6.

Enzyme and Biocatalysts’

 Enzymes organic molecules with specific catalytic
functions i.e., enzymes are biological catalysts.
 A catalyst is a substance which speeds up
(increases) the rate of a chemical reaction and is not
itself consumed during the reaction.
 Catalysts are effective in very small amounts.

 Enzymes speed up, or increases the rates of

chemical reactions.
What are enzymes made of?
Generally All enzymes are proteins,
proteins which are
polymers of amino acids. But except RNA which acts
as an enzyme and is known as a ribozyme
Some enzymes also require a cofactor.
The reactants of an enzyme-catalysed reaction are
called substrates and the products are called
products (obvious really).
 Substrate-binding site
The area of the enzyme to which the substrate
binds is called the substrate-binding site(active
site).
site)
The substrate is bound to this site by a number of
non-covalent bonds
 Types of bonds involved
• Hydrogen bonds
• Ionic bonds (salt
bridges)
• Hydrophobic
interactions
• van der Waals
interactions
Chemical Nature of Enzymes
• Most enzymes are proteins Enzymes may require a
non-peptide component as a cofactor.
• The peptide component is called the apoenzyme,
apoenzyme
the cofactor is called as the coenzyme and the
combined functional unit is the holoenzyme.
holoenzyme
• Apoenzyme + Cofactor = Holoenzyme

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 Chemical Nature of Enzymes
• The cofactor may be:
– A coenzyme a non-protein organic substance and
loosely attached to the protein part.
– A prosthetic group -an organic substance and
thermostable which is firmly attached to the protein
– A metal ion activator -these include K+, Fe++, Fe+++,
Cu++, Co++, Zn++, Mn++, Mg++, Ca++,and Mo+++.
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 Specificity of Enzymes
• Absolute specificity -the enzyme will catalyze only
one reaction.
• Group specificity -the enzyme will act only on
molecules that have specific functional groups, such
as amino, phosphate and methyl groups.
• Linkage specificity -the enzyme will act on a
particular type of chemical bond regardless of the
rest of the molecular structure.
• Stereochemical specificity -the enzyme will act on
a particular steric or optical isomer.
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 How are enzymes named?
• Enzymes are classified according to the reactions they carry out.

• Except for some of the originally studied enzymes such as pepsin, rennin, and
trypsin, most enzyme names end in "ase".
• Enzyme classification Enzymology : the systematic naming and classification of
enzymes based on the 1972 recommendations of the Nomenclature Committee of
the International Union of Biochemistry.
• Each enzyme is assigned a number consisting of four figures; the first figure
designates one of six main divisions: oxidoreductases, transferases, hydrolases,
lyases, isomerases, and ligases; the second figure denotes the subclass, the
third denotes the sub-subclass, and the fourth is the serial number of the enzyme
in its sub-subclass; the enzyme number is preceded by the abbreviation EC.
 The Six Classes of Enzymes
• There are six major groups of enzymes
– Oxidoreductases
– Transferases
– Hydrolases
– Lyases
– Isomerases
– Ligases
 The Six Classes of Enzymes

1. Oxidoreductases (dehydrogenases)
• Catalyze oxidation-reduction reactions

2. Transferases

• Catalyze group transfer reactions

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 3. Hydrolases

• Catalyze hydrolysis reactions where water is the

acceptor of the transferred group

4. Lyases
Catalyze lysis of a substrate, generating a double bond in a
nonhydrolytic, nonoxidative elimination (Synthases catalyze the
addition to a double bond, the reverse reaction of a lyase)

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5. Isomerases
• Catalyze isomerization reactions

6. Ligases (synthetases)
Catalyze ligation, or joining of two substrates
Require chemical energy (e.g. ATP)

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Enzyme Kinetics
Basic Enzyme Reactions
• The basic enzymatic reaction can be represented as
follows where E represents the enzyme catalyzing the
reaction, S the substrate, the substance being changed,
and P the product of the reaction.

