ENZYMES

LECTURE 1

Daniel A. Abera
(DBE, BSc, MPhil.)
PhD Biochemistry researcher, KNUST.
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course content
 Enzymes and their Reactions.
 Food nutrients: proteins, carbohydrates, fats, vitamins and
minerals, metabolism, digestion, hydrocarbons

 Natural product chemistry: lipids, glycosides, nucleic acids

 Alcohols, colloids, liquids and solutions and fluid balance.

 Blood and its components, other body liquids.

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 Course content cont…
 Radioactivity; Organic Chemistry
 The Chemical World
 Matter and Energy
 Gas and Gas Laws
 Atomic structures
 Properties of electrolyte and non-electrolytes

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 Content
 Introduction
 Chemical properties of enzymes
 Characteristics of enzymes
 Enzyme specificity
 Activation energy
 Enzyme inhibition
 Factors affecting enzyme action

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ENZYMES
 Enzymes are basically proteins that are produced by living organisms
 to bring about certain metabolic and biochemical reactions in the body.

 They are biological catalysts that speed up reactions inside the body.

 There are two fundamental conditions for life. The living entity
must be able to

 Self-replicate.
 Catalyze chemical reactions efficiently and selectively.

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Enzymes catalyze hundreds of stepwise reactions that
degrade nutrient molecules, conserve and transform chemical
energy, and make biological macromolecules from simple
precursors.

Enzymes are important practical tools in,

 medicine,
 chemical industry,
 food processing, and
 agriculture.
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• The orderly course of metabolic processes is only possible
because each cell is equipped with its own genetically
determined set of enzymes

• It is only this that allows coordinated sequences of reactions -

metabolic pathways

• Involved in many regulatory mechanisms.

• Almost all enzymes are proteins except catalytically active

ribonucleic acids, the ribozymes
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Characteristics of Enzyme
• Enzymes are characterized by three distinctive features:
• Catalytic Power
– Ability to catalyzes biochemical reaction
– Accelerating reaction rates as much as 10 16 over uncatalyzed
levels – far greater than any synthetic catalysts
• Specificity
– A given enzyme is very selective
– Both in the substances with which it interacts and in the
reaction that it catalyzes
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Regulation
– Metabolic inhibitors and activators
Enzymes activity can be regulated, that is enzymes can be activated or
inhibited so that the rate of product formation responds to the needs of
the cell

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 Difference from catalysts
• Like catalysts, the enzymes do not alter the chemical
equilibrium point of a reversible reaction but only the speed of
the reaction is changed

• Differ from catalysts in being the biological products, i.e.,

produced from the living cells.

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Difference from catalysts
•The enzymes are protein and, unlike catalysts, cannot last
indefinitely in a reaction system since they, being colloidal in
nature, often become damaged or inactivated by the reactions
they catalyze. they must be replaced constantly by further
synthesis in the body.

• Unlike catalysts, most individual enzymes are very specific in

that they act either on a single or at the most on some
structurally related substrates.
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Chemical properties of enzymes
• Most of enzymes carry out their functions relying solely on their protein
structure

• Many others require non-protein components – cofactors

• Conjugated proteins function only in the presence of specific non-
protein molecules or metal ions called prosthetic groups.

– If the non-protein component is tightly bound, and forms an integral part

of the enzyme structure, it is a true prosthetic group.

– If the non-protein component is weakly bound, and easily separated

from the rest of the protein, it is called a cofactor.
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• When the cofactor is an organic substance, it is a coenzyme. The
cofactor may also be an inorganic ion (usually a metal cation, such as
Mg2+, Zn2+, or Fe2+).
• The protein portion is called an apoenzyme:

apoenzyme + cofactor (coenzyme or inorganic ion) active enzyme

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• Many organic coenzymes are derived
from vitamins.

– For example, nicotinamide adenine

dinucleotide (NAD+) is a necessary part
of some enzyme-catalyzed redox reactions.
It is formed from the vitamin precursor
nicotinamide.

– In this reaction, NAD+ is the oxidizing agent

and accepts hydrogen from lactate. It can then
transfer the H+ to other compounds in
Subsequent reactions.

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According to Holum, the cofactor may be:

1. A coenzyme – a non-protein organic substance which is dialyzable,

thermostable and loosely attached to the protein part.

2. A prosthetic group – an organic substance which is dialyzable and

thermostable which is firmly attached to the protein or apoenzyme
portion.

