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ENZYMES

Rey John D. Caballero
1
, Jules Carl R. Celebrado
1
, Charlene Z. Diocancil
1
, and
Kathleen V. Manuel
1
1
Biology Student, Department of Biology, College of Science, Polytechnic University of the Philippines


ABSTRACT
An enzyme is a protein that catalyzes or speed up chemical reactions. The
optimum reaction conditions are different for each enzyme. The correct environmental
conditions, proper substrates, and, often, particular cofactors associated with an enzyme are
needed. Denaturation occurs when it is subjected to excessive heat or extremely high or low pH
(denaturing conditions). This laboratory activity will help to develop a concise understanding of a
specific enzymatic reaction and to understand more which factors that affect enzyme activity
that could be biologically important. To test the activity of amylase, sucrase, hydrolase and
catalase various techniques are conducted to show the presence or absence of a material that
reacts in the specific enzyme. Enzymatic proteins are fundamental to the survival of any living
system and are organized into a number of groups depending on their physiological processes
they are involved.
Keywords: Enzymes, catalyes, amylase, sucrase, hydrolase, catalase
INTRODUCTION
An enzyme is a protein that serves as a biological catalyst (Denniston, 2007). A catalyst
is any substance that increases the rate of a chemical reaction (by lowering the activation
energy of the reaction) (Denniston, 2007).
They are large protein molecules, folded so that they have very specifically shaped
substrate binding sites. These binding sites make substrates go into the transition state. To
catalyze the reaction, several regions of the binding site must be precisely positioned around
the substrate molecules. Any change in the shape of the overall folded enzyme molecule can
change the shape of the binding site.
Enzymes are highly specific for certain reactants; the compound acted upon by the
enzyme is known as the substrate. The names of enzymes most commonly end in the suffix -
ase, which is sometimes appended to the name of the substrate or the type of reaction. Some
Group 6

December 12, 2013

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enzymes function properly only in the presence of cofactors or coenzymes. Cofactors are
inorganic, often metallic, ions such as Mg
++
and Mn
++
, while organic molecules such as NAD,
NADP, and some vitamins are coenzymes. Both cofactors and coenzymes are loosely
associated with enzymes; however, prosthetic groups are nonprotein molecules that are
attached to some enzymes and are necessary for enzyme action.
Enzymes speed up chemical reactions by lowering activation energy (that is, the energy
needed for a reaction to begin). In every chemical reaction, the starting materials (the
substrate(s) in the case of enzymes) can take many different paths to form products. For each
path, there is an intermediate or transitional product between reactants and final products. The
energy needed to start a reaction is the energy required to form that transitional product.
Enzymes make it easier for substrates to reach that transitional state. The easier it is to reach
that state, the less energy the reaction needs.
. The optimum reaction conditions are different for each enzyme. The correct
environmental conditions, proper substrates, and, often, particular cofactors associated with an
enzyme are needed. Enzyme denaturation occurs when it is subjected to excessive heat or
extremely high or low pH (denaturing conditions). When an enzyme is denatured it loses its
quaternary, tertiary and secondary structure and becomes a chain of amino acids linked by
peptide bonds (or covalent bonds that occur between adjacent amino acids).
Enzymatic proteins are fundamental to the survival of any living system and are
organized into a number of groups depending on their specific activities. Catalytic enzymes that
break down proteins, which are called proteases, are found in many organisms; one example is
bromelain, which comes from pineapple and can break down gelatin and is often an ingredient
in commercial meat marinades. Anabolic enzymes are equally vital to all living systems. One
example is ATP synthase, the enzyme that stores cellular energy in ATP by combining ADP and
phosphate.
In addition to making life possible, many enzymes have numerous applications that
affect our daily lives in other ways such as food processing, clinical diagnoses, sewage
treatment, and the textile industry.
This laboratory activity will help to develop a concise understanding of a specific
enzymatic reaction and to understand more which factors that affect enzyme activity that could
be biologically important.
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METHODOLOGY

