ENZYMES

ENZYMES

• Enzymes are biological catalyst which carry

out chemical reactions without having
themselves being changed in the process.

• Enzyme + Substrate  Enzyme substrate

complex  Enzyme + Product.
Why enzymes are necessary in living
organisms
• Enzymes speed up chemical reactions by
lower the amount of energy and time
required for reactions to occur. Without
enzymes, reactions would occur to slowly in
biological organisms for life to exist at its
current pace.
• Enzymes control the sequence of chemical
reactions.
• They allow for many reactions to occur
simultaneously.
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• Enzymes are globular 3D proteins.
• The slowest-acting known enzyme, lysozyme
(an anti-bacterial enzyme found in raw milk),
can process about thirty molecules a minute.
Carbonic anhydrase is one of the fastest-
acting enzymes and can turnover 600,000
molecules/second speed.
Hydrogen carbonate ion

CO2 + H2O <-----> HCO3- + H

 ENZYMES
• Metabolism is a term
used to describe
chemical reactions
occurring in the body.
• Anabolic reactions are
building up reactions
for example the
formation of glycogen.
• Catabolic reactions are
breaking down
reactions for example
the digestion of protein
Enzyme catalyzed reaction
 Structure of Enzymes
• Enzymes are 3D globular proteins folded in
such a way that they have a cleft which serves
as a binding site.
• Within the binding site are specific amino
acids with particular R-groups which can form
temporary bond with the substrate.
 How enzymes work
Lock and key model
• Theory proposed by Emil Fischer in 1894
• The binding site is specific and is complementary to the
shape of the substrate molecule.
• It will bind to that type of molecule and that type alone.
• The key is analogous to the enzyme and the substrate is
analogous to the lock.
• This hypothesis explains that enzymes are rigid and
cannot change their shape.
Diagram of lock & Key model
 How enzymes work
Induced-fit Hypothesis
• The induced fit model was suggested by Daniel Koshland in 1958 and is the more
accepted model for enzyme-substrate complex than the lock-and-key model.
• In this hypothesis the enzyme is said to be less rigid and sort of flexibility.
• The enzyme doesn’t have a fixed active site and takes the shape of the substrate
as it binds with it.
• The induce fit model explains how enzymes lower the activation energy of a
reaction. This is because in the induced fit model, as the substrate binds, to the
active site, the enzyme changes shape exposing the parts that react with the
substrate. This change creates stress, which weakens bonds in the substrate and
therefore reduces the activation energy required for the reaction to occur.
• The change in shape can also lengthen of shorten the gap between molecules
making it easier for the reaction to occur.
• Induced fit is observed in many enzymes, such as hexokinase and lactate
dehydrogenase (LDH).

• Hexokinase phophorylates six carbon sugars such as glucose during respiration.

• LDH catalyses the conversion of pyruvate to lactate during anaerobic respiration.
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Induced-fit Hypothesis
 How enzymes work
• The interaction between the enzyme and the substrate stresses or
weakens some of the chemical bonds in the substrate. These stresses
encourages a link between two molecules or breakage of bond between
the two molecules which leads to the formation of a different molecule(s)
called the product.
Activation energy
• In any chemical reaction, energy is needed to make it work. E.g. heat
• This energy needed is called the activation energy.
• All reactions also have a transition state. It is defined as the state
corresponding to the highest energy along which the reaction coordinates.
• The transition state is the transitory of molecular structure in which the
molecule is no longer a substrate but not yet a product.
• Enzymes lower the activation energy of the transition state.
 How enzymes work
• How enzymes lower the AE?
• Enzymes lower the activation energy by temporarily binding
to the substrate(s) and orienting them in the correct position
and order so that they can react.
• Enzymes may add a charge to the substrate to make it more
reactive.
• They also induce strain on chemical bonds in the substrate so
they are less stable and more reactive.
• As a result of these mechanisms, enzymes reduce the time
taken for the reaction as will as the AE which is required.
• Without enzymes, molecules will have to react by random
chance, use a lot more energy and take a lot more time.
How enzymes work
 Co-factors
• Co-factors are non-protein components which are
required by enzymes for efficient functioning.
• Co-factors may vary from simple inorganic ions to
complex organic molecules.
• There are three types of cofactors:
-Inorganic ions
-Prosthetic groups (organic) and
-Co-enzymes (organic)
• An inactive enzyme, without the cofactor is called an
apoenzyme
• The complete enzyme with its cofactor is called a
holoenzyme
Inorganic ions
• Enzymes sometimes are in an inactive form and
become active when they are needed.
• They are made active by the use of inorganic ions
which bind to the enzyme at a special place called
an allosteric site.
• The ions re-shape the enzyme by making the
active site open up or retain its correct shape so it
can bind to the substrate.
• For example, salivary amylase activity is increased
in the presence of chloride ions.

