Enzymes

Enzymes are complex protein molecules synthesized in the cells where they act as biological catalysts
that help increase the rate of physico-chemical reactions. The specificity and kinetics of enzymes
come from the great diversity of the protein structure. Enzymes are often known by common names
obtained by adding suffix ‘-ase’ to the name of the substrate or to the reaction that they catalyze. The
journey to discover enzymes was pioneered by Louis Pasteur between 1850 and 1860 with his
fermentation experiment. The term enzyme was coined by W. Kuhne in 1878 In 1897, Eduard
Buchner accidentally discovered that fermentation was catalyzed by a clear juice. It took Arthur
Harden and William Young to show yeast extracts contained two different types of molecules
necessary for fermentation to occur.
It was in 1927 that James Sumner succeeded in purifying and crystallizing the enzyme urease from
beans. It was the same year that investigators came to believing that enzymes were proteins. In the
1930s, John Northrop isolated and characterized a series of digestive enzymes to drive home
Sumner’s conclusion that enzymes are proteins. Thousands of enzymes have been purified and their
structures solved to atomic resolution since then. Although most are enzymes, recent studies have
shown some RNA molecules to exhibit enzymatic activity.

Properties of Enzymes
Enzymes have four important properties; these include:
i. Catalytic properties
ii. Specificity
iii. Reversibility
iv. Sensitivity to heat, temperature, and pH
All enzymes are proteins synthesized within the cell. As such, their physical and chemical properties
are similar to that of proteins. They become biologically inactive and denature when they come in
contact with strong acids, bases, organic solvents, heat, and agitation. One essential property of
enzymes is that they speed up the rate of biochemical reactions but remain unchanged without loss of
activity.
Enzymes have a preferred substrate on which they act. Some enzymes have absolute specificity,
certain enzymes exhibit organic group specificity, some show broad specificity, and some optical
specificity. An enzyme recognizes its specific substrate and reacts with it to form product and gets
regenerated at the end of the reaction.
Enzymes lower the activation energy required for a reaction to occur. This allows a larger number of
molecules to react at a given temperature. The efficiency with which an enzyme acts on its substrate
is known as its turnover rate. This is the number of substrate molecules converted into the product by
a single molecule of enzyme per unit time.
Enzymes catalyzes both forward and backward reactions to reach a state of equilibrium. They control
and regulate the cellular process at low temperatures with maximum output. The activity of enzymes
depends on the pH of the medium. Each is most active at a specific pH. Most intracellular enzymes
function at near neutral pH.

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 Classification of Enzymes
Although the International Enzyme Commission has established a uniform naming system for
enzymes, the common names do not follow a consistent pattern. With the exception of the names of
some older enzymes (trypsin and pepsin.), all enzymes have names ending with ‘-ase.’ Enzymes are
named according to their activity or function.
According to the International Enzyme Commission, enzymes are classified into six major classes as
tabulated below.
Table 1: Major Classes of Enzymes and the Types of Reactions Catalyzed
Enzyme class Nature of reaction Major type of enzymes with their specific reactions

Oxido-reductase Biological oxidation and reduction 1. Dehydrogenases: catalyze the removal of 2 atoms of
hydrogen
2. Oxidases: these catalyze reduction of oxygen
3. Oxygenases: catalyze incorporation of molecular
oxygen into the substrate
4. Oxidative deaminases: catalyze the oxidation of
amino compounds with the formation of NH3
5. Hydroxylases: these introduce OH groups
6. Peroxidases: they use H2O2 as oxidants
Transferase Effecting exchange of groups 1. Aminotransferases: catalyze exchange of amino and
between two substrates: keto groups between amino and keto acid
AB+CD↔AC+BD 2. Kinases: catalyze the transfer of PO4 radical
3. Acyltransferases: catalyze the transfer of acyl/acetyl
group to a suitable acceptor
4. Glycosyltransferases: they transfer glycosyl groups
Hydrolases They catalyze hydrolysis 1. Peptidases: catalyze hydrolysis of peptide bonds
reactions: 2. Glycosidases: catalyze hydrolysis of glycosidic
AB+H20↔AOH+HB bonds
3. Esterases: catalyze hydrolysis of carboxylic esters
4. Phosphatases: catalyze hydrolysis of phosphate
groups
5. Phosphodiesterases: catalyze hydrolysis of
phosphoric acid esters
6. Deaminases: catalyze hydrolysis of amines
7. Deamidases: catalyze hydrolysis of amides
Lyases Remove groups from substrates 1. Decarboxylases
non-hydrolytically 2. Aldolases
AB↔A+B 3. Dehydratases
Isomerases Catalyze isomerization of 1. Racemases
substrates 2. Epimerases

