ENZYMES

and FACTORS
AFFECTING ITS
ACTIVITY
EXPERIMENT 3A and 3B
 Importance
• All biochemical reactions are
enzyme catalyzed in the living
organism
• Enzymes play an important role in
metabolism, diagnosis, and
therapeutics.
– Level of enzyme in blood are of
diagnostic importance e.g. it is a good
indicator in disease such as myocardial
infarction.
– Enzyme can be used therapeutically
such as digestive enzymes.
 Definition
• Enzymes are biological catalysts
– They catalyze nearly all the chemical
reactions taking place in the cells of the
body.
– They are proteins that increase the rate of
reaction by looking for an alternative
pathway that has a low energy of activation
CHARACTERISTICS
– They are not altered or consumed during reaction.
– Reusable
– Enzymes show specificity to the reaction they control
– Enzymes are sensitive to their environment so they can
be controlled by adjusting the temperature, the pH or
the substrate concentration
– They do not initiate chemical reaction because they just
speed up the reaction that is already underway.
 Structure
• They have a
globular
shape
• A complex
3D structure

Human pancreatic
amylase
© Dr. Anjuman Begum
 Terms to understand
biochemical nature of enzymes
• Active Site - The area on the enzyme where
the substrate or substrates is/are attach to
• Enzymes are usually very large proteins and
the active site is just a small region of the
enzyme molecule.

• Substrate – the reactant in the chemical

reaction
 Terms to understand
biochemical nature of enzymes
• Apoenzyme – the enzyme without its non
protein moiety is termed as apoenzyme and
it is inactive.
• Holoenzyme – the active enzyme with its non
protein component.
 Terms to understand
biochemical nature of enzymes
• Cofactor
– A cofactor is a non-protein chemical
compound that is bound (either
tightly or loosely) to an enzyme and
is required for catalysis.

– Types of Cofactors:
– Coenzymes – examples: vitamins or
compound derived from vitamins
– Prosthetic groups – Zinc,
Manganese, iron
 EXPT 3A ENZYMES
• AMYLASE – breaks starch into sugar
– Salivary Amylase (Ptyalin)
• Present in saliva with optimum pH
of 6.7-7.0
• Hydrolyzes α-1,4-glycosidic bonds
in starch producing
oligosaccharides, maltose or
glucose depending on the time
spent in the mouth of the food.
• Benedict’s Test – test for reducing
sugar, contains CuSO4, Na2CO3 and
sodium citrate, that results to green,
yellow, orange, or red precipitate
(depends on the amount of sugar)
 Enzymes
• OXIDASE – an enzyme that protects
organisms from the effects of hydrogen
peroxide
2H2O2 → 2H2O + O2
• H2O2 is a powerful oxidizing agent and is
potentially damaging to cells
• Catechol oxidase – yellow to red orange
solution
– works best in pH 6-7 (optimum pH)
• For cathecolase action: Cathecol + oxygen
→ benzoquinone and water
 EXPT 3A ENZYMES
• PROTEASE – Pepsin and Rennin

• PEPSIN – an enzyme that hydrolyzes protein to smaller

peptides to amino acids, cleaving peptide bonds between
hydrophobic and aromatic amino acids.
– it is active in acidic environment, having an optimum
pH of 1.5-2.0

• RENNIN – an enzyme that hydrolyzes protein to smaller

peptides to amino acids, cleaving the peptide bonds
between phenylalanine and methionine resulting to
calcium phosphocaseinate.

Calcium Phosphocaseinate + (NH4)2C2O4 → CaC2O4 (white precipitate)

Enzymes

Pepsin hydrolyzed casein

more than rennin (curding
of milk)
 EXPERIMENT 3B
FACTORS AFFECTING
ENZYME ACTIVITY
 Enzymatic Action
• 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.

Figure of Reaction pathway

 Mechanism
• 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 look for a new reaction pathway “a short
cut”
• Enzyme controlled reactions proceed 108 to
1011 times faster than corresponding non-
enzymatic reactions.
 Effect of temperature
• Enzymes optimum temperature:
– Temperature increases enzyme
activity up to its optimum
temperature

Q10 Denaturation
Enzyme
activity

0 1 2 3 4 5
0 Temperature
0 0 / °C
0 0
 RESULTS
STARCH SAMPLE
SAMPLES
+ IODINE

A1 = Saliva in ice bath Light purple

A2 = Saliva in warm bath

Colorless
(40oC)

A3 = Saliva in boiling water Dark violet

 Effect of pH
• Depends on the enzyme’s optimum
pH but extreme pH levels will cause
denaturation.
• Salivary amylase has 6.8 optimum
pH
 RESULTS
STARCH SAMPLE
SAMPLES
+ IODINE

B1 = Saliva in pH 4 Light purple

B2 = Saliva in pH 7 Colorless

B3 = Saliva in pH 10 Light Purple

 Effect of Substrate
Concentration
• The increase in velocity is proportional to the
substrate concentration but when enzyme is
no longer available, V max is reached –
saturation point

Vmax

Reaction
velocity

Substrate concentration
 Effect of Enzyme
Concentration
• Increasing enzyme concentration will
increase rate of reaction but when
enzyme is no longer available, it
reaches a maximum point.
 RESULTS
STARCH SAMPLE
SAMPLES
+ IODINE

C1 = 3 drops Saliva Slowest

C2 = 6 drops Saliva Mid

C3 = 7 drops Saliva Fastest

EXPERIMENT 4A
NUCLEIC ACIDS
 Importance
• DNA stores the genetic
information of an organism and
transmits that information from
one generation to another.

• RNA translates the genetic

information contained in DNA
into proteins needed for all
cellular function.
 Definition
• A nucleic acid is an unbranched
polymer in which the monomer
units are nucleotides.

• All nucleic acid molecules are

unbranched polymers.
 Structure
A nucleotide is composed
of:
• Nitrogen-containing bases
(amines)
• Sugar
– Ribose for RNA
– Deoxyribose for DNA
• Phosphate
 Experiment
• Blender - crush cell wall
• Add Homogenizing solution
– dishwashing liquid - removes the
lipids and push DNA to the salt
solution
– sodium chloride - increases
separation of DNA from
hydrophobic layer; aggregates DNA
• Add cold ethanol
– promotes aggregation of DNA and
wash the DNA from the salt
• Dische diphenylamine test