Determining Activation Energy for an Enzymatic Reaction Catalyzed by

Alkaline Phosphatase
Molecule to Organism 2017-2018

Kat Dyer

Partner: Trevor McCliment

1
Results:
Standard Curve:
Absorbance of 4-nitrophenol at 400 nm
Solution Name Concentration (M) Absorbance
Blank 0 0
Solution 2 0.000040256 0.742
Solution 3 0.000030192 0.493
Solution 4 0.000020128 0.376
Solution 5 0.000010064 0.194
Solution 6 6.70933E-06 0.131
Table 1: Absorbance of 4-nitrophenol at maximum wavelength of 400 nm.

Standard Curve of 4-nitrophenol at 400 nm

0.8

0.7 y = 17642x + 0.007

0.6

0.5
Absorbance

0.4
Absorbance
0.3 Linear (Absorbance)

0.2

0.1

0
0.00E+00 5.00E-06 1.00E-05 1.50E-05 2.00E-05 2.50E-05 3.00E-05 3.50E-05 4.00E-05 4.50E-05
Concentration (M)

Figure 1: Standard curve showing absorbance of 4-nitrophenol at 400 nm.

2
Absorbance at 1°C:
Production of 4-nitrophenol at 1°C
Time (min) Time (sec) Absorbance at 400 nm Concentration of 4-nitrophenol (M) Reaction Rate (M/s)
0 0 0 0 0
0.5 30 -0.3 -1.74017E-05 -5.80055E-07
1 60 -0.3 -1.74017E-05 0
2 120 -0.069 -4.3079E-06 2.18229E-07
3 180 -0.023 -1.70049E-06 4.34569E-08
4 240 -0.047 -3.06088E-06 -2.26732E-08
5 300 -0.227 -1.32638E-05 -1.70049E-07
10 600 0.273 1.50777E-05 9.44715E-08
15 900 0.247 1.36039E-05 -4.91252E-09
20 1200 0.159 8.6158E-06 -1.6627E-08
Overall Reaction Rate (M/s): 2.128E-08
Rate Constant (mol/mg s): 8.11285E-08
Table 2: Absorbance of enzyme solution at 1°C at a wavelength of 400 nm.

Production of 4-nitrophenol at 1°C

0.00002
y = 2.128E-08x - 9.707E-06

0.000015
Concentration of 4-nitrophenol (M)

0.00001

0.000005

0 Reaction Rate (M/s)

0 200 400 600 800 1000 1200 1400 Linear (Reaction Rate (M/s))
-0.000005

-0.00001

-0.000015

-0.00002
Time (sec)

Figure 2: Graph showing production of 4-nitrophenol at 1°C.

3
Absorbance at 23°C:
Production of 4-nitrophenol at 23°C
Time (min) Time (sec) Absorbance at 400 nm Concentration of 4-nitrophenol (M) Reaction Rate (M/s)
0 0 0 0 0
0.5 30 0.017 5.66829E-07 1.88943E-08
1 60 0.03 1.30371E-06 2.45626E-08
2 120 0.056 2.77746E-06 2.45626E-08
3 180 0.081 4.19454E-06 2.36179E-08
4 240 0.106 5.61161E-06 2.36179E-08
5 300 0.131 7.02868E-06 2.36179E-08
10 600 0.25 1.37739E-05 2.24842E-08
15 900 0.362 2.01224E-05 2.11616E-08
20 1200 0.47 2.62442E-05 2.04058E-08
25 1500 0.571 3.19692E-05 1.90832E-08
30 1800 0.668 3.74674E-05 1.83275E-08
Overall Reaction Rate (M/s): 2.11E-08
Rate Constant (mol/mg s): 8.04422E-08
Table 3: Absorbance of enzyme solution at 23°C at a wavelength of 400 nm.

Production of 4-nitrophenol at 23°C

0.000045
y = 2.110E-08x + 4.029E-07
0.00004

0.000035

0.00003
Concentration (M)

0.000025

0.00002 Reaction Rate (M/s)

Linear (Reaction Rate (M/s))
0.000015

0.00001

0.000005

0
0 200 400 600 800 1000 1200 1400 1600 1800 2000
Time (sec)

Figure 3: Graph showing production of 4-nitrophenol at 23°C.

