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,Assistant Professor,
Department of Pharmaceutical Chemistry.
Introduction, properties, nomenclature and IUB
classification of enzymes
Enzyme kinetics (Michaelis plot, Line Weaver Burke plot)
Enzyme inhibitors with examples
Regulation of enzymes: enzyme induction and repression,
allosteric enzymes regulation
Therapeutic and diagnostic applications of enzymes and
isoenzymes
Coenzymes –Structure and biochemical functions
All living things require energy.
Nutrients are one source of energy, as well as being
molecules organisms require
to grow, reproduce or repair.
Biochemical reactions are the
processes used for the
formation, breakdown and
rearrangement of molecules to
provide organisms with energy.
Activation Energy is the required input of energy to make
a reaction start
A catalyst is a chemical that speeds up the reaction but is
not used up in the reaction
◦ Lowers the activation energy needed to start a reaction
◦ Is not used up during the reaction
◦ Is unchanged after a reaction
Enzymes act as bio-catalyst.
Enzymes are proteins that
speed up a rate of reaction
Found in cells throughout the body
Lowers activation energy
Increasing the temperature make molecules move faster
Biological systems are very sensitive to temperature
changes.
Enzymes can increase the rate of reactions without
increasing the temperature.
They do this by lowering the activation energy.
They create a new reaction pathway “a short cut”
Enzyme controlled reactions proceed 108 to 1011 times
faster than corresponding non-enzymic reactions.
The substrate
The substrate of an enzyme are the reactants that are
activated by the enzyme
Enzymes are specific to their substrates
The specificity is determined by the active site
The active site
One part of an enzyme, the active site, is particularly
important
The shape and the
chemical environment
inside the active site
permits a chemical
reaction to proceed
more easily
Cofactors
An additional non-protein molecule that is needed by some
enzymes to help the reaction
Tightly bound cofactors are called prosthetic groups
Cofactors that are bound and released easily are called
coenzymes
Many vitamins are coenzymes
Specificity
1. Absolute Specificity – the characteristic that an enzyme
acts on only one substrate.
Eg: Glucokinase - Phosphorylates only glucose,
Urea is the only substrate for urease.
Specificity
2. Relative Specificity – the characteristic that an enzyme
acts on several structurally related substrates.
Eg: Hexokinase can catalyze phosphorylation of glucose,
galactose & mannose.
Specificity
3. Stereochemical Specificity – an enzyme's ability to
distinguish between stereoisomers
Eg: L-Amino acid oxidase – oxidises only L-amino
acids into α-keto acid and ammonia.
D-Amino acid oxidase – oxidises only D-amino acids
into α-keto acid and ammonia.
Denaturation - The process of unfolding
Most named for substrates & for reactions, with suffix “ase”
The name of an enzyme in many cases end in –ase
For example, sucrase catalyzes the hydrolysis of sucrose
The name describes the function of the enzyme
For example, oxidases catalyze oxidation reactions
Sometimes common names are used, particularly for the
digestion enzymes such as pepsin and trypsin
Some names describe both the substrate and the function
For example, alcohol dehydrogenase oxides ethanol
EC 1. Oxidoreductases
EC 2. Transferases
EC 3. Hydrolases
EC 4. Lyases
EC 5. Isomerases
EC 6. Ligases
PRINCIPLE OF THE INTERNATIONAL
CLASSIFICATION
6 CH2OH 6 CH OPO 2
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H OH
Mg2+
OH OH OH OH
3 2 3 2
H OH Hexokinase H OH
glucose glucose-6-phosphate
CLASSIFICATION
MECHANISM OF ACTION
Michaelis and Menten Concept
When the enzyme and substrate are connected, it is
known as enzyme-substrate complex
The binding site is where the enzyme physically attaches
itself to the substrate
The active site is where the enzyme will cause a specific
part of the substrate to change
MECHANISM OF ACTION
When a substrate (S) fits properly in an active site, an
enzyme-substrate (ES) complex is formed:
E + S ES
THEORIES
Fischer’s Lock and Key Model
+ S +
S
P
E + S ES complex E + P
The Induced Fit Hypothesis
Some proteins can change their shape (conformation)
When a substrate combines with an enzyme, it induces a
change in the enzyme’s conformation
The active site is then moulded into a precise conformation
Making the chemical environment suitable for the reaction
The bonds of the substrate are stretched to make the
reaction easier (lowers activation energy)
Koshland’s Induced Fit Model
P
S
S
P
E + S ES complex E + P
Enzyme Concentration
Substrate Concentration
pH
Temperature
Inhibitors
1. Enzyme Concentration
The increase in velocity is proportional to the enzyme concentration
Reaction
velocity
Enzyme concentration
Faster reaction but it reaches a saturation point when all the
enzyme molecules are occupied.