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(E.g. chloride ion)
 Co-enzymes
• Co-enzymes are cofactors which bound and released easily. They do not
remain attached to the enzyme like prosthetic groups.
• ‘Co` means they work with enzymes.
• All co-enzymes are derived form vitamins e.g. B1, B6 & B12. Coenzymes
are organic molecules.
• They bind temporarily to the enzyme (e.g the active site) and participate in
catalysis e.g. they function as intermediate carriers of electrons, specific
atoms or functional groups that are transferred in the overall reaction.
• Coenzymes also aid in recognizing, attracting or repulsing a substrate or
product.
• Good example include NAD, NADP, Co-enzyme A and ATP.
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Prosthetic group
• A prosthetic group is the cofactor which is tightly or
permanently bounded to an enzyme.
• Like co-enzymes, prosthetic groups are organic.
• They bind tightly to the enzyme and participate in catalysis
e.g they function as intermediate carriers of electrons,
specific atoms or functional groups that are transferred in
the overall reaction.
• A good example is FAD which contains vitamin B2. FAD
functions as a hydrogen acceptor during the process of
respiration.

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 Co-factor binding to an enzyme

Substrate

Allosteric
site

Enzyme
 Factors affecting enzyme activity
• Temperature
• pH
• Substrate concentration
• Enzyme concentration
• Presence of inhibitors.
 Factors affecting enzyme activity
Temperature
• As the temperature increases so too does the
rate of reaction.
• This is because heat provides kinetic energy
which makes the molecules move faster and
increases the chance of an enzyme binding to a
substrate.
• At a certain temperature the enzyme will
function best. This is known as the optimum
temperature.
• Beyond this optimum temperature enzyme
activity begins to decrease. This is because of
denaturation of enzymes by excess heat.
• Important bonds such as hydrogen bonds,
hydrophobic interactions and disulphide
bridges which hold the enzyme in its tertiary
structure are broken, distorting the enzyme’s
shape and preventing any more substrates
from binding.
 Temperature
They thrive in
temperature
ranging from 60 -
108 degrees C

Mesophile

Enzyme model.swf
 Temperature
• The Q10 temperature coefficient is a measure of the rate of
change of a biological or chemical system as a consequence of
increasing the temperature by 10°C.
• The Q10 is calculated as:
where
R is the rate T is the temperature in Celsius degrees or kelvins.
• For biological systems, the Q10 value is generally between 1
and 3.
• Many enzymes have a Q10 of between 2 and 3. In other
words, provided that the temperature is not so high that it
causes denaturation, an increase in temperature of 10°C will
speed up the reaction by a factor of 2-3, that is it will double
or triple it.
• Thermophiles can survive in the
extreme temperatures of deep-sea
hydrothermal vents .
• Scientists have found a thermophile,
Methanopyrus kandleri, that can
survive in temperatures in excess of
121 degrees C.
• A thermophile, Thermus aquaticus,
found in the hot springs at Yellow
Stone National Park, has been used
in the PCR, or Polymerase Chain
Reaction process, which uses
heating and cooling to make billion
of copies of a DNA section for
example in DNA fingerprinting.
• Another thermophile, Bacillus
stearothermophilus (temperature
maximum 75oC) has been grown
commercially to obtain the enzymes
used in 'biological' washing powders.
• Thermophiles are able to survive at
high temperature because they
change there amino acids so there
can be more ionic, hydrogen and
disulphide bonds. The also make
their enzymes which more
hydrophobic amino acids which will
help to repel water.
• Psychrophiles (cold-loving) can grow at 0oC, and
some even as low as -10oC; their upper limit is often
about 25oC
• They are present in alpine and arctic soils, high-
latitude and deep ocean waters, polar ice, glaciers,
and snowfields. They are of particular interest to
astrobiology, the field dedicated to the formulation
of theory about the possibility of extraterrestrial
life, and to geomicrobiology, the study of microbes
active in geochemical processes.
 pH
• All enzymes function best between a
certain pH range.
• At the optimum pH enzymes function
best
• Outside the pH range enzymes begin to
denature.
• The ionic bonds that help to determine
the 3-D shape of the protein can be
altered
• Changes in pH may not only affect the
shape of an enzyme but it may also alter
the charged properties of the substrate
so that either the substrate cannot bind
to the active
site or it cannot undergo catalysis.
pH
Alkaline phosphatase is a
responsible for removing groups
from many types of molecules
such as and .

