Introduction to enzymes comprised of the various properties of enzymes, their functions and properties

1. Program: B.Sc Biotechnology/ Microbiology
Semester 4, Second Year
BSBT/MB 403: Enzymology
Unit-I
Introduction to Enzymes
Lecture-1
Dr. Rita Sharma
Assistant Professor/School of Life Science
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2. • Introduction
• Cofactors & co enzymes
• Classification
• Lock and Key hypothesis
• Factors affecting enzyme activity
• Applications
• Exercise
• SELO
• References
Contents
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3. • To understand the basic nature of enzymes, its various
classes and applications.
Objective
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4. Apple Experiment
Come down and get an apple and a slice of lemon.
When you get back to your seat:
1. Take a big bite of your delicious apple.
2. Immediately squeeze lemon juice over the apple flesh that is now exposed
from the bite.
3. IMPORTANT! Don’t get lemon juice all over the apple. Make sure that it
is ONLY on the area that you just bit!
4. Set the lemon aside and wipe any lemon juice off of your hands with a
napkin.
5. Take another bite from the opposite side of your apple.
6. Set your apple aside.

5. What are
enzymes?
Enzymes are
proteins
(tertiary and
quaternary
structures).
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6. Introduction
• Proteinaceous substances that catalyses the
chemical reactions without undergoing any
change in themselves.
• Term enzymes was coined by “Kuhne” in 1878.
• Increases the rate of reaction by increasing
the activation energy.
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7. Cofactors & Coenzymes
• Non-protein substances (zinc, iron,
copper, vitamins) are sometimes need
for proper enzymatic activity are
cofactors.
• Organic cofactor = coenzymes
• Cofactors+ coenzymes = Apoenzymes
(prosthetic group)
• Complex of apoenzyme+ cofactor =
Holoenzyme
• Many vitamins are coenzymes
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8. Enzymes v/s catalysts
• biological origin
• Highly specific
• Enhanced rate of reaction by 10³
• Subjected to variety of
regulations which increase or
decrease the rate of reaction
• Maximum reaction rate
• Proteinaceous nature
• Mild, biologically compatible
• Side reactions do not occur
• Chemical origin
• Non-specific
• Just a fraction of that
• Not subjected to regulation
• Do not show substarte
saturation
• Metal & non- metal inorganic
molecules
• Often high temp. & pressure
• Occurs
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9. Classification & nomenclature
• Based on enzyme commision (EC)
• A 4 no. enzyme code following the letters
EC e.g. EC1.1.1.3
• 1st no. refers to the enzyme class
• 2nd- sub-class, 3rd – subclass
• 4th to the serial no.of enzyme within a
subclass
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10. Class Name Description Example
1 Oxidoreductases Redox reactions, transfer of
hydrogen & oxygen atom b/w m
molecules. Dehydrogenas,
oxidase,peroxidase etc.
Glucose oxidase
(EC 1.1.3.4)
2 Transferases Transfer of an atom or gp of atom
(alkyl,glycosyl)
Aspartate
aminotransferase
(EC 2.6.1.1)
3 Hydrolases Catalyses hydrolytic reactions.
Esterases, glycosidases, lipases,
proteases
Chymosin or renin
(EC 3.4.23.4)
4 Lyases Elimination reactions,removal of a
group of atoms from substrate
molecules. Aldoses, dehydratases.
Ammonia lyase
(EC 4.3.1.3)
5 Isomerses Catalyses the formation of isomers
of molecules.
Epimerases,racemases.
Xylose isomerases
(EC 5.3.1.5)
6 Ligases Formation of covalent bonds b/w 2
molecules utilizing the energy
obtained from hydrolysis of NTP.
Glutathione
synthetases
(EC 6.3.2.3)
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11. BSBT/MB 403 11

12. BSBT/MB 403 12

13. What do enzymes do?
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”
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14. The Lock and Key Hypothesis
Enzyme may
be used again
Enzyme-
substrate
complex
E
S
P
E
E
P
Reaction coordinate
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15. Fit between the substrate and the active site of the
enzyme is exact Like a key fits into a lock very precisely
The key is analogous to the enzyme and the substrate
analogous to the lock.
Temporary structure called the enzyme-substrate
complex formed Products have a different shape from
the substrate
Once formed, they are released from the active site
leaving it free to become attached to another substrate
The Lock and Key Hypothesis
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16. How do you stop
an enzyme?
Irreversible
egg protein
denaturation
caused by high
temperature
(while cooking
it).
• Alteration of a protein shape through
some form of external stress
• Example, by applying heat or changing
pH.
• Denatured protein can’t carry out its
cellular function .
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17. 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.
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18. Factors affecting Enzymes
• substrate concentration
• pH
• temperature
• inhibitors
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19. Substrate concentration:
Non-enzymic reactions
Reaction
velocity
Substrate concentration
• The increase in velocity is proportional to the
substrate concentration
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20. Substrate concentration:
Enzymic reactions
Reaction
velocity
Substrate concentration
Vmax
• 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. BSBT/MB 403 20

