MIC 414 MOLECULAR GENETICS NOTES 1

Biological catalysts

Definition: A chemical that speeds up a reaction without undergoing any permanent change.
Biological catalysts are known as enzymes.
How?
Enzymes can accelerate reactions in several ways, all of which lower the activation
energy (ΔG‡, Gibbs free energy). This is achieved through:
1. Stabilizing the transition state:
 Creating an environment with a charge distribution complementary to that of the
transition state to lower its energy
2. Providing an alternative reaction pathway:
 Temporarily reacting with the substrate, forming a covalent intermediate to provide a
lower energy transition state
3. Destabilising the substrate ground state:
 Distorting bound substrate(s) into their transition state form to reduce the energy
required to reach the transition state
4. Orienting the substrates into a productive arrangement:
 Reduces the reaction entropy change (the contribution of this mechanism to catalysis
is relatively small)

Characteristics of enzymes
Most enzymes are proteins and follow the physical and chemical reactions of proteins.
 Sensitive and labile to heat.
 Water soluble.
 Some enzymes can be precipitated by protein precipitating agents such as
ammonium sulfate and trichloroacetic acid.
 Speed up chemical reactions.
 Enzymes work in either direction i.e. they catalyse the forward and backward
reaction to some extent. The direction of the reaction depends on the relative
amounts of substrate and products present
 Are not used up during the reaction
 Are not destroyed at the end of the reaction and remain unchanged after a reaction
 They catalyze nearly all the chemical reactions taking place in cells and are re-
usable.
 There are thousands of different enzymes each controlling a specific chemical
reaction.

Discovery of enzymes
Enzymes were discovered in early 1700.
 1850: Louis Pasteur conducted experiment on sugar fermentation using yeast;
Conclusion: fermentation of sugar to alcohol was catalysed by chemicals from yeast. These
chemicals were known as ferments and were inseparable to the yeast cells. He called this the
vitalism theory.
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 Vitalists hold that living organisms are fundamentally different from non-living entities
because they contain some non-physical element or are governed by different principles than
are inanimate things. In its simplest form, vitalism holds that living entities contain some
fluid, or a distinctive ‘spirit’. In more sophisticated forms, the vital spirit becomes a
substance infusing bodies and giving life to them.
 1897: Eduard Buchner discovered that yeast extract could ferment sugar to alcohol. He
concluded that ferments were indeed separate from yeast cells hence disproving the vitalism
theory.
 Fredrick Kühne: named the ferments enzyme, the name that is currently used
 1926: James Sumner isolated and crystallised urease enzyme which was composed of
proteins. He concluded that all enzymes are made up of proteins.
 1930: John Northrop and Moses Kunitz isolated and crystallised pepsin, trypsin and other
digestive enzymes which were also composed of proteins
 Haldane made the remarkable suggestion that weak bonding interactions between an enzyme
and its substrate might be used to catalyze a reaction.
Many other enzymes have since been isolated and characterised.
Dynamics (activity, energetic, vibrance)
Enzymes are not rigid, static structures; they have complex internal dynamic motions. These
include
 Movements of parts of the enzyme's structure such as individual amino acid residues
 Groups of residues forming a protein loop or unit of secondary structure, or even an
entire protein domain
These motions give rise to a conformational group of slightly different structures that
interconvert with one another at equilibrium. Different states within this group may be associated
with different aspects of an enzyme's function. For example, different conformations of the
enzyme dihydrofolate reductase are associated with the substrate binding, catalysis, cofactor
release, and product release steps of the catalytic cycle.

Allosteric modulation
Allosteric sites are pockets on the enzyme, distinct from the active site, that bind to molecules in
the cellular environment. These molecules then cause a change in the conformation or dynamics
of the enzyme that is transduced to the active site and thus affects the reaction rate of the
enzyme. In this way, allosteric interactions can either inhibit or activate enzymes. Allosteric
interactions with metabolites upstream or downstream in an enzyme's metabolic pathway
cause feedback regulation, altering the activity of the enzyme according to the flux through the
rest of the pathway.

STRUCTURE OF ENZYMES
 With the exception of a small group of catalytic RNA molecules, all enzymes are proteins.
 Their catalytic activity depends on the integrity of their initial protein conformation.
 If an enzyme is denatured or dissociated into its subunits or is broken down into its
component amino acids, catalytic activity is usually lost.
 Enzymes, like other proteins, have molecular weights ranging from about 12,000 to more
than 1 million. The high molecular weight compounds are made up principally of chains of
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amino acids linked together by peptide bonds (molecular weight is referred to as atomic
mass).

