higher binding affinity).

3. Non-competitive inhibition
In non-competitive inhibition, the binding of the inhibitor to the enzyme reduces its activity but does not
affect the binding of substrate. As a result, the extent of inhibition depends only on the concentration of the
inhibitor. Vmax will decrease due to the inability for the reaction to proceed as efficiently, but Km will
remain the same as the actual binding of the substrate, by definition, will still function properly.

4. Mixed inhibition
In mixed inhibition, the inhibitor can bind to the enzyme at the same time as the enzyme's substrate.
However, the binding of the inhibitor affects the binding of the substrate, and vice versa. This type of
inhibition can be reduced, but not overcome by increasing concentrations of substrate. Although it is possible
for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from an allosteric
effect where the inhibitor binds to a different site on an enzyme. Inhibitor binding to this allosteric site
changes the conformation (i.e., tertiary structure or three-dimensional shape) of the enzyme so that the
affinity of the substrate for the active site is reduced.

These types can also be distinguished by the effect of increasing the substrate concentration [S] on the degree
of inhibition caused by a given amount of inhibitor. For competitive inhibition the degree of inhibition is
reduced by increasing [S], for noncompetitive inhibition the degree of inhibition is unchanged, and for
uncompetitive (also called anticompetitive) inhibition the degree of inhibition increases with [S].
Irreversible inhibitors
Irreversible inhibitors usually covalently modify an enzyme, and inhibition can therefore not be reversed.
Irreversible inhibitors often contain reactive functional groups such as nitrogen mustards, aldehydes,
haloalkanes, alkenes, Michael acceptors, phenyl sulfonates, or fluorophosphonates. These nucleophilic
groups react with amino acid side chains to form covalent adducts. The residues modified are those with side
chains containing nucleophiles such as hydroxyl or sulfhydryl groups; these include the amino acids serine
(as in DFP, below), cysteine, threonine, or tyrosine

Reaction of the irreversible inhibitor diisopropylfluorophosphate (DFP) with a serine protease

Irreversible inhibition is different from irreversible enzyme inactivation. Irreversible inhibitors are generally
specific for one class of enzyme and do not inactivate all proteins; they do not function by destroying protein
structure but by specifically altering the active site of their target. For example, extremes of pH or
temperature usually cause denaturation of all protein structure, but this is a nonspecific effect. Similarly,
some non-specific chemical treatments destroy protein structure: for example, heating in concentrated
hydrochloric acid will hydrolyse the peptide bonds holding proteins together, releasing free amino acids.
Uses of inhibitors
Enzyme inhibitors are found in nature and are also designed and produced as part of pharmacology
and biochemistry. Natural poisons are often enzyme inhibitors that have evolved to defend a plant
or animal against predators. These natural toxins include some of the most poisonous compounds
known. Artificial inhibitors are often used as drugs, but can also be insecticides such as malathion,
herbicides such as glyphosate, or disinfectants such as triclosan. Other artificial enzyme inhibitors
block acetylcholinesterase, an enzyme which breaks down acetylcholine, and are used as nerve
agents in chemical warfare.
1. For chemotherapy
2. For Antibiotics
3. For Metabolic control
4. As Pesticides
5. As natural poison for defense against predators