Corn (Zea mays) seedlings, germinated mungbean (Vigna radiata) sprouts and potato
(Solanum tuberosum) used as a botanical material in this experiment.
In examining different hydrolases in the plant, two enzymes under hydrolases are
observed and experimented (Amylase and Sucrase). In detecting the presence of amylase on
Zea mays, two test tubes are prepared and labeled that contained 10mL of 0.1% starch
solution. Two freshly detached root systems from the corn seedlings are soaked in the starch
solution of test tube 1, and then both test tubes are incubated overnight at room temperature. A
drop of I
2
Kl was added in each tube after incubation (root systems removed). Test tubes are
shook and compared according to their color intensities.
For sucrase, two test tubes are set up and labeled and then each test tube were added
with 5mL of 1.0% sucrose solution. Two freshly detached root systems of corn seedlings are
dipped in the sucrose solution of test tube 1, and then both tubes are incubated at room
temperature overnight. After incubation, each tube (root systems removed) are added with
same volume of Benedicts solution and heated in water bath for testing reducing sugars in the
solution.
In investigating different oxidoreductases in plants, two (2) enzymes under
oxidoreductases are used and experimented (Dehydrogenases and Catalases).
In observing dehydrogenases in Vigna radiata, two (2) big test tubes are marked and
filled up to its brim with 0.001% of methylene blue. 10g of freshly germinated Vigna radiata
(seed coat removed) was then added to test tube 1 and then each test tubes are sealed with a
stopper (make sure that the set up have no air bubbles). Both tubes are incubated overnight at
room temperature and observed for any change in color with test tube 2 as control.
In observing catalases in Solanum tuberosum, two (2) test tubes are labeled and
pipetted with 5mL of 3% hydrogen peroxide. Six (6) 2cm thin strips of freshly peeled Solanum
tuberosum are prepared; the first three strips are boiled for 3 minutes while the other three are
raw. After boiling, the strips are drop at the same time in both tubes (3 potato strips per test
tubes), and then observed for gas evolution after 5 to 10 minutes.
The results were presented using tables and pictures.
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RESULTS AND DISCUSSIONS
A. Hydrolases

A.1 Amylase




















After incubating the two test tubes at room temperature overnight, the root systems are
removed from the starch solution. In test tube 2, after adding I
2
KI, purple precipitate form below
while in test tube 1 where detached corn root systems was placed showed no product or
precipitate form after incubation.
I
2
KI or potassium iodide is used to test for starches. In our observation as we dropped
Potassium iodide in the test tubes, the test tube 1 that has a root system has no changes in
color, it is just low and blurry and there is no presence of purple particles at the bottom indicates
the absence of starch within the set-up. On the test tube 2 that is a control, has a color violet
Table 1 Reaction of 0.1% starch solution in with and without detached root of corn seedling
after 24 hours
Original After 24 hours
(after adding I
2
KI)
With corn root No reaction takes place No purple precipitate forms
(negative in starch)
Without corn root No reaction takes place Forms purple precipitate
(positive in starch)
Figure 1 (A-B): Enzyme activity of Amylase; both test tubes contains 0.1% starch solution.
In Test tube 1 having a detached corn seedling root (fig. A.1) while test tube 2 served as
the control (fig A.2); In figure B. There is a precipitate present in test tube 2
A B
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precipitate at the bottom which indicates the presence of starch that is due to the reaction of
I2KI. We can infer that the starch in the solution in test tube 1 was broken down into usable
sugar by the amylase present in the roots and stored for necessary energy and as food storage.
Amylase is present in the roots of the newly germinated corn seedlings. It broke down the starch
with the presence of water in the solution by the addition of the hydrogen and hydroxyl ions of
water to a molecule with its consequent splitting into simpler sugar molecules which are stored
in the roots as food and energy for plant. Test tube 1 was negative in I2KI test because it has no
reaction or changes in color that indicates the presence of starch.
Amylase is an enzyme that catalyses the hydrolysis of starch into sugars. Amylase in
plants assists in the initial development of the plant, before it is able to use energy from
photosynthesis. The amylase enzymes begin their role in plant development as the plant's seed
begins to germinate, root, and sprout. As diastase amylase was the first enzyme to be
discovered and isolated by Anselme Payen in 1833. All amylases are glycoside hydrolases and
act on -1,4-glycosidic bonds. Amylase is an enzyme that acts with the presence of water
molecules to hydrolyze carbohydrates. The role of amylase in plants is to break down starch
molecules. Starches are usually processed in this way during seed germination, and turned into
sugar which provides sources of energy for the plant during its early development. Plants are
able to store energy from the sun by creating sugar. As baird and Arevalo said, Without the
presence of amylase, a seedling would not be able to grow to reach the sunlight needed for
photosynthesis and healthy growth.
A.2 Sucrase (Invertase)