General structure of amino acid

Examples of amino acids

Enzyme (and protein) structure

 Some enzymes need an additional molecule called co-factors to carry out their activity.
These cofactors serve many purposes; for instance, metal ions can help in stabilizing
nucleophilic species within the active site. Co-factors are inorganic ions (eg metal
ions and iron-sulfur clusters) or organic molecules(e.g., flavin and heme) that serve an
enzyme helpers.
Organic cofactors can be either:
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  Co-Enzymes, which are released from the enzyme's active site during the reaction
or,
 Prosthetic Groups, which are tightly bound to an enzyme. Organic prosthetic
groups can be covalently bound (e.g., biotin in enzymes such as pyruvate
carboxylase).
Carbonic anhydrase is an example of an enzyme that contains a cofactor, which uses a
zinc cofactor bound as part of its active site and is involved in catalysis. For example,
flavin and heme cofactors are often involved in redox reactions
Some enzymes require a cofactor but do not have one bound. These are
called apoenzymes or apoproteins. An enzyme together with the cofactor(s) required for
activity is called a holoenzyme (or haloenzyme). The term holoenzyme can also be
applied to enzymes that contain multiple protein subunits, eg the DNA polymerases; here
the holoenzyme is the complete complex containing all the subunits needed for activity.
Some enzymes however require no chemical groups for their activity other than their
amino acid residues.
Structure of an enzyme

Enzyme Specificity

Enzymes have varying degrees of specificity for substrates: they may recognize and catalyze:
 a single substrate (absolute specificity)
 a group of similar substrates
 a particular type of bond

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Shared properties between biological catalysts with chemical catalysts
a. Enzymes are neither consumed nor produced during the course of a reaction.
b. Enzymes do not cause reactions to take place, but they greatly enhance the rate of
reactions that would proceed much slower in their absence. They alter the rate but not
the equilibrium constants of reactions that they catalyze.

Differences between enzymes and chemical catalysts

a. Enzymes are proteins.
b. Enzymes are highly specific and produce only the expected products from the given
reactants, or substrates (i.e., there are no side reactions).
c. Enzymes may show a high specificity toward one substrate or exhibit a broad specificity,
using more than one substrate.
d. Enzymes usually function within a moderate pH and temperature range.
Enzymes have two basic functions in biological:
They are catalytic and regulation.
1. Catalytic efficiency
Most enzyme-catalyzed reactions are highly efficient, proceeding from 10 3 to 108 times
faster than uncatalyzed reactions. Typically, each enzyme molecule is capable of
transforming 100 to 1000 substrate molecules into product each second. The number of
molecules of substrate converted to product per enzyme molecule per second is called the
turnover number.
2. Regulation
Enzyme 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.

Measures of Enzyme Activity

Enzymes are never expressed in terms of their concentration (as mg or µg etc.), but are
expressed only as activities.
This is measured in either of two ways:
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 Specific activity is usually expressed as mol of substrate transformed to product
per minute per milligram of enzyme under optimal conditions of measurement
 The rate of appearance of product or the rate of disappearance of substrate
 Test the absorbance: spectrophotometer
 Turnover number, or kcal, is the number of substrate molecules metabolized per
enzyme molecule per unit time with units of min-1 or s-1.
Measuring enzymatic rate ideally done with a system where the product or substrate
absorb a particular wavelength of light which depends on enzyme reaction and be
monitored with spectrophotometer by measuring either the appearance of product or
disappearance of substrate.
NOMENCLATURE AND CLASSIFICATION OF ENZYMES
Enzymes are classified into six functional classes (EC number classification) by the International
Union of Biochemists (I.U.B.) on the basis of the types of reactions that they catalyze
These major classes with their subclasses are:
a. Oxidoreductases- are involved in oxidation and reduction –EC1
b. Transferases -transfer functional groups (e.g., amino or phosphate groups) - EC 2
c. Hydrolases- transfer water; that is, they catalyze the hydrolysis of a substrate- EC 3
d. Lyases -add (or remove) the elements of water, ammonia, or carbon dioxide (CO 2) to (or
from) double bonds –EC 4
e. Isomerases -catalyze rearrangements of atoms within a molecule- EC 5
f. Ligases- join two molecules –EC 6