21. The effect of pH
Enzyme
activity Trypsin
Pepsin
pH
1 3 5 7 9 11
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22. The effect of pH
• 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.
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23. The effect of temperature
• Q10 (the temperature coefficient) = the
increase in reaction rate with a 10°C rise in
temperature.
• For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or triples
with every 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.
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24. Temperature / °C
Enzyme
activity
0 10 20 30 40 50
Q10 Denaturation
The effect of temperature
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25. The effect of temperature
• For most enzymes the optimum temperature
is about 30°C
• Many are a lot lower, cold water fish will die
at 30°C because their enzymes denature
• A few bacteria have enzymes that can
withstand very high temperatures up to 100°C
• Most enzymes however are fully denatured at
70°C
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26. 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)
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27. Enzyme Inhibitors
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28. Inhibitors
• 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.
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29. Types of inhibition
• Competitive inhibition
• Non-competitive inhibition
• Uncompetitive inhibition
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30. •Inhibitor compete with substrate for active sites.
•Structural analogue of substrate/ looks like a
substrate.
•Inhibition is reversible
•Inhibitor does not bind with ES complex
•Inhibitor’s action is proportional to the substarte
concentration
Competitive inhibition
Example
•Lactate dehydrogenase: lactate
,oxamate
•Succinate dehydrogenase : succinate,
malonate
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31. Non-competitive inhibition
•Not resemble substrate
•Binds to site other than active site
•Can bind with es complex
•Usually irreversible
•Increasing substrate concentration will not abolish inhibition
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.
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32. •Inhibitor has no affinity for free enzyme
•Binds to ES complex
•No product is produced
•Example : Inhibition of placental alkaline
phosphatase (regan isoenzyme) by phenylalanine
Uncompetitive Inhibition
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33. Meet the Enzyme: Catecholase
lemon juice and other acids are used to preserve
color in fruit, particularly apples, by lowering the pH
and removing the copper (cofactor) necessary for
the enzyme to function.
Reaction:
catecholase
catechol + O2 ----- polyphenol
colorless substrate brown product
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34. Enzyme kinetics
• Basic reaction
S + E ES E + P
Where
• S= substrate
– Substance on which the enzyme acts
• E= Enzyme
• ES= enzyme-substrate product
– Physical binding of a substrate to the active site of
enzyme
• P= Product
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35. •No matter how large the substrate concentration,reaction rate
can never exceed vmax.
•Km is the substrate concentration at which reaction rate is half
maximal.
•Km reflects the binding affinity of the enzyme for the substrate;
the higher the affinity, the smaller is km.
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36. Vo = VMAX
[S]
[S] + KM
Vo = VMAX
[S]
[S] + KM
•Km IS Called Michaelis constant & it is independent of enzyme
conc.
•Km is constant for enzyme.
•It is expressed in moles/l.
MICHAELIS-MENTEN EQUATION
•Where
–V0: velocity/rate of enzymatic activity
–Vmax: The maximal rate of reaction when the enzyme
is saturated
–Km: (constant)the substrate concentration that
produces ½ of the maximal velocity
–S: substrate concentration
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37. Properties of enzymes
• Proteinaceous nature
• High specificity
• High reactivity
• Denaturation
• Catalytic nature
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38. Applications of enzymes
Biological Functions of Enzymes:
• Enzymes perform a wide variety of functions in
living organisms.
• They are major components in signal transduction
and cell regulation, kinases and phosphatases help in
this function.
• They take part in movement with the help of the
protein myosin which aids in muscle contraction.
• Also other ATPases in the cell membrane acts as ion
pumps in active transport mechanism.
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39. • Enzymes present in the viruses are for infecting cell.
• Enzymes play a important role in the digestive
activity of the enzymes.
• Amylases and proteases are enzymes that breakdown
large molecules into absorbable molecules.
• Variuos enzymes work together in a order forming
metabolic pathways. Example: Glycolysis.
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40. Industrial Application of Enzymes:
•
Food Processing - Amylases enzymes from fungi and plants are
used in production of sugars from starch in making corn-syrup.
• Catalyze enzyme is used in breakdown of starch into sugar, and
in baking fermentation process of yeast raises the dough.
• Proteases enzyme help in manufacture of biscuits in lowering
the protein level.
• Baby foods - Trypsin enzyme is used in pre-digestion of baby
foods.
• Brewing industry - Enzymes from barley are widely used in
brewing industries.
• Amylases, glucanases, proteases, betaglucanases,
arabinoxylases, amyloglucosidase, acetolactatedecarboxylases
are used in prodcution of beer industries.
• Fruit juices - Enzymes like cellulases, pectinases help are used
in clarifying fruit juices.
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41. • Dairy Industry - Renin is used inmanufacture of
cheese. Lipases are used in ripening blue-mold cheese.
Lactases breaks down lactose to glucose and
galactose.
• Meat Tenderizes - Papain is used to soften meat.
• Starch Industry - Amylases, amyloglucosidases and
glycoamylases converts starch into glucose and
syrups.
• Glucose isomerases - production enhanced sweetening
properties and lowering calorific values.
• Paper industry - Enzymes like amylases, xylanases,
cellulases and liginases lower the viscosity, and
removes lignin to soften paper.
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42. • Biofuel Industry - Enzymes like cellulases are used in
breakdown of cellulose into sugars which can be fermented
• Biological detergent - proteases, amylases, lipases,
cellulases, asist in removal of protein stains,
oily stains and acts as fabric conditioners.
• Rubber Industry - Catalase enzyme converts latex
into foam rubber.
• Molecular Biology - Restriction enzymes, DNA ligase
and polymerases are used in genetic engineering,
pharmacology, agriculture, medicine, PCR techniques,
and are also important in forensic science.
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43. Assignment
• At the end of most lectures,
I will give you some type of
in-class assignment or
homework to evaluate your
understanding of today’s
topic.
• This assignment will always
be open-book.
• Today you may be completing
an experiment on the topic
of Enzymes.
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44. Assignment
• Define Enzymes.
• Give the different classes of enzymes.
• Discuss Lock and key hypothesis.
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45. SELO
1. Lifelong learning ability.
2. Design thinking ability
3. Application of concerning standards realistically to design a
solution component or a product
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46. Thank you
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