3. A metal-ion-activator – these include K+, Fe2+, Fe3+, Cu2+, Co2+,

Zn2+, Mn2+, Mg2+, Ca2+, and Mo3+.
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Classification of Enzyme
Biochemists, by international agreement, have
classified enzymes in to six classes, each with
subclasses, based on the type of reaction catalyzed
– Oxidoreductases
– Transferases
– Hydrolases
– Isomerases
– Lyases
– Ligases
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Oxidoreductases – To this class belong all enzymes catalysing oxidation
reduction reactions. The substrate that is oxidized is regarded as
hydrogen donor.
• This class comprises the enzymes which were earlier called
dehydrogenases, oxidases, peroxidases, hydroxylases, oxygenases etc

• The group, in fact, includes those enzymes which bring about oxidation-
reduction reactions between two substrates

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• Catalyze redox reaction and can be categorized into oxidase and
reductase

• More precisely, they catalyze electron transfer reactions. In this class

are included the enzymes catalyzing oxidoreductions of CH—OH, C=O,
CH—CH, CH—NH2 and CH=NH groups

• Alcohol dehydrogenase, Acetyl-CoA dehydrogenase, Cytochrome

oxidase, Catalase

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Transferases – Transferases are enzymes transferring a group, e.g. a
methyl group or a glycosyl group, from one compound (generally regarded
as donor) to another compound (generally regarded as acceptor).

• Catalyze the transfer or exchange of certain groups among some

substrates

• In these are included the enzymes catalyzing the transfer of one-carbon

groups, aldehydic or ketonic residues and acyl, glycosyl, alkyl, phosphorus
or sulfur-containing groups

• Choline acetyltransferase, Phosphorylase, Hexokinase

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• General equation :
A-X + B ↔ BX + A
• Eg. :
transaminase (transfer amino group from one molecule to another)

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Hydrolases – These enzymes catalyse the hydrolytic cleavage of C-O, C-
N, C-C and some other bonds, including phosphoric anhydride bonds.
• Accelerate the hydrolysis of substrates
• These catalyze the hydrolysis of their substrates by adding constituents
of
• water across the bond they split
• The substrates include ester, glycosyl, ether, peptide, acid-anhydride, C
—C, halide and P—N
bonds
• Lipase, Beta-galactosidase, Arginase, Trypsin. Pepsin, plasmin,

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 • catalyze hydrolysis of substrate by addition of water
• General equation :
A-X + H2O ↔ X-OH + HA
• Eg. :
maltase ( maltose broken to 2 glucose)
lipase (lipid broken to fatty acid and glycerol)

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Lyases –
Lyases are enzymes cleaving C-C, C-O, C-N, and other bonds by
elimination, leaving double bonds or rings, or conversely adding
groups to double bonds.
• Promote the removal of a group from the substrate to leave a
double bond reaction or catalyze its reverse reaction

• In these are included the enzymes acting on C—C, C—O, C—N,

C—S and C—halide bonds

• Aldolase, Fumarase, Histidase

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• breaks chemical bonds without adding water

• General equation : A-B → A=B + X-Y

• Eg.,
decarboxylases ( remove carboxyl group from
respiratory substrates to release carbon dioxide)

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Isomerases – These enzymes catalyse
geometric or structural changes within one
molecule. According to the type of
isomerism, they may be called racemases,
epimerases, cis-trans-isomerases,
isomerases, tautomerases, mutases or
cycloisomerases.

• catalyzes conversion of one isomer to

another by transferring a group of atoms
from one molecule to another

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Ligases – Ligases are enzymes catalysing the joining together of two
molecules coupled with the hydrolysis of a diphosphate bond in ATP or a
similar triphosphate