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Enzyme Kinetics
Energy Levels
• for most chemical reactions to proceed, some
form of energy is needed "the energy of
activation."
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.

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Enzyme Kinetics

15
Enzyme Kinetics
The Enzyme Substrate Complex
• the substrate and enzyme formed some
intermediate substance which is known as the
enzyme substrate complex.
complex

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Enzyme Kinetics

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Factors affecting enzyme activity
1. Enzyme concentration
2. Substrate concentration
3. Product concentration
4. Specific activity
5. Cofactors
6. Temperature
7. Hydrogen ion concentration (pH)
8. Ionic strength
9. Solvents
10. Inhibitors and activators
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Factors affecting enzyme activity
• Temperature i.e. supplying
energy to the system (reaction).
• As energy is supplied there is
an increase in the reaction rate.
As the temperature continues to
increase the rate of the reaction
falls off.
• Can you think why?

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• As temperature increases, more bonds, especially the
weaker hydrogen and ionic bonds, will break as a result of
this strain. breaking bonds within the enzyme will cause the
active site to change shape.
• This change in shape means that the Active Site is less
complementary to the shape of the Substrate, so that it is
less likely to catalyse the reaction.
• Eventually, the enzyme will become denatured and will
no longer function.
• The temperature at which the maximum rate of reaction
occurs is called the enzymes Optimum Temperature. This
is different for different enzymes.
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Factors affecting enzyme activity
• The hydrogen ion
concentration (pH)
pH
also affects the rate of
an enzyme catalysed
reaction.
• Can you think why?
(Hint - charged side
groups.)

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 At the optimum pH, the rate of reaction is at an optimum.

 Any change in pH above or below the optimum will quickly cause a

decrease in the rate of reaction, since more of the enzyme molecules
will have active sites whose shapes are not (or at least are less)
complementary to the shape of their substrate.
 For example, the enzyme pepsin functions best at around pH-2 and is
found in the stomach, which contains hydrochloric acid.

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Factors affecting enzyme activity
Substrate concentration

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 Factors affecting enzyme activity
Substrate concentration
• It is theorized that when this
maximum velocity had been
reached, all of the available
enzyme has been converted to ES
• This point on the graph is
designated Vmax.
Vmax
• The Michaelis constant Km is
defined as the substrate
concentration at 1/2 the
maximum velocity.
velocity

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 Factors affecting enzyme activity
Substrate concentration
• Using this constant and the fact that Km can also
be defined as:

25
Factors affecting enzyme activity
Substrate concentration
• Michaelis constants have been determined for many of the
commonly used enzymes. The size of Km tells us several things
about a particular enzyme.
– A small Km indicates that the enzyme requires only a small
amount of substrate to become saturated.
– A large Km indicates the need for high substrate
concentrations to achieve maximum reaction velocity.
– The substrate with the lowest Km upon which the enzyme
acts as a catalyst is frequently assumed to be enzyme's natural
substrate

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 Factors affecting enzyme activity
Cofactors

Many enzymes need

factors other than
polypeptide molecules
for full activity.

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 Factors affecting enzyme activity
Cofactors
1. Prosthetic groups
Organic molecules tightly bound to the enzyme
Flavin adenine dinucleotide (FAD) of succinate
dehydrogenase.
Biotin of carboxylases e.g. pyruvate carboxylase.
2. Coenzymes
Organic molecules loosely associated with the
enzyme.
NAD+ or NADP+ in enzymes catalysing redox
reactions e.g. dehydrogenases. 28
 Factors affecting enzyme activity
Cofactors

3. Metal ions

Zn2+ in carboxypeptidase A.

Cu2+ in cytochrome oxidase.

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 Factors affecting enzyme activity
Inhibitors, inducers and activators
• Activators:
Activators increase the activity of an enzyme
e.g. ADP is an activator of PFK.
• Inducers:
Inducers switch on (induce) the synthesis of
an enzyme e.g. lactose induces the enzyme -
galactosidase.
• Inhibitors:
Inhibitors inhibit (decrease) the activity of
enzyme molecules.

