EXPERIMENT 6: ENZYMES o Brown discoloration produces by

Enzymes: fruits and vegetables.

 Acts as catalyst (speed up the reaction).  Phenols
o Substate that acts upon the
 Neither consumed nor permanently altered in
enzyme.
their participation in the reaction.
 Phenolases
 Most enzymes are globular proteins
o An enzyme that acts on the
 Undergo all reaction including denaturation
substrate to produce the brown
Factors Affecting Enzyme Reaction:
discoloration.
 Temperature
 pH
 Substrate concentration SAMPLE USED: TIME OXIDIZED:
 Enzyme concentration
APPLE 2 MINUTES

Nomenclature and Classes of Enzymes BANANA 15 MINUTES

 Enzymes are grouped into classes based on
the types of reactions they catalyze POTATO 16 MINUTES

Reaction GUAVA 32 MINUTES

Class
Catalyzed

Oxidation–reduction POTATO OXIDASE

1.Oxidoreductases  Potato extract is a source of Oxidase
reactions

Results:
Functional group transfer  Tube 1: Extract + Phenol: Brown solution
2. Transferases
reactions  Tube 2: Extract + Catechol/ Pyrogallol:
Brown solution
3. Hydrolases Hydrolysis reactions  Tube 3: Extract + Guiac: Blue solution

Reactions involving addition

4. Lyases or removal of groups to
form double bonds

5. Isomerase Isomerization reactions

 Phenol + O2 + Oxidase = Quinone
Reactions involving bond
6. Ligases formation with the
participation of ATP

Oxidases
 Enzymatic discoloration
o A chemical process involving  Catechol + O2 + Oxidase = Benzoquinone
phenolases and other enzymes that
produce benzoquinones and
melanins from natural phenols
resulting in brown discoloration of
some fruits and vegetables.
 Polyphenol oxidase
o Initiates discoloration of fruits
 Benzoquinones
  Gum guiac reacts with the peroxidase
present in potatoes
POTATO PEROXIDASE
 Peroxidase
o Enzyme that decomposes H2O2 w/
Guiac or Benzidine
 Potato extract
o Contains substances that can be
oxidized.
 Potato peroxidase
o Decomposes H2O2 and organic
peroxides to produce Oxygen.
 Peroxidase facilitates oxidation with the use
of H2O2
 Potatoes are naturally high in
catalase/peroxidase
 Presence of enzyme can be checked by
ability to convert H2O2 to O2 and water
 As O2 is produced with the interaction of disk
containing the enzyme and H2O2, the disk in
tube will rise.
 As catalase is inactivated, O2 production
decreases and the ascent of the disk slows
down
 As the extract is heated, the enzyme gets
denatured (So enzyme in “denatured
catalase” tube is dysfunctional)
 No enzyme is present in water
 Tube “with catalase” = fastest time
 Tube “denatured catalase” and “no
catalase”= slowest
Effect of heat on peroxidase
 Heat destroys peroxidase
 Enzymes get denatured when they go higher
than their optimal temp.