• catalyzes the synthesis of new chemical bonds, using ATP

• Eg.,
DNA ligase is involved in DNA synthesis

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Translocases – Catalysing the translocation of hydrogen ions, inorganic
cations and anions, amino acids, carbohydrates or other compounds. A
new EC class was created in 2018.
• Catalyze the movement of ions or molecules across membranes or their
separation within membranes
• the reaction is designated as a transfer from “side 1” to “side 2”
• Translocases are the most common secretion system in Gram positive
bacteria
• Translocase of the outer membrane (TOM) can work in conjunction with
translocase of the
inner membrane (TIM) to transport proteins into the mitochondrion
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Intracellular and extracellular enzymes
o Intracellular
o enzymes are synthesized and retained in the cell for the use of cell itself.
o They are found in the cytoplasm, nucleus, mitochondria and
chloroplast.
Example: Oxydoreductase catalyses biological oxidation, Enzymes involved
in reduction in the mitochondria.
o Extracellular
o enzymes are synthesized in the cell but secreted from the cell to work
externally.
Example : Digestive enzyme produced by the pancreas, are not used by
the cells in the pancreas but are transported to the duodenum.
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Basic Enzyme Reactions
Enzymes are catalysts that increase the speed of a chemical reaction
without themselves undergoing any permanent chemical change. They are
neither used up in the reaction nor do they appear as reaction products.

The basic enzymatic reaction can be represented as follows:

S+E P+ E

E represents the enzyme catalyzing the reaction; S, the substrate, the

substance being modified; and P, the product of the reaction.

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The Enzyme Substrate Complex
A theory to explain the catalytic action of enzymes was proposed by the Swedish
chemist Savante Arrhenius in 1888.

He proposed that the substrate and enzyme formed some intermediate substance which
is known as the enzyme/substrate complex (ES). The reaction can be represented as:
S+E ES
S = Substrate; E = Enzyme; ES = Enzyme/Substrate Complex

If this reaction is combined with the original reaction equation, the following results:
S+E ES P+ E
S = Substrate; E = Enzyme; ES = Enzyme/Substrate Complex; P = Product

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Enzyme specificity
 There are two main theories seeking to explain the
enzyme-substrate interaction.
 Lock and key model for substrate binding – proposed by
Fischer

 Induced fit model for substrate binding

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Lock and Key Model
• also known as template model - proposed by Emil Fischer in 1898

• the union between the substrate and the enzyme takes place at the
active site more or less in a manner in which a key fits a lock and results in
the formation of an enzyme substrate complex

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• In fact, the enzyme-substrate union depends on a reciprocal fit between
the molecular structure of the enzyme and the substrate
• And as the two molecules (that of the substrate and the enzyme) are
involved, this hypothesis is also known as the concept of intermolecular
fit
• The enzyme-substrate complex is highly unstable and almost
immediately this complex decomposes to produce the end products of
the reaction and to regenerate the free enzyme.
• The enzyme-substrate union results in the release of energy. It is this
energy which, in fact, raises the energy level of the substrate molecule,
thus inducing the activated state
• In this activated state, certain bonds of the substrate molecule become
more susceptible to cleavage.
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Induced-Fit Theory
• unfortunate feature of Fischer’s model is the rigidity of the active site
• Koshland presumed that the enzyme molecule does not retain its
original shape and structure. But the contact of the substrate induces
some configurational or geometrical changes in the active site of the
enzyme molecule.

• Consequently, the enzyme molecule is made to fit completely the

configuration and active centres of the substrate

• At the same time, other amino acid residues may become buried in the
interior of the molecule
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• As to the sequence of events during the conformational changes, 3
possibilities exist
1. The enzyme may first undergo a conformational change, then bind
substrate
2. An alternative pathway is that the substrate may first be bound and
then a conformational change may occur
3. Both the processes may occur simultaneously with further
isomerization to the final conformation
• Koshland’s model has now gained much experimental support.
Conformational changes during substrate binding and catalysis have been
demonstrated for various enzymes such as phosphoglucomutase, creatine
kinase, carboxypeptidase
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Activation energy
Chemists have known for almost a century that for most chemical reactions
to proceed, some form of energy is needed.

They have termed this quantity of energy, “the energy of activation.”

It is the magnitude of the activation energy which determines just how fast
the reaction will proceed.

It is believed that enzymes lower the activation energy for the reaction they
are catalyzing. The enzyme is thought to reduce the “path” of the reaction.

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This shortened path would require less energy for each molecule of
substrate converted to product.
Given a total amount of available energy, more molecules of substrate
would be converted when the enzyme is present (the shortened “path”)
than when it is absent.

Hence, the reaction is said to go faster in a given period of time.

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 Activation energy

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Inhibition
Enzyme inhibitors are molecular agents that interfere with
catalysis, slowing or halting enzymatic reactions

Knowledge of enzyme-inhibition mechanism is important in

understanding the control of metabolism and the design of
useful drugs.