4
Absorbance at 25°C:
Production of 4-nitrophenol at 25°C
Time (min) Time (sec) Absorbance at 400 nm Concentration of 4-nitrophenol (M) Reaction Rate (M/s)
0 0 0 0 0
0.5 30 0.03 1.30371E-06 4.34569E-08
1 60 0.075 3.85444E-06 8.50244E-08
2 120 0.098 5.15815E-06 2.17285E-08
3 180 0.137 7.36878E-06 3.68439E-08
4 240 0.166 9.01258E-06 2.73967E-08
5 300 0.193 1.0543E-05 2.55073E-08
10 600 0.328 1.81952E-05 2.55073E-08
15 900 0.542 3.03254E-05 4.04338E-08
20 1200 0.647 3.62771E-05 1.9839E-08
25 1500 0.787 4.42127E-05 2.6452E-08
30 1800 0.949 5.33953E-05 3.06088E-08
Overall Reaction Rate (M/s): 2.906E-08
Rate Constant (mol/mg s): 1.10789E-07
Table 4: Absorbance of enzyme solution at 25°C at a wavelength of 400 nm.

Production of 4-nitrophenol at 25°C

0.00006

y = 2.906E-08x + 1.521E-06
0.00005
Concentration (M)

0.00004

0.00003
Reaction Rate (M/s)
Linear (Reaction Rate (M/s))
0.00002

0.00001

0
0 200 400 600 800 1000 1200 1400 1600 1800 2000
Time (sec)

Figure 4: Graph showing production of 4-nitrophenol at 25°C.

5
Absorbance at 30°C:
Production of 4-nitrophenol at 30°C
Time (min) Time (sec) Absorbance at 400 nm Concentration of 4-nitrophenol (M) Reaction Rate (M/s)
0 0 0 0 0
0.5 30 0.024 9.6361E-07 3.21203E-08
1 60 0.029 1.24702E-06 9.44715E-09
2 120 0.07 3.57102E-06 3.87333E-08
3 180 0.104 5.49824E-06 3.21203E-08
4 240 0.131 7.02868E-06 2.55073E-08
5 300 0.167 9.06927E-06 3.40097E-08
10 600 0.315 1.74583E-05 2.79636E-08
15 900 0.435 2.42603E-05 2.26732E-08
20 1200 0.545 3.04954E-05 2.07837E-08
25 1500 0.642 3.59937E-05 1.83275E-08
30 1800 0.727 4.08117E-05 1.60602E-08
Overall Reaction Rate (M/s): 2.337E-08
Rate Constant (mol/mg s): 8.90965E-08
Table 5: Absorbance of enzyme solution at 30°C at a wavelength of 400 nm.

Production of 4-nitrophenol at 30°C

0.00005
y = 2.337E-08x + 1.204E-06
0.000045

0.00004

0.000035
Concentration (M)

0.00003

0.000025
Reaction Rate (M/s)
0.00002 Linear ( Reaction Rate (M/s))

0.000015

0.00001

0.000005

0
0 500 1000 1500 2000
Time (sec)

Figure 5: Graph showing production of 4-nitrophenol at 30°C.

6
Absorbance at 37°C:
Production of 4-nitrophenol at 37°C
Time (min) Time (sec) Absorbance at 400 nm Concentration of 4-nitrophenol (M) Reaction Rate (M/s)
0 0 0 0 0
0.5 30 0.018 6.23512E-07 2.07837E-08
1 60 0.047 2.26732E-06 5.47935E-08
2 120 0.103 5.44156E-06 5.29041E-08
3 180 0.145 7.82224E-06 3.9678E-08
4 240 0.197 1.07698E-05 4.91252E-08
5 300 0.233 1.28103E-05 3.40097E-08
10 600 0.451 2.51672E-05 4.11896E-08
15 900 0.657 3.68439E-05 3.89223E-08
20 1200 0.846 4.7557E-05 3.57102E-08
25 1500 1.012 5.69663E-05 3.13645E-08
Overall Reaction Rate (M/s): 3.871E-08
Rate Constant (mol/mg s): 1.47579E-07
Table 6: Absorbance of enzyme solution at 37°C at a wavelength of 400 nm.