Ligases Catalyze joining together of two Synthases; bring about the formation of C-O, C-S, C-N, or
molecules coupled with the C-C bonds.
breakdown of a pyrophosphate Reactions require expenditure of energy with simultaneous
bond in ATP cleavage of ATP

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Mechanism of Enzyme Reaction
The ability of enzymes to lower the activation energy of a biochemical reaction is due to their
structure. Enzymes are large proteins with complex, highly ordered, 3D shapes produced by physical
and chemical interactions between their amino acids. Each enzyme has its own conformation with
ridges, grooves, and pockets that are lined with specific amino acids. The particular pockets involved
in catalyzing a reaction are called the active sites of the enzyme. The active site is the catalytic site
which reacts with the substrate.
According to Koshland, an enzyme consists of essentially four categories of amino acids, including:
a. Catalytic residues: these are the amino acids at the catalytic site which make and break
chemical bonds. They participate in the catalytic activity.
b. Binding residues: these amino acids hold the substrate in place while catalysis is taking place.
c. Structural residues: these amino acids hold the active site in the correct shape so that it can
function properly.
d. Non-essential residues: these amino acids have no specific function. They are often near the
surface of the enzyme and can be removed or replaced without loss of function.
The substrates have shapes that allow them fit into the active sites of the enzyme. The fit may not be
perfect at first, but as the substrate gradually slips into the active site, it is induced (induced-fit theory).
This induced fit, together with temporary bonds that form between the substrate and the amino acids
lining the active site, weakens the existing bonds within the substrate molecules and allows them to
be easily broken. New bonds are easily formed as substrates are brought close in the right orientation.
The enzyme-substrate complex, formed temporarily in the course of the reaction, then dissociates to
yield products and the free unaltered enzyme. This model of how enzymes work is known as the lock-
and-key model of enzyme activity. The following Table shows the factors that contribute to lowering
of the activation energy and accelerating rate of biochemical reaction.
Table 2: Mechanisms that Contribute to the Catalytic Efficiency of Enzymes
Mechanisms Description of catalysis

Proximity effects Temporary binding of substrates close to each on an enzyme increases the chance of a
reaction

Orientation effects Reactions are held by the enzyme in such a way that the bonds are exposed to attack
and a transition is readily achieved

Strain effects Enzyme may induce strain or distortion in the susceptible bond of the substrate
molecule, making the bond easier to break

Acid-base catalysis Acidic and basic amino acids in the enzyme facilitate the transfer of electrons to and
from the substrates

Covalent catalysis Enzyme may combine with the substrate to form an unstable covalent intermediate
that readily undergoes reactions to form the products

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 Microenvironment Hydrolytic amino acids create a water-free zone in which non-polar substrates may
effects react more easily

The Catalytic Cycle of Enzyme (from Campbell & Reece)

Enzyme Inhibition
Most enzymes are sensitive to inhibition by specific agents that interfere with the binding of a
substrate at the active site or with the conversion of the enzyme-substrate complex into products.
These agents may include drugs, poisons, etc. Inhibition occurs in a variety of ways, but is broadly
classified into: reversible and irreversible inhibitions.
1. Irreversible Inhibition: Some enzymes have a thiol (SH) group at the active site making them
reactive to compounds such as iodoacetate (CH2I.COOH) or mercurial thereby forming covalent
derivatives. This kind of bonding causes inactivation of the enzyme. It is irreversible as the
inhibitor cannot be released by any means. The inhibition is proportional to the concentration of
the inhibitor.
E-S + ICH2COOH → E-S-CH2COOH + HI
Examples of this inhibitor are shown in the following Table:

Inhibitor Enzyme group that combines with inhibitor

Cyanide Fe, Cu, Zn, other transition metals

p-Mercury benzoate Sulfhydryl

Diisopropylfluorophosphate Serine hydroxyl

Iodoacetate Sulfhydryl, imidazole, carboxyl, thioether

Irreversible inhibitors often provide clues to the nature of the active site. For example, enzymes
inhibited by iodoacetamide frequently have a cysteine in the active site, and the cysteinyl sulfhydryl
group often plays an important role in the catalytic mechanism.
2. Reversible Inhibition: Many inhibitors bind with enzymes reversibly. They affect the
equilibrium constant of the reaction. There are three types of reversible inhibition.
a. Competitive Inhibition: In this type of inhibition, both the inhibitor and the substrate compete
for the same active site of the enzyme, but the inhibitor has greater affinity. The effect of
inhibition can be overcome by increasing the substrate concentration. In such cases, the
inhibitor is structurally related to the substrate and bind with the enzyme decreasing the
effective concentration of the enzyme. The inhibitor is prevented from binding if the active
site is already occupied by the substrate. An example of this type of inhibition is succinic
dehydrogenase which converts succinate to fumaric acid. If malonic acid (analogous to the
structure of succinic acid) is added to the reaction, the activity of succinic dehydrogenase falls.
The activity can be restored by increasing the concentration of succinic acid.

