December 12, 2013Group 6ENZYMES
Rey John D. Caballero1, Jules Carl R. Celebrado1, Charlene Z.
Diocancil1, and Kathleen V. Manuel1 1Biology 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,
catalaseINTRODUCTIONAn 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 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.METHODOLOGYCorn (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 I2Kl
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.RESULTS AND DISCUSSIONSA. Hydrolases
A.1 Amylase
BA
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
Table 1 Reaction of 0.1% starch solution in with and without
detached root of corn seedling after 24 hours
OriginalAfter 24 hours(after adding I2KI)
With corn rootNo reaction takes placeNo purple precipitate
forms(negative in starch)
Without corn root No reaction takes placeForms purple
precipitate(positive in starch)
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 I2KI, purple precipitate form below
while in test tube 1 where detached corn root systems was placed
showed no product or precipitate form after incubation.I2KI 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 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.Amylaseis
anenzymethatcatalysesthehydrolysisofstarchintosugars. 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. Asdiastase amylase was the first
enzyme to be discovered and isolated byAnselme Payenin 1833. All
amylases areglycoside hydrolasesand 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)
Figure 2 After Adding Benedicts Solution and Heating
Table 2 Presence of Reducing Sugars and Enzymatic Activity of
Sucrase
Test TubeResults
1Green (positive)
2Blue (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.
B. Oxidoreductases B.1 Dehydrogenases
CBA
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
Table 3 Reaction of Methylene Blue on with and without Mung bean
Seedlings
Test tube with 0.001% methylene blueColor
Original After 24 hoursAfter Aeration
With germinated Mung bean seedlingsBlueColorlessBlue
without germinated Mung bean seedlingsBlueBlueBlue
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 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
+ Bwhere 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.
B. 2 Catalases
Figure 4: from L-R, reactions of potatoes between raw and boiled
after 5 minutes; reaction of potatoes raw and boiled after 10
minutes
Table 4: Reactions of raw and boiled potato to hydrogen
peroxides (H2O2)
Potato stripsObservation
Boiled potato strips No reaction upon contact with hydrogen
peroxide (H2O2)
Raw potato stripsProduce bubbles upon contact with hydrogen
peroxide (H2O2)
The raw potato strips produce foam (see test tube 1) while the
boiled potato strips do not react upon contact with hydrogen
peroxide (H2O2) (see test tube 2). The raw potato strips produce
foam upon contact with hydrogen peroxide (H2O2), because the
catalase in the potato strips react with hydrogen peroxide (H2O2)
and change it into water (H2O) and oxygen gas (O2). 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 (H2O2) because boiling the potato denatures the protein
enzyme (catalase) in the potato.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 (H2O2) 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
(H2O2) (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 (H2O2) 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 (H2O2) 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
QUESTIONS1. 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+ + CO2
(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 respirationB. Ribulose- 1,5
bisphosphate + CO2 2 (3- phosphoglyceric acid)
(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: Km=K-1 + K2 /
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.
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)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 reactionThe 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 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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