The of phosphatase. Enzymes are biological catalysts which are

The purpose
of this investigation was to determine the effect of
phenolphthalein phosphate on the enzyme activity of phosphatase. Enzymes are
biological catalysts which are large complex proteins shaped in living cells which
are essential in our daily lifestyles and perform a wide range of important
functions. They are proteins that comprise chains of amino acids connected by
peptide bonds. As a biological catalyst, the enzyme’s role depends on its
structure, predominantly the substrate’s binding site known as the active site.
The active site is the 3D structure complementary to the substrate’s shape and
is the specific part of the enzyme to which the substrate will bind, usually a
cleft or crevice. By a combination of many weak non-covalent bonds, the
substrate is held within the active site, by Van der Waals, hydrogen bonds, hydrophobic
interactions and electrostatic interactions.

Enzymes
significantly speed up the rate of chemical reactions providing an alternative
reaction pathway of lower activation energy. Thus, reactions are able to
proceed rapidly at relatively low temperatures. However, they do not undertake
permanent changes and thus remains unchanged at the end of the reaction.

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

The pH at
which an enzyme works most effectively is known as its optimum. By altering the
charge of the enzyme results in a change in the overall shape of the enzymes
active site. This affects the ability of the enzyme to interact with the
substrate, extreme pH’s can result in the enzyme becoming denatured. The
optimum pH normally reflects the environment in which the enzyme works.

The
kinetic energy within the reaction between the enzyme and substrate, as the
temperature increases will increase the rate of the chemical reaction. When the
temperature exceeds the optimum temperature the enzymes active site will
denature causing a conformational change in shape leading to a loss of 3D
structure and the substrate will no longer bind with the enzyme resulting a
decrease in catalytic activity.

The
opposite effect dominates, at very cold temperatures where particles move more
slowly, reducing the amount of enzyme-substrate collisions and therefore
decrease the enzyme activity. The enzymes shape is not affected i.e. denaturing,
however because of the lower temperature the enzymes and molecules will have
less energy to move around and reaction rates decreases. The enzymes and molecules
can be regenerated by reheating it back to its optimum temperature.

Enzymes
bind with organic molecules called substrates. For each type of enzyme, there
may be one or more substrates, depending on the chemical reaction.

There are
two theories explaining the enzyme-substrate interaction; the lock and key
model and the induced fit model. In the lock-and-key model, the active site of
an enzyme is specifically shaped to bind to specific substrates to form a
reaction intermediate.

In the
induced-fit model, Enzymes are not necessarily a perfect sit to substrate. The
enzyme changes shape temporarily because of the active site’s structure,
therefore increasing the binding and interaction with the substrate. If a
catalyst is absent, the energy of activation is quite large and the rate of the
reaction is extremely slow. The presence of a catalyst ensures that the
activation energy is lowered and that the reaction takes place faster.

Enzyme
Inhibitors are substances which decrease the frequency of an enzyme controlled
reaction by altering the catalytic action of the enzyme in some way and thus
slow down, or occasionally, stop catalysis. This effect may be permanent or
temporary. There are two types of inhibitors; Competitive Inhibitors and Non-Competitive
Inhibitors.

Competitive
Inhibitors are molecules that have a complementary shape to the active site,
similar to the substrate. Therefore, competition will take place between the
substrate and the inhibitor for the active site. If the inhibitor binds to the
active site, the substrate can no longer bind which reduces the rate of
reaction of the enzyme. If the enzyme substrate concentration increases
competition in favour of the substrate and so reaction increases.  Competitive inhibitors bind reversibly to the
active site and so the action of the competitive inhibitor can be overcome by
increasing the concentration of the substrate. Hence, the Vmax value is
unchanged, however at a higher substrate concentration it is required to
achieve this Vmax therefore Km is increased.

Non-Competitive
Inhibitors bind to an enzyme at a site other than the active site and when it
binds it causes a conformational change in shape to the active site. Hence, it
can no longer bind to the substrate. Increasing the substrate has no effect on
the rate of reaction because the substrate and the inhibitor are not in
competition. Substrate binding to the enzyme is unaffected (Km remains
unchanged) however the binding of the inhibitor prevents the formation of
product. This in turn decreases the catalytic activity of the enzyme. i.e. Vmax
is decreased. Since the non-competitive inhibitor binds at a different site to
the substrate, the enzyme can bind both the substrate and the inhibitor at the
same time.