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 b. Noncompetitive Inhibition: Here, there is no competition between the substrate or the
inhibitor. The inhibitor can bind with the enzyme or the ES complex. It binds to the enzyme
whether or not the active site is occupied by the substrate. This type of inhibition cannot be
fully reversed even at high substrate concentration. It lowers the rate of reaction but not the
equilibrium constant.
c. Uncompetitive Inhibition: Some inhibitors binds only to the ES complex and to the free
enzyme. These inhibitors are called uncompetitive inhibitors. This type of inhibition is rare in
reactions involving a single substrate but more common in those with multiple substrates.

Enzyme Cofactors
Many enzymes are completely inactive when they are isolated in a pure state. This is because some
of the ions and smaller organic molecules removed during the purification process play an essential
role in enzyme activity. These ions and smaller organic molecules needed for the activity of specific
enzymes are called cofactors. They include metal ions such as Ca++, Mg++, Mn++, Cu++, Zn++, and
selenium. Some enzymes with a cofactor requirement do not have a properly shaped active site in the
absence of the cofactor. In these enzymes, the attachment of cofactors causes a conformational change
in the protein that allows it to combine with its substrate. The cofactors of other enzymes participate
in the temporary bonds between the enzyme and its substrate when the ES complex is formed. For
example, the enzyme carboxypeptidase digests proteins by employing a zinc ion (Zn++) in its active
site to remove electrons from the bonds joining amino acids. Like zinc, these substances are required
in the diet in small amounts.
Coenzymes are cofactors that are organic molecules derived from niacin, riboflavin, and other water-
soluble vitamins. They participate in enzyme-catalyzed reactions by transporting hydrogen atoms and
small molecules from one enzyme to another. In numerous oxidation-reduction reactions that are
catalyzed by enzymes, the electrons pass in pairs from the active site of the enzyme to a coenzyme
that serves as the electron acceptor. The coenzyme then transfers the electrons to a different enzyme,
which releases them (and the energy they bear) to the substrates in another reaction. Often, the
electrons pair with protons (H+) as hydrogen atoms. In this way, coenzymes shuttle energy in the form
of hydrogen atoms from one enzyme to another in a cell. One of the most important coenzymes is the
hydrogen acceptor nicotinamide adenine dinucleotide (NAD+). The two nucleotides that make up
NAD+, nicotinamide monophosphate (NMP) and adenine monophosphate (AMP), are joined head-
to-head by their phosphate groups. The two nucleotides serve different functions in the NAD+
molecule: AMP acts as the core, providing a shape recognized by many enzymes; NMP is the active
part of the molecule, contributing a site that is readily reduced (that is, easily accepts electrons). When
NAD+ acquires an electron and a hydrogen atom (actually, two electrons and a proton) from the active
site of an enzyme, it is reduced to NADH. The NADH molecule now carries the two energetic
electrons and the proton. The oxidation of energy-containing molecules, which provides energy to
cells, involves stripping electrons from those molecules and donating them to NAD+. As we’ll see,
much of the energy of NADH is transferred to another molecule.

Factors affecting rate of Enzyme Action

The rate of an enzyme-catalyzed reaction is affected by the concentration of substrate, and of the
enzyme that works on it. In addition, any chemical or physical factor that alters the enzyme’s 3D
shape- such as temperature, pH, salt concentration, and the binding of specific regulatory molecules-
can affect the enzyme’s ability to catalyze the reaction.
I. Temperature