Six Major Classes of Enzymes and Examples of Their Subclasses

Classification Distinguishing Feature
1. Oxidoreductases Аred +Вox → Аоx + Вred
Oxidases Use oxygen as an electron acceptor but do not incorporate it into
the substrate
Dehydrogenases Use molecules other than oxygen (e.g., NAD +) as an elec- tron
acceptor
Oxygenases Directly incorporate oxygen into the substrate
Peroxidases Use H2O2 as an electron acceptor
2. Transferases А-В+С  А+В-С
Methyltransferases Transfer one-carbon units between substrates
Aminotransferases Transfer NH2 from amino acids to keto acids
Kinases Transfer PO3~ from ATP to a substrate
Phosphorylases Transfer PO3~ from inorganic phosphate (P,) to a substrate
3. Hydrolases А-В+Н2О  А-Н + В-ОН
Phosphatases Remove PO3~ from a substrate
Phosphodiesterases Cleave phosphodiester bonds such as those in nucleic acids

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 Proteases Cleave amide bonds such as those in proteins
4. Lyases А(ХН)-В  А-Х+В-Н
Decarboxylases Produce CO2 via elimination reactions
Aldolases Produce aldehydes via elimination reactions
Synthases Link two molecules without involvement of ATP
5. Isomerases Catalyze changes within one molecule. They convert one isomer to
another, meaning that the end product has the same molecular
formula but a different physical structure
Racemases Interconvert L and D stereoisomers
Mutases Transfer groups between atoms within a molecule
6. Ligases A+B+ATP  A-B+ADP+Pi
Carboxylases Use CO2 as a substrate
Synthetases Link two molecules via an ATP-dependent reaction
Enzymes have been named by:
i. Adding the suffix “-ase” to the name of their substrate or to a word or phrase describing their
activity. eg urease catalyses hydrolysis of urea, and DNA polymerase catalyses the
polymerization of nucleotides to form DNA
ii. The people who discovered them for a broad function, before the specific reaction was
known. For example, an enzyme known to act in the digestion of foods was named pepsin,
from the Greek pepsis, “digestion”
iii. Their source: eg trypsin, named in part from the Greek tryein, “to wear down”, was obtained
by rubbing pancreatic tissue with glycerin
*Different enzymes that catalyze the same chemical reaction are called isozymes.*

Each enzyme is assigned two names:

 Recommended name- short and convenient for everyday use
 Systematic name - more complete which is used when the enzyme must be identified
without ambiguity.
A. Recommended name
Most commonly used enzyme names have the suffix "- ase" attached to the substrate of the
reaction, for example, glucosidase, urease, sucrase; or to a description of the action
performed, for example, lactate dehydrogenase
[Some enzymes retain their original trivial names, which give no hint of the associated
enzymic reaction, for example, trypsin and pepsin.]
A. Systematic name
The International Union of Biochemistry and Molecular Biology (IUBMB) developed a
system of nomenclature in which enzymes are divided into six major classes, each with
numerous subgroups. The suffix "-ase" is attached to a fairly complete description of the
chemical reaction catalyzed, for example D-glyceraldehyde 3- phosphate:NAD
oxidoreductase. The IUBMB names are unambiguous and informative, but are sometimes too
cumbersome to be of general use.

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 Principle of the International Classification
Each enzyme has a classification number consisting of four digits e.g., EC: (2.7.1.1)
hexokinase
EC: (2.7.1.1) these components indicate the following groups of enzymes:
 2-class (transferase)
 7-subclass (transfer of phosphate)
 1-sub-sub class (alcohol is phosphate acceptor)
 1-specific name atp,d-hexose-6-phosphotransferase (hexokinase)

Hexokinase catalyzes: Glucose + ATP à glucose-6-P + ADP

ENZYME KINETICS

Enzyme kinetics is the quantitative measurement of the rate of enzyme catalyzed reactions and
the systematics study of factors that affect these rates
 Enzyme kinetics began in 1902 when Adrina Brown reported an investigation of the rate of
hydrolysis of sucrose as catalyzed by the yeast enzyme inveratase
 Brown demonstrated-when sucrose concentration is much higher than that of the enzyme,
reaction rate becomes independent of sucrose concentration.
 Brown proposal-overall reaction is composed of two elementary reaction in which the
substrate forms a complex with the enzyme that subsequently decomposes to products and
enzymes