o
Optimal temp = 55 C
Enzymes, Cofactors, Apoenzyme, Prosthetic
group, & Holoenzyme
 Enzymes are biomolecules, usually proteins,
that facilitate or increase the rate of a
reaction
 Cofactors are the molecules needed for an
enzyme to effectively function (Coenzymes
and Activators)
 Apoenzyme is the protein part of an enzyme
 Prosthetic group are the parts of an enzyme
that are non-protein
 Holoenzyme is both the protein and non-
protein parts of an enzyme together
Enzyme structure
• Simple enzyme: Enzyme composed only of
protein (amino acid chains)
• Conjugated enzyme: Enzyme that has non-
protein and protein parts
– Apoenzyme: Protein part of a
conjugated enzyme
– Cofactor: Non-protein part of a
conjugated enzyme
– Holoenzyme: Biochemically active
conjugated enzyme
• Apoenzyme + cofactor =
holoenzyme
Denaturation
 Denaturation is when the secondary, tertiary,
and quaternary structures of a protein is
broken leaving only the original primary
structure intact.
Effect of Heat/pH to Denaturation
 Extreme pH change or an extreme increase
in temperature can irreversibly denature
proteins
What are the substrates of Catecholase? What is
the color of the product produced? Write the
reaction. Correlate this with the browning of fruits.
 The substrates of catecholase are catechol
and oxygen.
 The products formed are benzoquinone and
water
 In fruits, catechol oxidase acts upon alcohols
present in the fruit in the presence of oxygen,
forming Benzoquinone. This product inhibits
the growth of microorganisms and prevents
damaged fruit from rotting.
What is the reaction of peroxidase?
 Basically, OXIDATION IN THE PRESENT
OF H2O2
What is blanching and what is its purpose?
 Blanching is a mild heat treatment (scalding
quickly in boiling water or steam) done to
vegetables to inactivate enzymes prior to
freezing that might cause loss of flavor,
color, and texture.
 It also cleanses the surface of dirt and
organisms, brightens the color, and helps
slow down the loss of vitamins
What factors affect the rate of enzymatic browning
in fruits/vegetables?
 The most important factors that determine
the rate of enzymatic browning of vegetables
and fruits are the concentration of both active
polyphenol oxidase and phenolic compounds
in the fruit/vege, the pH, the temperature,
and the oxygen availability of the tissue.
 The optimum pH and/or temp of PPO activity
varies with the source.
EXPERIMENT 7: FACTORS
AFFECTING ENZYMATIC ACTIVITY
 pH
 Temperature
 Enzyme Concentration
 Substrate Concentration
Catalytic action of an enzyme is influenced by several
factors:
 Heavy metals
 Radiation,
 Water
 Activators or inhibitors
 Heavy metal acts as a form of “poison” to
enzymatic activity.
 Heavy metal ions react with S-H group of
cysteine bonds, forming a covalent bond with
sulfur atom and displacing the hydrogen ion.
 This causes the enzyme to loses it ability to
catalyze reactions.
 Radiation can also cause decrease in activity
 Water : Enzymes are large proteins that
catalyze chemical reactions. That means
they assist in the formation or disruption of
atomic bonds. Enzymes, like other proteins,
get their properties from their shapes.
Anything that disrupts the shape of an
enzyme -- including boiling and freezing --
will make it inactive.
Heating:
 Proteins hold their shapes because regions
with different electric charges attract each
other, bending the protein into a particular
shape.
 When enzymes boil, they irreversibly
denature and become inactive.
 The higher the temperature, the more an
enzyme will vibrate. If it vibrates enough it
will distort out of shape, or denature.
 If the temperature goes up even more, the
different regions of the protein will swing so
far apart from each other that they can't
come back.
Cooling:
 As enzymes cool, they vibrate less. They
don't lose their shape when that happens,
but the regions around their active sites get
frozen in place.
  Some enzymes in specialized bacteria called
extremophiles can function at very high and
low temperatures.
 That prevents the enzyme from reacting. In
general, freezing temperatures will make
enzymes inactive -- although they can
recover their activity when the temperature
rises.
Potato oxidase:
 Wash and peel a medium-sized camote/
potato.
 Grate and place the grated potato/ camote in
an evaporating dish with 100mL of H2O.
 Transfer the resulting mixture onto a clean
cheesecloth.
 Squeeze the grated potato and filter.
 Transfer the filtrate into a beaker and label it
“potato oxidase”
 pH: Each enzyme has an optimum pH range.
Changing the pH outside of this range will
slow enzyme activity. Extreme pH values can
cause enzymes to denature.
 HCl is an acid (low pH)
 Sodium Carbonate is a base (high pH)
2. Temperature
 Too high temperature alters the enzyme
conformation causing a decrease in function.
 Small increase is reversible.
 TOO HIGH = DENATURATION
 Low temperature is reversible.

o
37 C (Most enzymes)

PPO Activity:

 Low temp: INACTIVATION/PRESERVATION


o
Optimum temp is 55 C
Expected result:

o
Most intense color is seen at 70 C

nd o
2 : 37 C/ Optimum temperature

o
Least intense: 0 C

3. Substrate concentration
 As substrate concentration increases, rate of
reaction increases until such time that ALL
ENZYMES ARE SATURATED (it will not
1. PH increase further).
 Small pH changes = Reversible
 Large pH changes can cause denaturation =
irreversible
 Every enzyme has an optimum pH

Expected result:
 Most intense color: Undiluted Sample 1 and
3
 Least intense: Diluted Sample 2 and 4
Expected result:  Brown color
 Most intense color is seen at pH 7.