Production of 4-nitrophenol at 37°C

0.00007

y = 3.871E-08x + 6.977E-07
0.00006

0.00005
Concentration (M)

0.00004
Concentration of 4-nitrophenol (M)
0.00003
Linear (Concentration of 4-nitrophenol
(M))
0.00002

0.00001

0
0 200 400 600 800 1000 1200 1400 1600
Time (sec)

Figure 6: Graph showing production of 4-nitrophenol at 37°C.

7
Absorbance at 42°C:
Production of 4-nitrophenol at 42°C
Time (min) Time (sec) Absorbance at 400 nm Concentration of 4-nitrophenol (M) Reaction Rate (M/s)
0 0 0 0 0
0.5 30 0.006 -5.66829E-08 -1.88943E-09
1 60 0.031 1.36039E-06 4.72358E-08
2 120 0.067 3.40097E-06 3.40097E-08
3 180 0.102 5.38488E-06 3.3065E-08
4 240 0.138 7.42546E-06 3.40097E-08
5 300 0.169 9.18263E-06 2.92862E-08
10 600 0.336 1.86487E-05 3.15535E-08
15 900 0.46 2.56774E-05 2.34289E-08
20 1200 0.57 3.19125E-05 2.07837E-08
25 1500 0.661 3.70706E-05 1.71938E-08
30 1800 0.744 4.17753E-05 1.56823E-08
Overall Reaction Rate (M/s): 2.43E-08
Rate Constant (mol/mg s): 9.2642E-08
Table 7: Absorbance of enzyme solution at 42°C at a wavelength of 400 nm.

Production of 4-nitrophenol at 42°C

0.00005
y = 2.430E-08x + 1.116E-06
0.000045

0.00004

0.000035
Concentration (M)

0.00003

0.000025
Reaction Rate (M/s)
0.00002
Linear (Reaction Rate (M/s))
0.000015

0.00001

0.000005

0
0 200 400 600 800 1000 1200 1400 1600 1800 2000
-0.000005
Time (sec)

Figure 7: Graph showing production of 4-nitrophenol at 42°C.

8
Summary of Absorbance Data:

1.4
Absorbance at 400 nm by Temperature

1.2
1C

1 23 C
25 C
0.8 30 C
Absorbance (nm)

37 C
0.6
42 C

0.4 Linear (1 C)
Linear (23 C)
0.2
Linear (25 C)
Linear (30 C)
0
0 200 400 600 800 1000 1200 1400 1600 1800 2000 Linear (37 C)
-0.2 Linear (42 C)

-0.4 Time (sec)

Figure 8: Graph showing absorbance at 400 nm for various temperatures.

Sample Calculations:
Reaction Rate:

To find concentration of 4-nitrophenol from absorbance data, the equation of the best-fit line from the standard curve
was used and solved for concentration.

𝑦 = 17642𝑥 + 0.007
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 = 17642 (𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛) + 0.007
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 − 0.007 = 17642(𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛)
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 0.007
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = ( )−( )
17642 17642
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = ( ) − (3.9678 ∗ 10−7 )
17642
For 23°C:
0.017
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = ( ) − (3.9678 ∗ 10−7 )
17642
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = (9.6361 ∗ 10−7 ) − (3.9678 ∗ 10−7 )
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = 5.669 ∗ 10−7
Concentration of 4-nitrophenol was then plotted against time in seconds and the slope of the best fit line was found.
9
Rate Constant:

