1. ENZYMES
Dr.Kamlesh shah
A protein with catalytic properties due to its
power of specific activation

2. Enzyme Fundamentals
• Enzymes are protein complexes that speed up biochemical
reactions by lowering the activation energy
• Enzymes accelerate reactions by facilitating the formation of the
transition state
• The position of the equilibrium, enthalpy of reaction, and free
energy of the reaction are unchanged by an enzyme
• The enzymes themselves are the same after the reaction as they
was before
• Enzymes are powerful and highly specific catalysts
• Free energy is a useful thermodynamic function for understanding
enzymes
• The Michaelis-Menten model accounts for the kinetic properties
of many enzymes
• Enzymes can be inhibited by specific molecules
• Vitamins are often precursors to coenzymes

3. Some Enzyme Terminology
• Enzyme – a biomolecule that catalyzes biochemical
reaction by lowering activation energy
• Substrate – the substance that undergoes a chemical
change by an enzyme
• Absolute Specificity – the characteristic that an enzyme
acts on only one substrate
• Relative Specificity – the characteristic that an enzyme
acts on several structurally related substrates
• Stereochemical Specificity – an enzyme's ability to
distinguish between stereoisomers
• Cofactor – a nonprotein molecule or ion required by an
enzyme for catalytic activity
• Coenzyme – an organic molecule required by an
enzyme for catalytic activity

4. More Enzyme Terminology
• Apoenzyme – a catalytically inactive protein formed by
removal of the cofactor from an active enzyme
• Active Site – the location on an enzyme where a
substrate is bound and catalysis occurs
• Enzyme Activity – the rate at which an enzyme
catalyzes a reaction
• Turnover Number – the number of molecules of
substrate acted upon by one molecule of enzyme per
minute
• Enzyme International Unit (IU) – a quantity of enzyme
that catalyzes the conversion of 1 micromole of
substrate per minute under specified conditions
• Optimum Temperature – the temperature at which
enzyme activity is highest

5. And More Enzyme Terminology
• Optimum pH - the pH at which enzyme activity is
highest
• Extremozyme – an enzyme that thrive in extreme
environments
• Enzyme Inhibitor – a substance that decreases the
activity of an enzyme
• Competitive Inhibitor – an inhibitor that binds to the
active site of an enzyme
• Noncompetitive Inhibitor – an inhibitor that binds at a
location other than the enzyme’s active site
• Zymogen (proenzyme) – the inactive enzyme precursor
• Modulator – a substance that binds to an enzyme at a
location other than the active site that alters the
enzyme's catalytic activity

6. And Yet More Enzyme Terminology
• Allosteric Enzyme – an enzyme with a quaternary
structure whose activity is changes by the binding of a
modulator
• Activator – a substance that binds to the allosteric
enzyme and increases its activity
• Feedback Inhibition – a process in which the end
product of a sequence of enzyme catalyzed reaction
inhibits an earlier step in the process
• Enzyme Induction – the synthesis of enzyme in
response to a cellular need
• Isoenzyme – a slightly different form of the same
enzyme produced by different tissues
• Holoenzyme – apoenzyme + cofactor

7. Two Fundamental conditions for life
 Ability to self replicate.
 Ability to catalyze chemical reactions efficiently and
specifically.
 Example

8. Enzymes
 Reaction catalysts of biological system.
 Extraordinary catalytic power.
 High degree of specificity.
 Function in aqueous solutions under mild conditions
of temperature and pH.

9. Practical Importance
 Inheritable genetic disorders (deficiency or total
absence of enzymes).
 Excessive activity of enzymes may cause diseases.
 Diagnosing certain illness.
 Many drugs exert their effect by interacting with
enzymes.
 Important practical tools in medicine, chemical
industry, food processing and agriculture.

10. History
 1850 – Louis Pasteur
Concluded that fermentation of sugar to alcohol by
yeast is catalyzed by “ferments”.
 1878 – The molecules extracted from cells
responsible for catalysis were named “Enzymes” by
Frederick Kuhne.
 1897 – Eduard Buchner
Disproved “Vitalism” and stated that fermentation
could be carried out by yeast extracts.