If you alter the concentration of the enzyme then Vmax will
change too. Vmax
Reaction
velocity
Substrate concentration
Extreme pH levels will produce denaturation
The structure of the enzyme is changed
The active site is distorted and the substrate molecules will
no longer fit in it
At pH values slightly different from the enzyme’s optimum
value, small changes in the charges of the enzyme and it’s
substrate molecules will occur
This change in ionisation will affect the binding of the
substrate with the active site.
Optimum pH values
Enzyme
activity Trypsin
Pepsin
1 3 5 7 9 11
pH
Q10 (the temperature coefficient) = the increase in
reaction rate with a 10°C rise in temperature.
Enzyme-controlled reactions follow this rule as they are
chemical reactions
BUT at high temperatures proteins denature
The optimum temperature for an enzyme controlled reaction
will be a balance between the Q10 and denaturation.
Q10 Denaturation
Enzyme
activity
0 10 20 30 40 50
Temperature / °C
Inhibitors are chemicals that reduce the rate of enzymic
reactions.
The are usually specific and they work at low
concentrations.
They block the enzyme but they do not usually destroy it.
Many drugs and poisons are inhibitors of enzymes in the
nervous system.
?
The number of molecules of substrate with which a
single enzyme can react at a given time (ex.
reactions/minute) is known as the turnover number
◦ Can be quite large compared to uncatalyzed
reeactions
◦ Can depend on the environment
Order of reaction : When the velocity of the reaction is
almost proportional to the substrate concentration( i.e. [S] is
less than Km), the rate of reaction is said to be first order
with respect to substrate. When the [S] is much greater than
Km, the rate of reaction is independent of substrate
concentration, and the reaction is said to be zero order.
Enzyme kinetics and Km value : The enzyme (E) and
substrate (S) combine with each other to form an unstable
enzyme-substrate complex (ES) for the formation of
product (P).
The process of inhibition of enzymes activity.
1. Reversible Enzyme Inhibition:
a. Competitive Reversible Enzyme Inhibition
b. Noncompetitive Reversible Enzyme Inhibition
2. Irreversible Enzyme Inhibition
3. Allosteric Inhibition
These can be washed out of the solution of enzyme by dialysis.
There are two categories.
a. Competitive - mimic the substrate and bind to the active site.
b. Noncompetitive - bind to some other part of the enzyme.
These compete with the substrate molecules for the active site.
The inhibitor’s action is proportional to its concentration.
Resembles the substrate’s structure closely.
Succinate Fumarate + 2H++ 2e-
Succinate dehydrogenase
CH2COOH CHCOOH
CH2COOH CHCOOH
COOH
COOH
CH2
COOH COOH
Oxalate Malonate
These are not influenced by the concentration of the
substrate. It inhibits by binding irreversibly to the enzyme
but not at the active site.
Examples - Cyanide combines with the Iron in the enzymes
cytochrome oxidase.
Heavy metals, Ag or Hg, combine with –SH groups.
These can be removed by using a chelating agent such as
EDTA.
Combine with the functional groups of the amino acids in
the active site, irreversibly.
Examples: nerve gases and pesticides, containing
organophosphorus, combine with serine residues in the
enzyme acetylcholine esterase.
Allosteric Enzyme – an enzyme with a quaternary structure
whose activity is changes by the binding of a modulator
Cell’s can’t have enzymes turned on all the time
The control of an enzyme complex by the binding of a
regulatory molecule.
Regulatory molecule may stimulate or inhibit the enzyme
complex by causing it to change shape.
Works like a reversible non-competitive inhibitor
Allosteric regulation
Heterotropic ligand binding modulates substrate binding and
catalysis.
Feedback Regulation
Homotropic regulation – Multisubunit
Covalent modification – Reversible
Phosphorylation, nucleotides, lipid anchors
The end product of a metabolic pathway affects the function
of an enzyme
A metabolic pathway is switched off by the inhibitory
binding of its end product to an enzyme that acts early in
the pathway
Usually allosteric regulation
The enzyme without its non protein moiety is termed as
apoenzyme and it is inactive.
Holoenzyme is an active enzyme with its non protein
component.
Some enzymes need an additional molecule like co-factors /
co-enzymes to carry out the process.
Cofactors are molecules that serve an enzyme helpers.
A cofactor can be a metal ion or an organic molecule.
A cofactor that is an organic molecule is called a
coenzyme.
Vitamin derived Co-enzymes
I. Enzymes as Therapeutic Agents (Drugs)
S. Enzyme Disease/therapy
No
1 Streptokinase Clot lysis in myocardial infarction,
trauma, bleedings
2 Aspariginase Acute lymphocytic leukemia