When the substrate concentration becomes high enough to entirely convert the enzyme to ES
form, the second step of the reaction becomes the limiting step. The overall reaction rate
becomes insensitive to further increase of the substrate concentration.
ENZYME ACTIVE SITE
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This is the area of an enzyme that binds to the substrate. The structure has a unique geometric
shape that is designed to fit the molecular shape of the substrate.
 Each enzyme is substrate specific
 Thus the active site is complementary to the geometric shape of a substrate molecule
There is two models of active site:
 Lock and key model
 Induced fit model

Lock and key model

In the lock-and-key model of enzyme action:
 the active site has a rigid shape
 only substrates with the matching shape can fit
 the substrate is a key that fits the lock of the active site

 This explains enzyme specificity

 This explains the loss of activity when enzymes denature

Induced fit model

 Enzyme structure is flexible, not rigid
 Enzyme and active site adjust shape to bind substrate
 Increases range of substrate specificity
 Shape changes also improve catalysis during reaction
 Transition-state like configuration

Enzyme catalysed reaction

 When a substrate (S) fits properly in an active site, an enzyme-substrate (ES) complex
is formed:
 E + S D ES
 Within the active site of the ES complex, the reaction occurs to convert substrate to
product (P):
 ES ® E + P
 The products are then released, allowing another substrate molecule to bind the enzyme
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  this cycle can be repeated millions (or even more) times per minute
 The overall reaction for the conversion of substrate to product can be written as
follows:
 E + S D ES ® E + P

The steps in enzyme catalysed reactions are:

Step 1: Enzyme substrate complex

Enzyme and substrate combine to form complex
E + S ES
Enzyme Substrate Complex

+
Step 2: Enzyme product complex is formed
ES EP

Step 3: The enzyme and product separate

EP E+P Product is made

FACTORS THAT INFLUENCE ENZYME ACTION

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1. Enzyme concentration

At low enzyme concentration there is great competition for the active sites and the rate of
reaction is low. As the enzyme concentration increases there are more active sites and the
reaction can proceed at a faster rate. Eventually, increasing enzyme concentration beyond a
certain point has no effect because the substrate concentration becomes limiting.

An increase in enzyme concentration;

 Binds more substrate with enzyme
 Increases the rate of reaction at constant substrate concentration

2. Substrate concentration

When more substrate molecules are added, more enzyme-substrate complexes can be formed
as there are more active sites and the rate of reaction increases. Eventually increasing the
substrate concentration yet further will have no effect. The active sites will be saturated so no
more substrate complexes can be formed.
An increase in substrate concentration:
 Increases the rate of reaction at constant enzyme concentration
 Eventually saturates an enzyme with substrate to give maximum activity

3. CHANGES IN TEMPERATURE

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 Enzymes work best at an optimum temperature (usually 37 0C in humans). Different enzymes
have different temperature optima (the point when maximum activity occurs). An increase in
temperature provides more kinetic to the molecules involved. The number of collisions
between enzyme and substrate will increase so the rate. Too high a temperature above the
optimum temperature causes the enzyme to be denatured. Bonds holding the structure
together will be broken and the active site loses its shape and will no longer work.

4. pH

All enzymes have a certain narrow range of pH at which they perform best, usually 4.0 – 8.0.
Extremes of pH can affect the enzyme by denaturing or affecting the change of critical amino
acids in its site or change in the substrate.

5. WATER
ACTIVITY
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 Water can influence enzyme activity in a number of ways. It is critical for:
 Substrate Product reaction (eg hydrolysis)
 Solubilization of the substrate and product
 Flexibility of the enzyme
The content of water can be varied in foods to slow down enzyme activity

6. INHIBITORS
These are chemical compounds that inhibit or slow down the activity of enzymes.
 Competitive inhibitors:- compete with the substrate for the active site. The enzyme can
only bind to either the substrate or inhibitor at any one time
 Non-competitive inhibitors:- Bind to the enzyme at another site than the active site.
Enzyme can bind to both substrate and inhibitor at the same time
 Un- competitive inhibitors:- Can only bind to the enzyme/substrate complex (the
intermediate state). The enzyme binds first to the substrate and then the inhibitor.
Inhibitions can be reversible or irreversible.

Examples of inhibitors:
 Antibiotics affecting bacterial metabolism by inhibiting enzymes
 Nerve gases cause irreversible inhibition
 Insecticides eg choline esterase
 Many heavy metal poisons that irreversibly inhibit enzymes.

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