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.

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 by Them

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
Transferasse Effecting exchange of groups 1. Aminotransferases: catalyze exchange
between two substrates: of amino and keto groups between
AB+CD↔AC+BD amino and keto acid
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 reactions: 1. Peptidases: catalyze hydrolysis of
AB+H20↔AOH+HB peptide bonds
2. Glycosidases: catalyze hydrolysis of
glycosidic 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 non- 1. Decarboxylases
hydrolytically 2. Aldolases
AB↔A+B 3. Dehydratases
Isomerases Catalyze isomerization of substrates 1. Racemases
2. Epimerases

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

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 Acidic and basic amino acids in the enzyme facilitate the transfer of electrons
catalysis 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
Microenvironment Hydrolytic amino acids create a water-free zone in which non-polar substrates
effects may 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 (CH 2I.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-Mercuribenzoate 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.
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
susbstrates.

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
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.
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.

Short Test