11. Contd….
 1926 James Sumner
Isolated and crystallized Urease enzyme and
postulated that “ All Enzymes are Proteins”.
 John Northrop and Moses Kunitz crystallized
pepsin, trypsin and other digestive enzymes.

12. Enzyme structure
 All Enzymes are Proteins
 Exception – Catalytic RNA
molécules
 Molecular Weight – 12,000
to >1 million.
 They have a globular shape.
 A complex 3-D structure.
 1o,2o,3o, and 4o structures of
protein enzymes are essential
for their Catalytic activity.
Human pancreatic amylase

13. Enzymes
 Endoenzymes or
Intracellular
 Act within the cells
in which they are
produced
 Metabolic enzymes
 Plant enzymes
 Exoenzymes or
extracellular
 Liberated by living
cells and act outside
in its environment
 Chiefly act as
digestive enzymes
 Microbial enzymes

14. Cofactors
 An additional non-protein
molecule that is needed by
some enzymes to help the
reaction.
 Example:
• Inorganic ions such as Fe2+,
Mg2+, Mn2+, or Zn2+
• Complex organic or
metalloorganic molecule
called a Coenzyme.
 Some enzymes require both
Coenzymes as well as one
or more metal ions for their
activity. Nitrogenase enzyme with Fe, Mo and ADP cofactors

15. Cofactors…..
 Tightly bound cofactors are called Prosthetic groups.
 Cofactors that are bound and released easily are
called coenzymes
 Many vitamins are coenzymes.
 Complete catalytically active enzyme together with
bound coenzyme/ metal ion – Holoenzyme.
 Protein part of the enzyme - Apoenzyme or
Apoprotein.

16. 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

17. Enzyme Classification

19. Nomenclature
 Enzyme commission - Six classes
– 1st Main class, 2nd Sub class, 3rd Sub subclass
– 4th molecule
1.Oxidoreductases 2. Transferases 3. Hydrolases
4. Lyases 5. Isomerases 6. Ligases
Eg: Histidine carboxylyase: EC. 4.1.1.22
Lyase C - C
C-COOH
Histidine

20. Chemical reactions
 Chemical reactions need an initial input of energy =
THE ACTIVATION ENERGY
 During this part of the reaction the molecules are
said to be in a transition state.

21. Reaction pathway

22. An enzyme controlled pathway
 Enzyme controlled reactions proceed 108 to 1011 times faster
than corresponding non-enzymic reactions.

23. Making reactions go faster
 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”

24. 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

25. The Lock and Key Hypothesis
 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

26. The Lock and Key Hypothesis
Enzyme may
be used again
Enzyme-
substrate
complex
E
S
P
E
E
P
Reaction coordinate

27. The Lock and Key Hypothesis
 This explains enzyme specificity
 This explains the loss of activity when enzymes
denature

28. 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)

29. The Induced Fit Hypothesis
 This explains the enzymes that can react with a
range of substrates of similar types
Hexokinase (a) without (b) with glucose substrate
http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

30. Factors affecting Enzymes
 substrate concentration
 pH
 temperature
 inhibitors

31. Substrate concentration: Non-enzymic reactions
 The increase in velocity is proportional to the
substrate concentration
Reaction
velocity
Substrate concentration

32. Substrate concentration: Enzymic reactions
 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.
Reaction
velocity
Substrate concentration
Vmax

33. The effect of pH
Optimum pH values
Enzyme
activity Trypsin
Pepsin
pH
1 3 5 7 9 11

34. 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.

35. 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.

36. The effect of temperature
Temperature / °C
Enzyme
activity
0 10 20 30 40 50
Q10 Denaturation

37. 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

38. 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.

39. The effect of enzyme inhibition
 Irreversible inhibitors: 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.

40. The effect of enzyme inhibition
 Reversible inhibitors: These can be washed out of
the solution of enzyme by dialysis.
There are two categories.