Phosphatase
enzymes are found in both plant and animal tissues and can be grouped as an
acid or alkaline depending on their optimum pH. These phosphates are essential
for synthesis of, for example, ATP, phospholipids and nucleotides.

In this investigation,
by grinding up the beansprouts the enzyme is obtained, and the liquid extract
is collected. An artificial substrate phenolphthalein phosphate (PPP) is used.
The enzyme and substrate were reacted with a buffer solution, resulting the
substrate being degraded to phosphate and free phenolphthalein.

The
phosphate from the reaction may possibly act as an enzyme inhibitor since the
phosphate is a product of phosphatase activity. End product inhibition is a
type of negative feedback commonly used to control the rate of a metabolic
pathway in living things. Phenolphthalein phosphate (PPP) is colourless in
alkaline solution, whereas free phenolphthalein produces a pink colour. After a
period of incubation any free phenolphthalein formed can be detected by adding
alkali (sodium carbonate) as at this pH phenolphthalein is pink.

The
intensity of colour produced can thus be estimated by the amount of enzymatic
activity from a standard reaction mixture when an alkali (sodium carbonate
solution) is added. The intensity of colour can be measured quantitatively
using a spectrophotometer. The sodium carbonate also denatures the phosphatase
and stops the reaction.

Procedure:

All of the
equipment required was gathered for the experiment. The beakers were labelled
with the solutions they will contain before the appropriate volume from the
communal stock of solutions were taken. The test tubes were labelled A-D and
1-6. The enzyme mixture was prepared by grinding approximately 40g of bean
sprouts in a pestle and mortar until a mushy paste was produced. The liquid was
filtered through a cloth into a clean centrifuge tube. The yield should be 14
cm3 of the extract – any extra liquid was discarded. The extract was
centrifuged at high speed for 10 minutes. The solution was separated into two
parts – supernatant and pellet. The liquid contained the enzyme phosphatase.
Care was taken to not disturb the pellet of the extract. While the enzyme
extract was in the centrifuge, the dilutions were prepared, and the reactions
were set up. The graduated glass pipettes were used to prepare the dilutions of
the inhibitor as shown in Table 1. Care was taken into consideration when
inserting the glass pipette into the pipette filler. The additions of inhibitor
were taken into account first then the water was added.

The enzyme
reactions in test tubes 1-6 were assembled as shown in Table 2. A different
transfer pipette was used for each different solution. Care was taken to
prevent any contamination or mix up of solutions.

The concentration
of inhibitor solution in each reaction was calculated by using the “C1V1 = C2V2” calculation before the
addition of enzyme and substrate. It was made sure no enzyme was added to test
tube 6 as this will act as a control. The test tubes containing the mixture was
mixed well by ‘flicking’ each tube. The reactions were incubated at 30oC
for 20 minutes in a water bath. After 20 minutes, 3cm3 of Sodium
Carbonate was added to each test tube. This raised the pH of the solution
allowing any phenolphthalein produced to turn pink. The test tubes were mixed
if necessary. 2/3 of the cuvette was filled with some solution from each test
tube. The spectrophotometer was blanked with water. The absorbance of light
with a 550nm filter was used to measure for each sample and values were
recorded in a table. Adjusted Absorbance was calculated and by subtracting the
absorbance in test tube 6 to the rest of the readings. A graph was then plotted
with the calculated inhibitor concentration against the adjusted absorbance.
The results were then compared and averaged with the rest of the class ensure
reliability and comparison of results.

Results:

To
calculate the Adjusted Absorbance, the absorbance at 550nm in test tube 6 was
subtracted from the rest of the readings. Any pink colour from the test tube
will have come from the degraded substrate and would artificially increase the
readings in each test tube. Hence, subtracting the absorbance value for test
tube 6 compensates this.

Discussion:

The
purpose of this investigation was to determine the effect of phenolphthalein
phosphate on enzyme activity of phosphatase. This investigation observed that
the greater intensity of pink detected in each test tube, the greater the
absorbance readings hence suggesting more substrates were broken down due to the
enzyme concentration present. It was predicted that an increase in inhibitor
concentration, lowers the enzymatic activity which lowers the absorbance
whereas a decrease in inhibitor concentration, raises the enzymatic activity
hence a larger absorbance. The results of the investigation were as predicted
as the results clearly show that when the inhibitor concentration is low, the
absorbance was higher due to higher enzyme catalytic activity. This can be seen
in test tube 2 of the individual results which had an inhibitor concentration
of 0.0357M with an adjusted absorbance of 1.018 A.U. It can also be seen in
test tube 2 of the group results which had an inhibitor concentration of 0.0357M
with an adjusted absorbance of 1.043 A.U. This suggest that the inhibitor may
be non-competitive as there was a higher absorbance reading on the solutions,
hence the enzyme concentration present is larger compared to the inhibitor
concentration. Non-competitive inhibition is where the inhibitor binds to any
part of an enzyme apart from its active site which causes a conformational
change in the active site hence the substrate can no longer bind. Hence, when
the absorbance is low there were more inhibitors present compared to the
enzymes suggesting that an inhibitor can prevent an enzyme complex reaction
from taking place. For the individual results, the lowest absorbance was
obtained at test tube 4 which had an inhibitor concentration of 0.1429M with an
adjusted absorbance of 0.824 A.U (Table 3) compared to the overall class
average group result, the lowest absorbance was obtained at test tube 5 which
had an inhibitor concentration of 0.2143M with an adjusted absorbance of 0.577
A.U (Table 4).

 

Conclusion:

Results
were obtained through the investigation between the effect of phenolphthalein
phosphate on enzyme activity of phosphatase. The experiment showed that as the
inhibitor concentration decreases the enzyme activity decreases as there is a
greater concentration of inhibitor suggesting that the substrate can no longer
bind to the enzyme and the enzyme-substrate complex is restricted. The results
clearly show that when the inhibitor concentration is low, the absorbance was
higher due to higher enzyme catalytic activity. Both results come to a
conclusion that there is a large reduction in enzyme activity due to an
increase in inhibitors resulting in a less likelihood of enzyme binding to the
substrate, so instead the inhibitors bind. These results are reliable as group
and individual results were gathered to ensure reliability and comparison of
results.

 

Evaluation:

The
procedure was made as reliable as possible as the measurements and preparations
of solutions and dilutions were taken using 3cm3 transfer pipettes
and 10cm3 glass pipettes. Deionised water was used at all times to
prevent contamination of the results. The mass of the sprouts was weighed using
a balance which measured to 2 significant figures. The volumes of solutions
were measured as accurately as possible with transfer pipettes and glass
pipette. However, as the volumes used were small, there could be some errors.
They were added to a water bath at 30oC to ensure that the
temperature was constant and incubated. All the solutions were kept the same
and at a constant temperature during the incubation stage, to avoid any
anomalies and keep the enzymes at an optimum temperature. Multiple water baths
could be used in future experiments as not all the test tubes containing the
mixtures fit into one water bath, so this could cause inconsistency as they may
not be extremely accurate. Also, different pipettes were used for different
solutions; enzyme extract, pH buffer, substrate, inhibitor solution, distilled
water and stop solution to prevent any cross contamination. The colour change
of the solution was also carefully and accurately monitored using a
spectrophotometer with a 550nm filter.

Enzyme
activity is pH dependent so adding a pH 5 buffer to moderate and keep the pH of
the solution constant. If the pH had been altered, this could have affected the
enzyme’s activity and the results in the same way as temperature due to enzymes
having a specific optimal pH to which they work best.

Using a
stop clock, time was taken for the mixture to incubate which may not have been
exact due to human error in reaction time. Also, the mixture may have been left
in the water bath longer than necessary and the length of time the reactants
were allowed to react was 20 minutes. However, only a few of the groups got their
solutions out at exactly 20 minutes and as the reaction occurred rapidly on a
molecular level hence the groups could have a product more or less than what would
actually be produced in the time which could affect the results. The time taken
for the sodium carbonate when added to the solution may not be consistent
between every group which may affect the results as the reaction continues to react
when taken out of the water bath.

The enzyme
concentration could have somewhat varied as 40g of each mung beans sample of may
not have exactly the same concentration of enzymes in it and not all of the
enzymes may have been extracted from each sprout. This means that in some mixtures
there may have had more enzyme which would increase the reaction rate and so
more phenolphthalein would be produced in the time altering the results. Some
solutions may also have had less enzyme in them and so the reaction rate would
be slower producing less phenolphthalein in the time which would alter the
results. Doing several replicates will allow to see the natural variance.

A control
was set up where the enzyme extract was not added to the test tube.

The
experiment could be repeated to get more accurate results. As only one trial
per group were achieved within the experiment so the procedure could have been
repeated again as there will be variation. Although, average class group
results were gathered to ensure reliability and comparison of results.

BACK TO TOP
x

Hi!
I'm Rhonda!

Would you like to get a custom essay? How about receiving a customized one?

Check it out