To find the rate constant, [ES] (presumed to be equal to [E] at steady state) was calculated.
1
[𝐸𝑆] = [𝐸𝑛𝑧𝑦𝑚𝑒 𝑠𝑡𝑜𝑐𝑘 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛] ∗ [𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟] ∗ 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑑𝑖𝑙𝑢𝑡𝑒𝑑 𝑒𝑛𝑧𝑦𝑚𝑒 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 ∗
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒
2.5𝑚𝑔 32𝑢𝐿 1𝑚𝐿 1𝑚𝐿 1
[𝐸𝑆] = ∗ ∗ ∗ 50𝑢𝐿 ∗ ∗
𝑚𝐿 5𝑚𝐿 1000𝑢𝐿 1000𝑢𝐿 3.00𝑚𝐿 + 50𝑢𝐿
8.00 ∗ 10−4 𝑚𝑔
[𝐸𝑆] =
3.05𝑚𝐿
𝑚𝑔 1000𝑚𝐿
[𝐸𝑆] = 2.623 ∗ 10−4 ∗
𝑚𝐿 1𝐿
𝑚𝑔
[𝐸𝑆] = 0.2623
𝐿
This value was used with the reaction rate to find k2 for 23°C.
𝑘2
𝑅𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 =
[𝐸𝑆]
𝑘2 = (𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒)([𝐸𝑆])
𝑀 𝑚𝑔
𝑘2 = (2.110 ∗ 10−8 ) (0.2623 )
𝑠 𝐿
𝒎𝒐𝒍
𝒌𝟐 = 𝟖. 𝟎𝟒𝟒 ∗ 𝟏𝟎−𝟖
𝒎𝒈 ∗ 𝒔𝒆𝒄

10
Summary Information:

Rate and Rate Constant By Temperature

Rate Constant k2
Temperature (°C) Temperature (K) 1/Temperature (1/K) Rate (M/s) (mol/mg s) ln(k2)
1 274.15 0.003647638 2.128E-08 8.113E-08 -16.33
23 296.15 0.003376667 2.110E-08 8.044E-08 -16.34
25 298.15 0.003354016 2.906E-08 1.108E-07 -16.02
30 303.15 0.003298697 2.337E-08 8.910E-08 -16.23
37 310.15 0.003224246 3.871E-08 1.476E-07 -15.73
42 315.15 0.003173092 2.430E-08 9.264E-08 -16.19
Table 8: Reaction rate and rate constant by temperature.

Rate Constant versus Temperature

1.600E-07
Rate Constant k2 (mol/mg s)

1.400E-07
1.200E-07
1.000E-07
8.000E-08
6.000E-08
4.000E-08
2.000E-08
0.000E+00
270 275 280 285 290 295 300 305 310 315 320
Temperature (K)

Figure 9: Graph showing rate constant versus temperature.

Natural Log of Rate Constant versus 1/Temperature

-15.60
0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 0.0037
Natural Log of Rate Constant

-15.70
-15.80
-15.90
-16.00
-16.10
-16.20
-16.30
-16.40
1/Temperature (1/K)

Figure 10: Graph showing rate constant versus temperature.

11
Calculation of Activation Energy:
To find activation energy, the two-point Arrhenius equation was used with the rate constant and temperature for the
two most linear data sets, 23°C and 37°C.
𝑘2 𝐸𝑎 1 1
𝑙𝑛 ( ) = − ( − )
𝑘1 𝑅 𝑇2 𝑇1
3.871 ∗ 10−8 𝐸𝑎 1 1
𝑙𝑛 ( )=− ( − )
2.110 ∗ 10 −8 𝐽 310.15𝐾 296.15𝐾
8.315
𝑚𝑜𝑙 𝐾
𝐸𝑎 0.000152
𝑙𝑛(1.8346) = − (− )
𝐽 𝐾
8.315
𝑚𝑜𝑙 𝐾
𝐸𝑎 0.000152
0.606825 = − (− )
𝐽 𝐾
8.315
𝑚𝑜𝑙 𝐾
𝐽
(0.606825) (8.315 )
−𝐸𝑎 = 𝑚𝑜𝑙𝐾
0.000152
(− 𝐾 )

𝐽
−𝐸𝑎 = −33195.7
𝑚𝑜𝑙
𝑬𝒂 = 𝟑𝟑. 𝟐 𝒌𝑱/𝒎𝒐𝒍

12