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Table 2 Presence of Reducing Sugars and Enzymatic Activity of Sucrase
Test Tube Results
1 Green (positive)
2 Blue (negative)

Color Range: (None) -----Blue-----Green----Yellow-----Orange-----Red----- (Abundant)

In the experiment, we put the fresh Zea maize roots with 5 ml of 1.0 % sucrose solution
in test tube 1. We filled up test tube 2 with sucrose solution only for it would be our control for
this activity. After incubating for 24 hours, we removed the roots in test tube 1 then added 5 ml
of Benedict's solution on both tubes proceeded by heating. In Test tube1 the resulting color is
green while in test tube 2 is blue.
The amount of glucose formed is directly proportional to the reaction rate. The more
active the sucrase (invertase), the more sucrose will be broken down, and the more glucose will
form. This will be indicated by the color change when testing with Benedict. Test tube 2, which
remained blue in color, implies that the result is negative, for sucrose is a non-reducing sugar.
(fig 2.2). Since test tube 1 reacted and showed a color change from blue to green (fig. 2.1),
reducing sugars were present. In testing with Benedicts reagent, there must be a color change
of orange-red. Using a color range, it was indicated that test tube 1 contained small amount of
reducing sugars since it only change its color partially.
Therefore, test tube 1 with Zea maize roots showed minimal enzymatic activity of
sucrase because small amount of reducing sugars were present. The enzyme sucrase was not
active enough to break down sucrose into glucose and fructose form.
Invertase is a key metabolic enzyme which hydrolyzes the disaccharide sucrose (the
major type of sugar transported through the phloem of higher plants) to glucose and fructose. In
higher plants, invertase exists in several isoforms with different biochemical properties and
subcellular locations. The specific functions of the different invertase isoforms are not clear, but
they appear to regulate the entry of sucrose into the different utilization pathways. Invertase,
alone or in combination with plant hormones, are involved in regulating developmental
processes, carbohydrate partitioning, as well as biotic and abiotic interactions.
Figure 2 After Adding Benedicts Solution and Heating
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B. Oxidoreductases
B.1 Dehydrogenases




Two test tubes were brim-filled with 0.001% methylene blue. The first test tube has a
Mung bean seedlings and the second test tube without mung bean seedlings served as the
control (see figure A). It was covered tightly with a stopper and was incubated overnight. After
24 hours there was a change of color in test tube 1. From blue it becomes colorless and the test
Table 3 Reaction of Methylene Blue on with and without Mung bean Seedlings
Test tube with
0.001% methylene blue
Color
Original After 24 hours After Aeration
With germinated
Mung bean seedlings
Blue Colorless Blue
without germinated
Mung bean seedlings
Blue Blue Blue
A B C
Figure A-C: Test tubes having a brim-filled of 0.001% Methylene blue; Fig. A with and without Mung
bean seedlings before incubation; Fig. B- after incubation within 24 hours and Fig. C-after aeration
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tube 2 remains unchanged (see figure B). After aeration, the solution in test tube 1 becomes
blue in color again (see figure C).
Methylene blue acts as an artificial electron acceptor (oxidizing agent). It is blue when
oxidized, but turns colorless when reduced due to the stopping of air to flow inside the test tube.
Methylene blue can, therefore, be used to show the presence of active dehydrogenase
enzymes by a color change. Dehydrogenase enzymes remove hydrogen from their substrate.
As a result, oxygen is liberated and is free for take up of the seedling. Methylene blue is
reduced and seed gets its needed oxygen. Presence of dehydrogenase in germinating mung
bean seedlings reduced the methylene blue. When methylene blue is substituted for NAD+, the
blue color of methylene blue will disappear as it is reduced, thus, change it from blue to
colorless. NADH or reduced methylene blue can be oxidized by the mitochondrial respiratory
electron transport system when oxygen is available. This will result in the blue color of
methylene blue reappearing upon reoxidation by the respiratory chain.
Oxidoreductases are a class of enzymes that catalyze oxidoreduction reactions.
Oxidoreductases catalyze the transfer of electrons from one molecule (the oxidant) to another
molecule (the reductant). Oxidoreductases catalyze reactions similar to the following, A + B
A + B where A is the oxidant and B is the reductant. It can be oxidases or dehydrogenases.
Oxidases are enzymes involved when molecular oxygen acts as an acceptor of hydrogen or
electrons. Whereas, dehydrogenases are enzymes that oxidize a substrate by transferring
hydrogen to an acceptor that is either NAD+/NADP+ or a flavin enzyme.
Although a great deal of information has been amassed concerning dehydrogenases in
animal tissues, there was for a long time little evidence that certain of these important enzymes
even existed in plants. Malic and citric dehydrogenases were reported in 1929 in cucumber
seeds (Thunberg 1929), but it was not until 1939 that succinic dehydrogenase was found, first in
pollen. (Okunuki 1939) and then in certain other tissues (Damoran1941). Nevertheless, the
apparent absence or near-absence of succinic dehydrogenase in some tissues (Bartlett 1943)
as well as the occasional reports of the presence of individual enzymes (Thunberg 1938)
seemed to indicate that the dehydrogenases, at least those of the 4 carbon and 6 carbon acids,
were distributed only sporadically. It was during this period that the tricarboxylic acid cycle of
Krebs (Krebs 1943), embodying many of these dehydrogenases, was becoming accepted as
the main pathway of respiration in animal tissues. Respiration studies in plants (Bonner 1948)
pointed in the same direction.
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B. 2 Catalases
