Inhibition studies often tell us something about the specificity

of an enzyme, the physical and chemical architecture of the
active site and the kinetic mechanism of the reaction
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Inhibition
Enzyme inhibitors can be drugs, antibiotics, preservatives and
poisons.
Enzyme inhibitors can be broadly classified as either
1. Reversible
2. Irreversible

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Reversible inhibition
These are inhibitors that rarely binds covalently to an enzyme
and may be easily removed by pressure such as dialysis, dilution,
or addition of excess substrate, with the restoration of the
enzyme activity.

The three type of reversible inhibitors are

1.Competitive
2.Noncompetitive
3.Uncompetitive.
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Competitive inhibition
A competitive inhibitor competes with the substrate for the
active site of an enzyme. While the inhibitor (I) occupies the
active site it prevents binding of the substrate to the enzyme.

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Noncompetitive inhibition/mixed inhibition
The substrate and the inhibitor binds reversibly, randomly,
and independently at different site on the enzyme.
Noncompetitive inhibitors is usually not structurally related
to the substrate. It binds to either E or ES.

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Uncompetitive inhibition
Uncompetitive inhibitors bind at a separate site, but bind only
to the ES complex.
Irreversible inhibition
These forms mostly covalent bonds
with the enzyme and are relatively
difficult to remove without destroying
the enzymes activity. Many deadly
poisons function as irreversible enzyme
inhibitors.
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Factors effecting enzyme activity
The contact between enzyme and substrate is the most essential
pre-requisite for enzyme activity. The important factors that
influence the enzyme reaction are
– Concentration of Substrate
– Concentration of Enzyme
– Temperature
– pH
– Product concentration
– Activators
– Light and radiation
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Substrate concentration
• The concentration of substrate, [S], also affects the rate of the reaction.
• Increasing [S] increases the rate of the reaction, but eventually, the rate
reaches a maximum (vmax), and remains constant after that.
• The maximum rate is reach when the enzyme is saturated with substrate,
and cannot react any faster under those conditions.

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Enzyme concentration
• The concentration of an enzyme, [E], is typically low compared to that of
the substrate. Increasing [E] also increases the rate of the reaction:

• The rate of the reaction is directly proportional to the concentration of the

enzyme (doubling [E] doubles the rate of the reaction), thus, a graph of
reaction rate vs. enzyme concentration is a straight line:

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Temperature
• Like all reactions, the rate of enzyme-catalyzed reactions increases with
temperature.
• Because enzymes are proteins, beyond a certain temperature, the enzyme
denatures.
• Every enzyme-catalyzed reaction has an optimum temperature at which
the enzyme activity is highest, usually from 25º-40ºC; above or below that
value, the rate is lower.

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The Effect of pH
• Raising or lowering the pH influences the acidic and basic side chains in
enzymes. Many enzymes are also denatured by pH extremes. (E.g., pickling
in acetic acid [vinegar] preserves food by deactivating bacterial enzymes.)
• Many enzymes have an optimum pH, where activity is highest, near a pH
of 7, but some operate better at low pH (e.g., pepsin in the stomach).

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Enzyme Regulation
• Enzymes work together to facilitate all the biochemical
reactions needed for a living organism.

To respond to changing conditions and cellular needs, enzyme

activity requires very sensitive controls:

– activation of zymogens
– allosteric regulation
– genetic control
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Allosteric Regulation
• Compounds that alter enzymes by changing the 3D conformation of the
enzyme are called modulators.
• They may increase the activity (activators) or decrease the activity
(inhibitors). (Noncompetitive inhibitors are examples of this activity.)
• Enzymes with quaternary structures with binding sites for modulators are
called allosteric enzymes.
• These variable-rate enzymes are often located at key control points in cell
processes.
• Feedback inhibition occurs when the end product of a sequence of
enzyme-catalyzed reactions inhibits an earlier step in the process. This
allows the concentration of the product to be maintained within very
narrow limits.
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Allosteric Regulation
• The synthesis of isoleucine from threonine is an
example of allosteric regulation.
– Threonine deaminase, which acts in the first step of the conversion
pathway, is inhibited by the isoleucine product.
– When isoleucine builds up, it binds to the allosteric site on threonine
deaminase, changing its conformation so that threonine binds poorly. This
slows the reaction down so that the isoleucine concentration starts to fall.
– When the isoleucine concentration gets too low, the enzyme becomes
more active again, and more isoleucine is synthesized.

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 TO BE CONTINUED

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