41. The effect of enzyme inhibition
1. Competitive: 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.
Enzyme inhibitor
complex
Reversible
reaction
E + I EI

42. The effect of enzyme inhibition
Succinate Fumarate + 2H++ 2e-
Succinate dehydrogenase
CH2COOH
CH2COOH CHCOOH
CHCOOH
COOH
COOH
CH2
Malonate

43. The effect of enzyme inhibition
2. Non-competitive: 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.

44. Applications of inhibitors
 Negative feedback: end point or end product
inhibition
 Poisons snake bite, plant alkaloids and nerve gases.
 Medicine antibiotics, sulphonamides, sedatives and
stimulants

45. Enzymes in medicine
Glucose oxidase
Glucose Hydrogen peroxide
peroxidase
Dye: Blue---Green---Brown
Dye changes according to
amount of glucose
Enzyme-linked immunosorbent assays (ELISAs) detect
antibodies to infections.
Glucose oxidase + peroxidase + blue dye on dipsticks to detect
glucose in urine:

46. Fermentation
Culture supernatant
Centrifugation
to remove cells
Liquid preparation
to animal feed
market
Fermentation
Culture supernatant
Fermentation
Cell pellet
Intracellular fraction
Animal feed enzyme Analytical enzyme Therapeutic protein
Centrifugation
to remove cells
Centrifugation
to remove
medium
Protein
precipitation
Cell
lysis Centrifugation
Protein fraction
Protein
precipitation
Protein fraction
1 or 2 purification
steps
Semi-purified
protein 3-4 purification
steps
Homogeneous
protein
Sterile
bottling
To pharmaceuticals market
Lyophilisation
Bottling
To chemicals market

47. Fungal Enzymes
Enzyme EC Sources Location Application
a-Amylase 3.2.1.1 Aspergillus E Baking
Catalase 1.11.1.6 Aspergillus I Food
Cellulase 3.2.1.4 Trichoderma E Waste
Dextranase 3.2.1.11 Penicillium E Food
Glucose oxidase 1.1.3.4 Aspergillus I Food
Lactase 3.2.1.23 Aspergillus E Dairy
Lipase 3.1.1.3 Rhizopus E Food
Rennet 3.4.23.6 Mucor miehei E Cheese
Pectinase 3.2.1.15 Aspergillus E Drinks
Protease 3.4.23.6 Aspergillus E Baking
E: extracellular enzyme; I: intracellular enzyme

48. Bacterial Enzymes
Enzyme Sources Application
a-Amylase 3.2.1.1 Bacillus E Starch
b-Amylase 3.2.1.2 Bacillus E Starch
Asparaginas
e
3.5.1.1
Escherichia
coli
I Health
Glucose
isomerase
5.3.1.5 Bacillus I
Fructose
syrup
Penicillin
amidase
3.5.1.11 Bacillus I
Pharmac
eutical
Protease 3.4.21.14 Bacillus E Detergent

49.  Amylases
• hydrolysis of starch (glucose polymer), one of the most readily available
plant polysaccharides
• production of sweeteners from starch: maltose or glucose syrups
(further transformation to high fructose syrup with glucose isomerase)
• starch hydrolyates used as additives in the manufacture of candies, baked
goods, canned goods, and frozen foods
 Proteases
• used in laundry detergents

50.  Glucose Isomerase
 D-glucose ketoisomerase: causes the isomerization of glucose to fructose
 since reaction is reversible the rtion of glucose and fructose depends on the
enzyme and reaction conditions
 high fructose corn syrup fructose 2x sweeter than sucrose
 Chymosin
 site-specific proteolysis by chymosin detaches hydrophilic “tails” of κ-
casein
 resulting in coagulation (curlding)
 calf chymosin (prochymosin) cloned and expressed in E. coli (first
genetically
 engineered protein approved for human consumption, 1990)

51.  Aspartic acid and phenylalanine are synthesized into
aspartame the artificial sweetener through enzyme
Thermolysin. Aspartame is a non-nutritive sweetener
of diet soft drinks and other foods sold as low-
calorie or sugar-free products.

52. Microbial enzymes and their Applications