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
• 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
• Enzymes are powerful and highly specific catalysts
• Free energy is a useful thermodynamic function for understanding
• 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
• 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
• Extremozyme – an enzyme that thrive in extreme
• 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
• 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
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
Important practical tools in medicine, chemical
industry, food processing and agriculture.
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
1897 – Eduard Buchner
Disproved “Vitalism” and stated that fermentation
could be carried out by yeast extracts.
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
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
Act within the cells
in which they are
Liberated by living
cells and act outside
in its environment
Chiefly act as
An additional non-protein
molecule that is needed by
some enzymes to help the
• Inorganic ions such as Fe2+,
Mg2+, Mn2+, or Zn2+
• Complex organic or
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
Tightly bound cofactors are called Prosthetic groups.
Cofactors that are bound and released easily are
Many vitamins are coenzymes.
Complete catalytically active enzyme together with
bound coenzyme/ metal ion – Holoenzyme.
Protein part of the enzyme - Apoenzyme or
16. The active site
One part of an enzyme,
the active site, is
The shape and the
inside the active site
permits a chemical
reaction to proceed more
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
Biological systems are very sensitive to temperature
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
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
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
be used again
27. The Lock and Key Hypothesis
This explains enzyme specificity
This explains the loss of activity when enzymes
28. The Induced Fit Hypothesis
Some proteins can change their shape
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
Making the chemical environment suitable for the
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
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
33. The effect of pH
Optimum pH values
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
36. The effect of temperature
Temperature / °C
0 10 20 30 40 50
37. The effect of temperature
For most enzymes the optimum temperature is about
Many are a lot lower,
cold water fish will die at 30°C because their
A few bacteria have enzymes that can withstand very
high temperatures up to 100°C
Most enzymes however are fully denatured at 70°C
Inhibitors are chemicals that reduce the rate of
The are usually specific and they work at low
They block the enzyme but they do not usually
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
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
Resembles the substrate’s
E + I EI
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.
Cyanide combines with the Iron in the enzymes
Heavy metals, Ag or Hg, combine with –SH groups.
These can be removed by using a chelating agent such
44. Applications of inhibitors
Negative feedback: end point or end product
Poisons snake bite, plant alkaloids and nerve gases.
Medicine antibiotics, sulphonamides, sedatives and
45. Enzymes in medicine
Glucose Hydrogen peroxide
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:
to remove cells
to animal feed
Animal feed enzyme Analytical enzyme Therapeutic protein
to remove cells
1 or 2 purification
protein 3-4 purification
To pharmaceuticals market
To chemicals market
47. Fungal Enzymes
Enzyme EC Sources Location Application
a-Amylase 126.96.36.199 Aspergillus E Baking
Catalase 188.8.131.52 Aspergillus I Food
Cellulase 184.108.40.206 Trichoderma E Waste
Dextranase 220.127.116.11 Penicillium E Food
Glucose oxidase 18.104.22.168 Aspergillus I Food
Lactase 22.214.171.124 Aspergillus E Dairy
Lipase 126.96.36.199 Rhizopus E Food
Rennet 188.8.131.52 Mucor miehei E Cheese
Pectinase 184.108.40.206 Aspergillus E Drinks
Protease 220.127.116.11 Aspergillus E Baking
E: extracellular enzyme; I: intracellular enzyme
48. Bacterial Enzymes
Enzyme Sources Application
a-Amylase 18.104.22.168 Bacillus E Starch
b-Amylase 22.214.171.124 Bacillus E Starch
126.96.36.199 Bacillus I
188.8.131.52 Bacillus I
Protease 184.108.40.206 Bacillus E Detergent
• hydrolysis of starch (glucose polymer), one of the most readily available
• 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
• 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
site-specific proteolysis by chymosin detaches hydrophilic “tails” of κ-
resulting in coagulation (curlding)
calf chymosin (prochymosin) cloned and expressed in E. coli (first
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.