&
Arginase Arginase is
involved in the
deamination of
excess amino acids
and hence the
formation of urea
• Sulfolobus species gain
their energy by
oxidising the sulphur
granules around hot
springs, generating
sulphuric acid and
thereby lowering the
pH.
• They also have a low pH
optimum (pH 2-3) so
they are termed
thermoacidophiles.
 Substrate concentration
• For a given enzyme concentration , the rate of an
enzyme reaction increases with increase of substrate
concentration.
• A point is eventually reached where there is no further
increase in reaction even if more substrates are added.
• This point is known as Vmax which is the maximum rate
of reaction.
• This is because all the active sites off all the enzymes
are saturated. Thus an extra substrate has to wait until
the enzyme/substrate complex has released the
product before it may itself enter the active site of the
enzyme.
 Substrate concentration

• Vmax

• Initial rate
of reaction

• Substrate concentration
 Enzyme concentration
• Providing that the substrate concentration is
maintained at a high level, and other factors
such as pH and temperature are kept
constant, the rate of reaction is proportional
to enzyme concentration.
• Thus as the enzyme concentration increases,
so to will the rate of the enzyme reaction.
 Enzyme concentration

• Initial rate
of reaction

• enzyme concentration
 Presence of inhibitors
• An inhibitor is a molecule which can reduce
the rate of enzyme reactions.
• Inhibition may be competitive or non-
competitive. Non-competitive may be
reversible or non-reversible.
 Competitive inhibition
• This type of inhibition occurs when another
molecule which has a similar shape to the
substrate binds to the enzyme, preventing the
substrate itself from binding.
• They therefore compete for the active site hence
why it is called competitive inhibition.
• This type is however reversible. By adding more
substrates, this will help to out compete the
inhibitors, and hence increase the rate of reaction.
Competitive inhibition
 Competitive inhibition of succinic
dehydrogenase
• Succinic acid is oxidized to
fumaric acid in the presence of
the enzyme succinic
dehydrogenase and a suitable
hydrogen acceptor (FAD) in the
process of respiration.
It is possible however, to block
the active site of the enzyme by
using a molecule which closely
resembles the substrate.
Malonic acid is such a molecule.
Another example of competitive inhibition is nicotine mimicking acetylcholine in synapses.
When a nerve impulse reaches the end of one neuron, it triggers the release of
neurotransmitters (acetylcholine) into the synapse.
The acetylcholine molecules which are release bind to the next nerve. This causes a nerve
impulse to again be generated, so that the nerve impulse in continued, and keep going
from one nerve to the next.
After the nerve impulse has passed, an enzyme called acetylcholinesterase breaks down all
the acetycholine , in order to prevent continuous nerve impulses from being sent.
Nicotine mimics acetycholine. This causes nerve impulses to be sent continuously.
 Non-competitive inhibition
Reversible
• In this type of inhibition, the inhibitor has no similar shape as
the substrate. It does not compete for the active site, so
increasing substrate concentration has no effect on the rate of
reaction.
• The inhibitor binds to the enzyme at a place other that the
active site, for example an allosteric site. This changes the
shape of the active site so substrates can no longer bind to the
enzyme.
• Examples of inhibitors include heavy metals such as lead and
mercury.
• This type of inhibition can be reversed by adding more
enzymes or by adding a molecule that has a higher affinity for
the allosteric site than the inhibitor.
 Non competitive inhibition

E.g. cyanide poison

 Non-competitive inhibition
Irreversible
• Sometime inhibitors bind to enzymes permanently
• This results the permanent denaturation of the enzyme.
• A prime example is the nerve gas (diisopropylphosphofluoridate ) DIPF.
This inhibitor permanently binds to the enzyme acetyl-cholinesterase
which is responsible for breaking down the neurotransmitter
acetylcholine after a nerve impulse has been transmitted
• Another example is cyanide poison.
• Cyanide poison inhibits the enzyme cytochrome c oxidase which is
responsible for combining electron, oxygen and hydrogen ions to form
water, in the last step of extracting energy from sugar (respiration).
• Organophosphates such as malathion and parathion, are used as
insecticides or as nerve gases (soman and sarin). They inhibit the enzyme
acetylcholinesterase and therefore interfere with normal neuro-
muscularactivity.
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End product inhibition (Negative feedback
inhibition)
• Usually before a substrate has to be converted into a product, a
number of intermediate products have to be formed before the final
product is made.
• It involves more than one enzymes in a series of steps called a
biochemical pathway.
• In end product inhibition, the final products act as an inhibitors to the
reaction.
• It occurs when the final products are in a high enough concentration,
and making more simply wouldn’t make any sense, as this will be
wasteful.
• The final product then binds to the allosteric site of enzyme one of the
biochemical pathway.
• This changes the shape of the active site of the enzyme which prevents
any further catalyst of the enzyme, and the reaction therefore stops.
 Substrate2

Substrate3 Substrate4
 End product

Active site
closed

Allosteric site
 Classification of enzymes
Enzymes can be classified according to the chemical reactions they
catalyze
• 1. Oxidoreductases oxidation/reduction
(eg. dehydrogenases)
• 2. Transferases group transfer
(eg. kinases)
• 3. Hydrolases hydrolysis
(eg. proteases)
• 4. Lyases lysis, generating double bond
(eg. synthases)
• 5. Isomerases rearrangement
(eg. racemases)
• 6. Ligases ligation requiring ATP
(eg. synthetases)