The raw potato strips produce foam (see test tube 1) while the boiled potato strips do not
react upon contact with hydrogen peroxide (H
2
O
2)
(see test tube 2).
The raw potato strips produce foam upon contact with hydrogen peroxide (H
2
O
2
),
because the catalase in the potato strips react with hydrogen peroxide (H
2
O
2
) and change it into
water (H
2
O) and oxygen gas (O
2
). The bubbles at top are pure oxygen bubbles while water
settles below while the boiled did not produce any bubbles upon contact with hydrogen peroxide
(H
2
O
2
) because boiling the potato denatures the protein enzyme (catalase) in the potato.
Table 4: Reactions of raw and boiled potato to hydrogen peroxides (H
2
O
2
)
Potato strips Observation
Boiled potato strips No reaction upon contact with hydrogen
peroxide (H
2
O
2
)
Raw potato strips Produce bubbles upon contact with
hydrogen peroxide (H
2
O
2
)
Figure 4: from L-R, reactions of potatoes between raw and boiled after 5 minutes; reaction of
potatoes raw and boiled after 10 minutes
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Catalases are produced inside the cell that is why the potato was cut to strips, to destroy
some cells first. Three strips are boiled and the others were not to test the presence of catalase
in different temperatures, the rapid production of bubbles in raw shows that the catalase is doing
the reaction fast. The cooked potato strips did not produce any bubbles because the structure of
the catalase was altered, heating, increasing salinity etc. denatures a protein, once the
structure is changed it will not work anymore; therefore no oxygen no bubbles.
The process of photorespiration can be explained as when the plants receives too much
light and not enough water, that results excessive production of hydrogen peroxide. Hydrogen
peroxide (H
2
O
2
) is a by-product of respiration and is made in all living cells. Hydrogen peroxide
is harmful and must be removed as soon as it is produced in the cell but left to its self hydrogen
peroxide will slowly lose the extra oxygen and change into water that is why cells make the
enzyme catalase to speed up the reaction and remove hydrogen peroxide (Hopkins and A.
Huner, 2008).
Catalases are protein enzymes that react with hydrogen peroxides (H
2
O
2
) (P. George,
1947), it can be found in animals it is mostly produced by the liver and heart, in plants some
catalases helps in the breakdown of the toxic hydrogen peroxide (H
2
O
2
) during respiration for
the production of glycine to serine during the glycolate cycle (Leegood et al., 2000), while some
chloroplastic enzymes helps in reducing hydrogen peroxides (H
2
O
2
) to water (Apel and H. Hirt,
2004). Plants use these kinds of chemicals to avoid oxidative damage or changing hydrogen
peroxide to a highly toxic hydroxyl radical (OH-) (Demmig-Adams et al., 2006).
STUDY QUESTIONS
1. Give the: (1) name of enzymes catalyzing the following chemical reactions, (2) their cellular
localization, and the (3) plant physiological process involved.
A. Pyruvate + NAD
+
+CoA Acetyl-CoA + NADH + H
+
+ CO
2


(1) Pyruvate Dehydrogenase catalyzes the reaction
(2) The reaction takes place in the mitochondrion.
(3) Krebs cycle is the process involved an event in cellular respiration
B. Ribulose- 1,5 bisphosphate + CO
2
2 (3- phosphoglyceric acid)

