Experiment 2 ENZYME KINETICS Beatriz E. Saldana

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Experiment 2 ENZYME KINETICS Beatriz E. Saldana
Experiment 2
ENZYME KINETICS
Beatriz E. Saldana Farias
8797
BIO 4110
Biochemistry 2 Laboratory
Experiment Performed: February 4, 2016
Report Submission Date: March 4, 2016
Instructor: Wade Dauberman
Beatriz E. Saldana Farias
8797
ABSTRACT
The purpose of this experiment was to test the reaction rate of the decomposition of pnitrophenyl glucopyranoside into glucose and p-nitrophenol, in the presence of an enzyme called
cellobiase. The experiment consisted of six different activities where the enzyme was exposed to
a variety of conditions and the difference in reaction rate was analyzed using a
spectrophotometer. In conclusion, the enzyme efficiency is increased by about two orders of
magnitude in the presence of cellubiase, and the enzyme activity in greately affected by the
environment it is exposed to due to the fact that it evolved to function under very specific
conditions.
INTRODUCTION
Enzymes are bundles of proteins with the function of catalyzing biological reactions.
Different types of enzyme are designed specifically for the substrate they bind to; the substrate
molecules bind to the active site of the enzyme. Enzymes create instability in the molecular
bonds of the substrate they are bound to, and therefore speed up the reaction rate
[3]
. Enzyme
activity can be drastically affected by the environment they evolved in, thus external factors such
as temperature, pH, and concentration change the efficiency of the enzyme.
Enzyme efficiency is examined by analytically measuring the changes in the reaction
rate. In this experiment, the reaction rate of cellobiase will be tested in a variety of different
conditions. Cellobiase is a class of enzymes produced by fungi and other organisms that are
involved in the decomposition of cellulose. The enzyme catalyzes the decomposition of pnitrophenyl glucopyranoside into glucose and p-nitrophenol. This enzyme is incredibly
significant to the process of decomposition on the food chain, due to the fact that most organisms
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on the planet cannot consume cellulose, if it were not for this enzyme, the cellulose content of
the plant would never be recycled back into the ecosystem. Although the dissociation of
cellulose would eventually occur, cellobiase significantly increases the rate of reaction so that the
matter can be recycled at a faster time scale. The study of cellobiase has become increasingly
significant to humanity in recent years due to the exponential growth in population and lack of
resources to sustain such a large population. If humans could find a way to consume cellulose,
the fear of famine would greatly decrease. Due to the fact that cellobiase is a key enzyme
involved in the decomposition of cellulose, it is crucial to fully understand their function.
The purpose of this experiment was to test the efficiency of cellobias in a variety of
conditions. The experiment consisted of six different activities where the enzyme was tested, the
first activity consisted of testing the reaction rate of a reaction with and without the enzyme, the
purpose of the activity was to determine that the enzyme does in fact increase the reaction rate
with increasing concentration. The rate of breakdown of p-nitrophenyl glucopyranoside into
glucose and p-nitrophenol in the presence and absence of cellobias would be analyzed using a
spectrophotometer. The second activity tested the efficiency of cellobiase in three different
temperatures, 0ºC, 22ºC, and 33ºC. Temperature tends to affect the rate of reaction of a variety of
enzymes due to the thermodynamic effects, and all enzymes are designed to function in different
temperature ranges
[7]
. The third experiment tested the abilities of cellobiase to function in
solutions of varying acidity levels. Enzymes are specifically designed for the environment they
function in, and therefore the pH of the solution in which they catalyze the reaction will affect
the reaction rate. The third experiment was designed to test exactly that pheonomenon and find
out in what environment cellobiase functions best in. The fourth and fifth activities consisted of
testing the effects of enzyme and substrate concentration on the reaction rate. The concentration
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can affect the rate of reaction by increasing or decreasing the statistical probability of a reaction
occurring
[4]
. The last activity in the series was more intricate and required the extraction of
mushroom enzymes. The first part of the activity was to successfully extract the accurate
compounds containing the enzyme. After the extraction the enzymes were analyzed using a
spectrophotometer. Mushrooms were used for the last activity because they are one of the
organisms that produce cellobiase, and thus it was important to learn the extraction process and
biochemical location of the enzymes inside an organism.