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 Enzyme Inhibitors
• Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action. (Denaturing agents are not

inhibitors)

• Irreversible inhibitors form covalent or very tight permanent bonds

with aa at the active site of the enzyme and render it inactive. 3

classes: group secific reagents, substrate analogs, suicide

inhibitors

• Reversible inhibitors form an EI complex that can be dissociated

back to enzyme and free inhibitor. 3 groups based on their mechanism

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of action: competitive, non-competitive and uncompetitive

 Factors affecting enzyme activity
Reversible Inhibitors
There are three types of reversible enzyme
inhibitors:
1. Competitive inhibitors
2. Non-competitive inhibitors
3. Un-competitive inhibitors
Excess substrate can inhibit enzyme activity - in
this case substrate molecules bind to another site
causing inhibition. 32
 Factors affecting enzyme activity
Competitive Inhibitors
• As the name suggests competitive inhibitors compete
with the substrate at the substrate-binding site.
• They increase the Km but leave the Vmax unaffected.
• E.g. malonate is a competitive inhibitor of succinate
dehydrogenase.

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 What effect does a competitive
inhibitor have on KM and VMAX?

• Competitive inhibitors
increase the KM.

• Competitive Inhibitors
have no effect on the
VMAX.

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 Factors affecting enzyme activity
Non-competitive Inhibitors
• Non-competitive inhibitors bind at a site away from the
substrate-binding site.
• They affect Vmax but do not affect Km.
• E.g. the heavy metal ions Hg2+ and Pb2+ are non-competitive
inhibitors. They are thought to bind to -SH groups within the
enzyme.
•The KM was not affected.
•The VMAX decreases.

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 Factors affecting enzyme activity
Un-competitive Inhibitors
• Uncompetitive inhibitor binds only to ES
complex and inactivates E.
• They affect Vmax and Km.

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Irreversible Enzyme Inhibition
• Irreversible inhibitors form stable covalent bonds with the enzyme
(e.g. alkylation or acylation of an active site side chain)
• There are many naturally-occurring and synthetic irreversible
inhibitors
• These inhibitors can be used to identify the amino acid residues at
enzyme active sites
• Incubation of I with enzyme results in loss of activity
• Examples of enzyme inhibitors:
Penicillin
Malthion
Glyphosphate
Diisopropylfluorophosphate*
Iodoacetate*
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 Substrate Binding models
Lock-and-key
It used to be thought that the substrate binding to
an enzyme’s substrate binding site was a bit like
an key ‘fitting’ a lock. Both are rigid structures
and the shape of one is complementary to the
other.

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 Substrate Binding
Induced Fit
 It is now though that as the substrate approaches the substrate-binding site of the
enzyme both the substrate molecule and (especially) the enzyme molecule change
their shapes.
 Induced-fit model: This updated model states that enzymes interact with substrates
and in the process change their conformation such that the enzyme is snug around
the substrate, sort of like a glove around a hand.

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 Allosteric Enzymes
• Allosteric enzymes have one or more allosteric
sites
• Allosteric sites are binding sites distinct from an
enzyme’s active site or substrate-binding site
• Molecules that bind to allosteric sites are called
effectors or modulators
• Binding to allosteric sites alters the activity of the
enzyme. This is called cooperative binding.
binding
• Effectors may be positive or negative
• Effectors may be homotropic or heterotropic
• Regulatory enzymes of metabolic pathways are 40
allosteric enzymes (eg: feedback inhibition)
 Allosteric inhibition
• These enzymes
have two receptor
sites
• One site fits the
substrate like other
enzymes Inhibitor
• The other site fits Substrate molecule
an inhibitor cannot fit into
the active site
molecule Inhibitor fits into
allosteric site
 Allosteric inhibition

Active
site

E Allosteric
Substrate site empty
Conformational
E
fits into change Inhibitor
the active molecule
Substrate
site is present
cannot fit
The inhibitor into the
molecule is active site Inhibitor fits
absent into allosteric
site