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(1) The reaction was catalyzed by RuBisCO or Ribulose 1,5-bisphosphate carboxylase
(2) It takes place in stromal space of the chloroplast
(3) It involves the process of Carbon Dioxide Fixation (as for Photosynthesis/ Calvin
Cycle)
C. fructose-6-phosphate + ATP fructose-1,6-bisphosphate + ADP
(1) Phosphofructokinase (PFK) catalyzes the reaction
(2) The reaction takes place in the cytosol of cells
(3) Glycolysis is the process involved (an event in cellular respiration)
2. Describe the rate of enzyme catalyzed reaction with increasing substrate concentration.
It has been shown experimentally that if the amount of the enzyme is kept constant and
the substrate concentration is then gradually increased, the reaction velocity will increase until it
reaches a maximum. After this point, increases in substrate concentration will not increase the
velocity (delta A/delta T). It is theorized that when this maximum velocity had been reached, all
available enzyme has been converted to enzyme substrate complex (Freshcorn et. al., 2008).
The Michaelis constant Km is defined as the substrate concentration at 1/2 the
maximum velocity. Using this constant and the fact that Km can also be defined as: K
m
=K
-1
+ K
2
/ K
+1
. Michaelis constants have been determined for many of the commonly used enzymes. The
size of Km tells us several things about a particular enzyme (Freshcorn et. al., 2008).
a. A small Km indicates that the enzyme requires only a small amount of substrate to
become saturated. Hence, the maximum velocity is reached at relatively low substrate
concentrations.
b. A large Km indicates the need for high substrate concentrations to achieve maximum
reaction velocity.
c. The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently
assumed to be enzyme's natural substrate, though this is not true for all enzymes.


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A simple chemical reaction with a single substrate shows a linear relationship between the rate
of formation of product and the concentration of substrate





Figure 4.1. Rate of Enzymatic reaction based on substrate concentration (Linear)
For an enzyme catalization reaction, there is usually a hyperbolic relationship between the rate of reaction
and the concentration of substrate.






Figure 4.2 Rate of Enzymatic reaction based on substrate concentration (Hyperbolic)
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At low concentration of substrate, there is a steep increase in the rate of reaction with
increasing substrate concentration. The catalytic site of the enzyme is empty, waiting for
substrate to bind, for much of the time, and the rate at which product can be formed is limited by
the concentration of substrate which is available. As the concentration of substrate increases,
the enzyme becomes saturated with substrate. As soon as the catalytic site is empty, more
substrate is available to bind and undergo reaction. The rate of formation of product now
depends on the activity of the enzyme itself, and adding more substrate will not affect the rate of
the reaction to any significant effect (Freshcorn et. al., 2008).





Figure 4. 3. Limitation of substrate concentration in an enzymatic reaction
The rate of reaction when the enzyme is saturated with substrate is the maximum rate of
reaction, V max. The relationship between rate of reaction and concentration of substrate
depends on the affinity of the enzyme for its substrate. This is usually expressed as the Km
(Michaelis constant) of the enzyme, an inverse measure of affinity. For practical purposes, Km
is the concentration of substrate which permits the enzyme to achieve half V max. An enzyme
with a high Km has a low affinity for its substrate, and requires a greater concentration of
substrate to achieve V max (Freshcorn et. al., 2008).
4. In what ways does hydrogen ion concentration affect enzyme activity?
The pH scale is the logarithm of the reciprocal of hydrogen-ion concentration in gram
atoms per liter; provides a measure on a scale from 0 to 14 of the acidity or alkalinity of a
solution (where 7 is neutral and greater than 7 is basic while lesser than 7 is acidic). Hydrogen
ion concentration affects enzyme activity by its relationship to pH. Changes in pH may not only
affect the shape of an enzyme but it may also change the shape or charge properties of the
14

substrate so that either the substrate can bind or cannot bind to the active site or it cannot
undergo catalysis. High hydrogen ion content caused the breaking of the ionic bonds that hold
the tertiary structure of the enzyme in place. The enzyme lost its functional shape, particularly
the shape of the active site, such that the substrate no longer fit into it, the enzyme is denatured.
The ions also affected the charges on the amino acids within the active site such that the
enzyme was unable to form an enzyme-substrate complex (Krause et. al, 1998). In general
enzymes have a pH optimum. However the optimum is not the same for each enzyme.
CONCLUSIONS




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