MATERIALS AND METHODS
This experiment consisted of six different activities that all consisted of testing the rate of
breakdown of p-nitrophenyl glucopyranoside into glucose and p-nitrophenol in the presence of
cellobias. The purpose of the first activity was to determine the reaction rate in the presence or
absence of cellobias. The first step was to label five corvettes E1 – E5 , and two others “Start”
and “End”, which served as the controls. Then 500ul of stop solution was pipetted into all seven
curvettes. Then two 15ml conical tubes were labeled “Enzyme Reaction” and “Control”, and 2ml
and 1ml of 1.5 mM substrate were pipetted into each respectively. 500ul of the buffer solution
were pipetted into the tube labeled “Control”, the solution was mixed, and 500ul of the solution
was pipetted into the curvet labeled “Start”. Then 1ml of the enzyme solution was pipetted into
the tube labeled “Enzyme Reaction”, the solution was mixed and the timer was started. After
1min 500ul of the “Enzyme Reaction” solution was pipetted into the curvet labeled E1, after
2min the solution was pipetted into the E2 curvet, then 4min into E3, 6min into E4, and 8min
into E5. Finally, 500ul from the control solution were pipetted into the curvet labeled “End”. The
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samples were then analyzed using a spectrophotometer at 410nm blanked with the curvet labeled
“S1”. The results were recorded on a table and the p-nitrophenol produced was determined using
a standard curve.
The second activity consisted of testing the enzyme reaction rate at different
temperatures, 0ºC, 22ºC, and 37ºC. To do that first three curettes were labeled 0, 22, and 37 and
500ul of stop solution was pipetted into each. Then six micro-centrifuge tubes were labeled 0E,
22E, 37E, 0S, 22S, and 37S and 250ul of Enzyme was pipetted into the microcentrifuge tubes
containing the letter “E” and 1.5 mM substrate was pipetted into the tubes containing the letter
“S”. The tubes with a “0” on it were placed in an ice cup, the ones with a “22” were left on the
lab bench, and the ones with a “37” were placed in a beaker with warm water; the tubes were left
there for about 5min. After the time went by, 250ul of the tubes containing the enzyme were
pipetted into the tubes containing the substrate of their same temperature, and the timer was
started. After two minutes 500ul from each tube containing the substrate and enzyme were
pipetted into the curvets labeled with their respective temperature. The samples were then
analyzed using a spectrophotometer at 410nm blanked with the curvet labeled “S1”. The results
were recorded on a table and the p-nitrophenol produced was determined using a standard curve.
The purpose of the third activity was to determine the effect of pH on the enzyme. The
activity was executed just like the second activity but instead of exposing the solution to three
different temperature conditions, it was exposed to different acidities of pH of 5, 6.3, and 8.6.
The resulting solutions were analyzed using a spectrophotometer. The fourth and fifth
experiments varied the enzyme and substrate concentration respectively. The sixth activity
consisted of first extracting enzymes from white mushrooms and then testing the efficiency of
the enzyme in a very similar manner than the first activity.
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RESULTS
Activity 1: Determine the Reaction Rate in the Presence or Absence of an Enzyme
Table 1: Absorbance values for standards
Standards
Amount of p-Nitrophenol (nmol)
Absorbence at 410 nm
S1
0
0.00
S2
12.5
0.111
S3
25
0.208
S4
50
0.427
S5
100
0.804
Table 2: Determining p-nitrophenol produced using a standard curve
Time (min)
Cuvette
Amount of p-nitrophenol (nmol) from the Absorbance at 410 nm
Standard Curve
0
Start
0.75
8
End
1
E1
12.5
0.100
2
E2
16.75
0.134
4
E3
29.25
0.234
6
E4
43.376
0.347
8
E5
54.875
0.439
0.006
-0.012
Activity 2: Determine the Effects of Temperature on the Reaction Rate
Table 3: P-nitrophenol produced at three different temperatures
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Temperature
Absorbance at 410nm
Amount of p-Nitrophenol Produced (nmol)
0ºC
0.081
10.125
22ºC
0.093
11.88
37ºC
0.230
28.75
Initial rate of product formation at 0ºC= 5.0625 nmol/min
Initial rate of product formation at 22ºC= 5.94 nmol/min
Initial rate of product formation at 37ºC= 14.375 nmol/min
Activity 3: Determine the Effects of pH on Reaction Rate
Table 4: p-nitrophenol produced at three different pH levels
pH
Absorbance at 410 nm
Amount of p-nitrophenol produced
5.0
0.380
47.50
6.3
0.477
59.63
8.6
0.072
9.00
Initial rate of product formation at pH 5.0= 23.75 nmol/min
Initial rate of product formation at pH 6.3= 29.80 nmol/min
Initial rate of product formation at pH 8.6= 4.50 nmol/min
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Activity 4: Determine the Effect of Enzyme Concentration on Reaction Rate