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Increasing the temperature of an un-catalyzed reaction will increase its rate because the additional
heat represents an increase in random molecular movement. The rate of an enzyme-catalyzed reaction
also increases with temperature, but only up to a point called the temperature optimum. Below this
temperature, the hydrogen bonds and hydrophobic interactions that determine the enzyme’s shape is
not flexible enough to permit the induced fit that is optimum for catalysis. Above the temperature
optimum, these forces are too weak to maintain the enzyme’s shape against the increased random
movement of the atoms in the enzyme. At these higher temperatures, the enzyme denatures. Most
human enzymes have temperature optima between 35°C and 40°C, a range that includes normal body
temperature. Bacteria that live in
hot springs have more stable enzymes (that is, enzymes held together more strongly), so the
temperature optimal for those enzymes can be 70°C or higher.
II. pH
Ionic interactions between oppositely charged amino acid residues, such as glutamic acid (–) and
lysine (+), also hold enzymes together. These interactions are sensitive to the hydrogen ion
concentration of the fluid the enzyme is dissolved in, because changing that concentration shifts the
balance between positively and negatively charged amino acid residues. For this reason, most
enzymes have a pH optimum that usually ranges from pH 6 to 8. Those enzymes able to function in
very acid environments are proteins that maintain their three-dimensional shape even in the presence
of high levels of hydrogen ion. The enzyme pepsin, for example, digests proteins in the stomach at
pH 2, a very acidic level.
III. Inhibitors and Activators
Enzyme activity is sensitive to the presence of specific substances that bind to the enzyme and cause
changes in its shape. Through these substances, a cell is able to regulate which enzymes are active
and which are inactive at a particular time. This allows the cell to increase its efficiency and to control
changes in its characteristics during development. A substance that binds to an enzyme and decreases
its activity is called an inhibitor. Very often, the end product of a biochemical pathway acts as an
inhibitor of an early reaction in the pathway, a process called feedback inhibition. An allosteric site
sites serve as chemical on/off switches; the binding of a substance to the site can switch the enzyme
between its active and inactive configurations. A substance that binds to an allosteric site and reduces
enzyme activity is called an allosteric inhibitor. Alternatively, activators bind to allosteric sites and
keep the enzymes in their active configurations, thereby increasing enzyme activity.

Regulation of Enzyme Activities

Enzymes regulate the chemistry of the cell, but what controls the enzymes? One regulatory
mechanism involves controlling the amount of enzyme produced. A specific gene directs the synthesis
of each type of enzyme. The gene, in turn, may be switched on by a signal from a hormone or by
some other signal molecule. When the gene is switched on, the enzyme is synthesized. The total
amount of enzyme present then influences the overall cell
reaction rate. If the pH and temperature are kept constant (as they are in most cells), the rate of the
reaction can be affected by the substrate concentration or by the enzyme concentration. If an excess
of substrate is present, the enzyme concentration is the rate-limiting factor. The initial rate of the
reaction is then directly proportional to the enzyme concentration. If the enzyme concentration is kept
constant, the rate of an enzymatic reaction is proportional to the concentration of substrate present.
Substrate concentration is the rate-limiting factor at lower concentrations; the rate of the reaction is
therefore directly proportional to the substrate concentration. However, at higher substrate
concentrations, the enzyme molecules become saturated with substrate; that is, substrate molecules
are bound to all available active sites of enzyme molecules. In this situation, increasing the substrate
concentration does not increase the net reaction rate.
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The product of one enzymatic reaction may control the activity of another enzyme, especially in a
sequence of enzymatic reactions. For example, consider the following metabolic pathway:

Enzyme 1 Enzyme 2 Enzyme 3 Enzyme 4

A B C D E

A different enzyme catalyzes each step, and the final product E may inhibit the activity of enzyme 1.
When the concentration of E is low, the sequence of reactions proceeds rapidly. However, an
increasing concentration of E serves as a signal for enzyme 1 to slow down and eventually to stop
functioning. Inhibition of enzyme 1 stops the entire reaction sequence. This type of enzyme
regulation, in which the formation of a product inhibits an earlier reaction in the sequence, is called
feedback inhibition.
Another method of enzymatic control focuses on the activation of enzyme molecules. In their inactive
form, the active sites of the enzyme are inappropriately shaped, so the substrates do not fit. Among
the factors that influence the shape of the enzyme are pH, the concentration of certain ions, and the
addition of phosphate groups to certain amino acids in the enzyme. Some enzymes have a receptor
site, called an allosteric site, on some region of the enzyme molecule other than the active site. When
a substance binds to an enzyme’s allosteric site, the conformation of the enzyme’s active site changes,
thereby modifying the enzyme’s activity. Substances that affect enzyme activity by binding to
allosteric sites are called allosteric regulators. Some allosteric regulators are allosteric inhibitors that
keep the enzyme in its inactive shape. Conversely, the activities of allosteric activators result in an
enzyme with a functional active site.

ENZYMOLOGY
Apoenzyme; protein portion of an enzyme (i.e., lacking a coenzyme)
• Enzyme; protein which catalyzes a biochemical reaction, an enzyme name ends in -ase (e.g.,
amylase and carbonic anhydrase)
• Holoenzyme; complete active enzyme (i.e., protein + coenzyme)
• Allosteric enzyme; a regulatory enzyme whose affinity for its substrate is affected by the presence
or absence of other molecules.

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Lock and Key Hypothesis or model
• This model assumes that the substrate and enzyme active site have complementary shapes in
which the substrate fits exactly into the active site.
• The enzyme’s active site represents the ‘lock’ while the substrate denotes the ‘key’. Catalysis
therefore only takes when a substrate with the right shape complements the active site shape.

The Induced Fit Hypothesis or 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.
✓ Hence, the configurations of both the enzyme and substrate are modified by substrate binding
and it is this conformational change that leads to product formation.