nd
2 : pH 4 and 0.1M Na2CO3 4. Enzyme concentration
 Least intense: 10M HCl  As enzyme concentration increases, rate of
reaction will also increase.
  Optimum pH is 6.0
Expected result:
 Most intense color: Tube 1

nd
2 : Tube 2
 Least intense: Tube 3
 Brown color
Data and results:
1. Substrate concentration:
SOLUTION: (Added with
Potato Oxidase)
a. 4mL Catechol
b. 3mL Catechol + 1mL distilled
water
c. 4mL Guiac solution
d. 3mL Guiac + 1mL distilled
water
2. ENZYME concentration:
SOLUTION: RESULT:
a. 5mL Potato Oxidase
b. 1mL Potato Oxidase +
0.5mL water
c. 1mL Potato Oxidase +
1mL water
3. ph
SOLUTION: (Added with
Potato Oxidase)
a. 10M HCl
b. pH 4 Phosphate buffer
c. pH 7 Phosphate buffer
d. 2mL 0.1 Na2CO3
 IF pH IS NOT MAINTAIN, THE ENZYME
STRUCTURE WILL BE DISRUPTED,
THEREFORE IT CANNOT FUNCTION
NORMALLY ANYMORE THUS
IRREVERSIBLE REACTION IS POSSIBLE.
4. TEMPERATURE
SOLUTION: (Added RESULT:
with Potato Oxidase)
o
a. 0 C LIGHT BROWN
o
b. 37 C LIGHT/DARK BROWN
o
c. 70 C DARK BROWN
d. Optimum DARK BROWN
Temperature
• Temperature had a great influence on PPO
activity, due to the high temperature would
destroy the enzyme protein which led to
inactivation.
• Low temp: INACTIVATION/PRESERVATION
o
RESULT: • Optimum temp is 55 C
DARK BROWN GUIDE QUESTIONS:
Enzyme kinetics, Zero-order kinetics, First order
LIGHT BROWN kinetics
 Enzyme Kinetics is the study of rates of
DARK BROWN enzymatic reactions.
LIGHT BROWN o Zero Order Kinetics is a state at
which the rate of enzyme reaction is
independent of the concentration of
the substrate administered.
o First Order Kinetics is occurring
when a constant proportion of the
DARK BROWN drug is eliminated per unit time.
LESS DARK BROWN Saturated level of substrate
 Enzyme saturation is the point at which, the
LIGHT BROWN rate of reaction reaches maximum with no
further increase at a particular
substrate concentration.
RESULT:
Effects of the following in enzymatic activity:
a. Heavy metals acts as a form of “poison” to
LIGHT BROWN enzymatic activity. This causes the enzyme
to loses it ability to catalyze reactions.
LIGHT BROWN a. A. Heavy metal - Reacts with S-H
group of cysteine bonds, forming a
LIGHT/ DARK BROWN covalent bond with sulfur atom and
displacing the hydrogen ion.
b. Inhibitors stop a substrate from entering
DARK BROWN
the enzyme's active site and/ hinder
the enzyme from catalyzing its reaction.
c. Activators binds to enzymes to increase
activity.
Maximum velocity?
  Maximum Velocity is a point when there are
enough substrate molecules to completely fill
(saturate) the enzyme's active sites.
The maximum rate of an enzymatic reaction
that can be achieved by progressively
increasing the substrate concentration.
the enzyme's shape changes, it
cannot bind to the substrate.
 Optimum pH and Temperature - The point at
which enzymes has their highest activity.

Different models that explain the mode of action

of enzymes
 Lock-and-key model
o Enzyme has a fixed, rigid
geometrical conformation
o Only substrates with a
complementary geometry can be
accommodated at the site
 Induced-fit model
o The active site allows for small
changes in space to
accommodate the substrate
o Example: How a hand fits into a
glove

Forces That Assist Substrate Binding

• Electrostatic interactions
• Hydrogen bonds
• Hydrophobic interactions

Effect of ph and temperature in enzymatic activity

Optimum ph and temperature
 pH - Extremely high or low pH values
generally result in complete loss
of activity for most enzymes.
 Temperature - Higher temperatures tend to
speed up the effect of enzyme activity, while
lower temperatures decrease the rate of
an enzyme reaction.
o Temperature - If the temperature is
too high, an enzyme will denature,
which causes the shape of
the enzyme to change. If