Table 5: P-nitrophenol produced using a high and low enzyme concentration based on a standard
curve
Cuvette
Absorbence at 410 nm
Amount of p-Nitrophenol Produced (nmol)
H1
0.719
89.875
H2
0.927
115.875
H3
1.372
171.5
L1
0.215
26.875
L2
0.320
40
L3
0.775
96.875
Activity 5: Determine the Effect of Substrate Concentration on Reaction Rate
Table 6: Determination of p-nitrophenol produced using a high and low substrate concentration
based on a standard curve
Cuvette
Absorbance at 410 nm
Amount of p-Nitrophenol Produced (nmol)
Hi
0.057
7.125
H2
0.154
19.250
H3
0.261
32.625
L1
0.008
1
L2
0.000
0
L3
0.097
12.125
Activity 6: Test Ability of Mushroom Extracts to Increase Reaction Rate
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Table 7: Determination of p-nitrophenol produced by the mushroom extract breaking down the
substrate based on a standard curve
Cuvette
Absorbance at 410 nm
Amount of p-Nitrophenol Produced (nmol)
1
0.008
1
2
0.053
6.625
3
0.030
3.75
4
0.065
8.125
5
0.098
12.25
6
0.00
---
DISCUSSION
The experiment results indicated a very specific niche in which the enzyme activity is
optimized. The first experiment results depicted an increase in absorbance and p-Nitrophenol
production with time. Therefore the longer an enzyme is exposed to substrate, the more substrate
that will be catalyzed. This happens due to the fact that substrate binds to enzyme only while it is
being catalyzed, and then the enzyme is freed, thus the more time an enzyme is allotted to
catalyze, the more it will catalyze, until all of the substrate has been disassociated. The second
experiment tested the reactive capability of the enzyme in a variety of temperatures. There is a
very evident positive correlation between temperature increase and absorption. As the
temperature increased, the catalytic rate of reaction drastically increased. This is due to the fact
that temperature directly affects the kinetic energy of a system, therefore as the kinetic energy
increases, the easier the activation energy of a reaction is achieved especially in the presence of
an enzyme [5]. Biological systems typically operate better at neutral pH, although some enzymes
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are specialized for more basic or acidic environments. According to the results of the third
activity, cellobiase is also one of the enzymes that preforms better in neutral conditions. Most of
the organisms that are capable of decomposing cellulose do not contain complex organs or
specialized acidic digestion, thus most of their enzymes work better in a neutral environment.
The fifth and sixth activities provided very expected results, the higher the concentration the
more p-Nitrophenol produced. That is due to the fact that the more substrate or enzymes present
the higher the binding probability. The last activity was very similar to the first one, but required
an extraction at first. The extraction was successful and thus the results were the same as the first
activity, and for the same reason. In conclusion, enzymes are vulnerable to the environment and
the rate of reaction can be drastically influenced by the conditions an enzyme is exposed to.
LITERATURE CITED
[1] Baars, Johan JP, et al. "Nitrogen assimilating enzymes in the white button mushroom
Agaricus bisporus." Microbiology 140.5 (1994): 1161-1168.
[2] Craig, Douglas B., et al. "Studies on Single Alkaline Phosphatase Molecules: Reaction Rate
and Activation Energy of a Reaction Catalyzed by a Single Molecule and the Effect of Thermal
Denaturation The Death of an Enzyme."Journal of the American Chemical Society 118.22
(1996): 5245-5253.
[3] Garcia-Viloca, Mireia, et al. "How enzymes work: analysis by modern rate theory and
computer simulations." Science 303.5655 (2004): 186-195.
Gong, Cheng‐Shung, Michael R. Ladisch, and George T. Tsao. "Cellobiase from Trichoderma
viride: purification, properties, kinetics, and mechanism."Biotechnology and Bioengineering 19.7
(1977): 959-981.
[4] Maguire, R. James. "Kinetics of the hydrolysis of cellobiose and p-nitrophenyl-β-d-glucoside
by cellobiase of Trichoderma viride." Canadian journal of biochemistry 55.1 (1977): 19-26.
[5] Segel, Irwin H. Enzyme kinetics. Vol. 957. Wiley, New York, 1975.
[6] Sengupta, Saswati, Anil K. Ghosh, and Subhabrata Sengupta. "Purification and
characterisation of a β-glucosidase (cellobiase) from a mushroom Termitomyces
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clypeatus." Biochimica et Biophysica
Enzymology 1076.2 (1991): 215-220.
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Acta
(BBA)-Protein
Structure
and
Molecular
[7] Wolfenden, Richard, et al. "The temperature dependence of enzyme
enhancements." Journal of the American Chemical Society 121.32 (1999): 7419-7420.
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