Evelin Isabel Falcão (335385)
ESTABLISHMENT OF KINETIC PARAMETERS FOR ALKALINE PHOSPHATASE
The purpose of this lab was to determine the activity of the enzyme alkaline phosphatase using the synthetic
substrate pNPP through a continuous assay. The amount of pNP or product was determined using
spectrophotometric measurement at A410.
I started by preparing 50mL of the reaction buffer and 6mL of substrate solution using the volumes and
concentrations shown on table 1.
The next step was the preparations of six cuvettes with increasing volumes of buffer and substrate solution,
these cuvettes were then inverted 3 times with parafilm covering the top to ensure that both solutions were
mixed.
After that I measured the absorbance of the first cuvette, added 100µL of enzyme solution to it, inverted it 3
times and very quickly put it back in the sprectrophotometer to measure the absorbance every 20s for 2
minutes. This process was repeated for the remaining 5 cuvettes.
The aborbance values I obtained throught this are shown on table 2.
I then plotted the graph (graph 1) time vs. Absorbance and found that the interval from 60s to 120s was the
most linear one, it was used for the calculation of V0 and others (table 3).
After obtaining V0 and the concentration of pNPP I plotted the graph V0 vs. [S] (graph 2). Observing the
graph I found that maintaining the concentration of enzyme constant while increasing that of the substrate
results in an increase in the velocity of the reacion but only until a certain maximum when all the ezyme has
formed an ES complex, therefore working at a constant rate.
Afterwards I calculated 1/ V0 and 1/[S], plotted it (graph 3) and generated the graph equation which allowed
me to calculate Vmax, Km and subsequently Kcat.
Following the procedure described above I prepared 6 other cuvettes, but this time I included 100µL of
enzyme inhibitor, which is a substance that stops the enzyme from carrying out its function, in each cuvette.
The absorbance values obtained from this are shown in table 4.
Looking at the resulting graph (graph 4) once again I chose the interval from 60s to 120s to perform the
calculation of V0 and others (table 5).
After plotting the graph V0 vs. [S] and overlaying it with the data obtained without the presence of an
inhibitor (graph 5), I concluded that the presence of the inhibitor drastically reduces the velocity of the
reaction regardless of the concentration of the substrate.
Assignment of inhibition mode:
To determine the type of inhibition occurring in my cuvettes, I calculated 1/ V0 and 1/[S] for the data set
containing the inhibitor and plotted it, also overlaying with the data set without the inhibitor (graph 6).
According to my data and graphs I can safely assume that the method of inhibition is non-competitive.
I set the regression on my graph to 18,0 on excel so that the intersection with the x axis is visible, and used
the Lineawaever-Burk method (graph 7).
A constant slope is expected in this type of inhibition, although my value for slope did not remain constant
there are other factors that lead me to my conclusion.
With this type of inhibition a decrease in the Vmax and a constant Km value is also expected when
comparing it with those of the reaction without the inhibitor, in my case the Vmax value did decrease and
the change in the Km value was of 0,01 so it is safe to say that it has remained somewhat constant.
The graph I plotted looks very similiar to what a graph demonstrating non-competitive inhibition looks like1
And I also found theory suggesting that L-Phenylalanine is a non competitive inhibitor.2
1
https://files.mtstatic.com/site_4463/5970/0?Expires=1666633314&Signature=GCJVzJaMYeYJahrsoMUpyaLcQ4vdEv~OpV8nctuvUQj7EOyu
H6HBNiz~zGLudNoV6HgV8ByeLS7AuVg9U-BvTL~B4KkNQzupYdkIlzceZHMDwSQE0F0o~BvEGtQMP0jpat5N2g~7gxEtafohonC6FsXLCtLpkyDktHE7rofN50_&Key-Pair-Id=APKAJ5Y6AV4GI7A555NA
2 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1270344/pdf/biochemj00745-0292.pdf
1
Preparation of 50 mL of the reaction buffer (0.5 M Tris-HCl pH 9.0 with 5 mM MgCl2)
Volume of 1 M Tris-HCl pH 9.0
25mL
Volume of 50 mM MgCl2
5mL
Volume of H2O
20mL
Preparation of the working substrate solution (6 mL of 1.35 mM pNPP in the reaction buffer)
Volume of 1 M Tris-HCl pH 9.0
3mL
Volume of 50 mM MgCl2
0,6mL
Volume of 10 mM pNPP
0,81mL
Volume of H2O
1,59mL
Table 1
A410 values recorded at indicated time intervals of the assay:
Sample no.
Time [s]
0
20
40
60
80
100
120
1
0,018
0,069
0,113
0,152
0,191
0,231
0,269
2
3
4
5
6
0,034
0,091
0,135
0,19
0,235
0,276
0,322
0,031
0,1
0,15
0,208
0,259
0,309
0,359
0,033
0,1
0,173
0,223
0,285
0,342
0,397
0,031
0,1
0,164
0,23
0,297
0,36
0,423
0,032
0,137
0,165
0,260
0,315
0,420
0,463
Table 2
Plot of ΔA410 vs. time [s]:
Graph 1
2
Example calculations for a sample 4:
A410 (120s) – A410 (60s) = 0.364 – 0.190 = 0.174
A= ε*c*l
0,174=18500*c*1
c=0,174/18500
c=9,4*10-6 M or 9,4 μM
ΔC/min = 9,4 μM/min
V0=9,4*2,5/1000
V0= 0,0235 μmol/min
Assay conditions:
Sample
no.
C pNPP
[mM]
1
0,08
0,108
0,162
0,216
0,270
0,540
2
3
4
5
6
ΔC/min
V0
ΔA410/min [µM/min] [µmol/min]
0,117
0,132
0,151
0,174
0,193
0,203
6,32
7,14
8,16
9,4
10,4
10,92
1,58*10-2
1,79*10-2
2*10-2
2,35*10-2
2,6*10-2
2,73*0-2
1/[S]
[1/mM]
1/V0
[min/µmol]
12,5
9,26
6,17
4,63
3,7
1,85
63,29
55,87
50
42,55
38,46
36,63
Table 3
Plot of V0 vs. [S]:
Graph 2
Plot of 1/V0 vs. 1/[S]:
Graph 3
3
Determination of Km and Vmax parameters (without inhibitor):
Formula generated from graph: y = 2,6439x + 31,007
Vmax= 1/b= 1/31,007= 0,08527 µmol/min
Km= slope*Vmax= 2,6439*0,03225= 0,03225 mM or 0,5375nmol/s
Slope: 2,64
Determination of kcat value:
nE= 0,0357 mols
Kcat= Vmax/nE= 0,5375/0,0357= 15 s-1
Inhibition of alkaline phosphatase by L-Phe
Determination of Km and Vmax parameters (with inhibitor):
Formula generated from the graph: 9,6939x + 114,5
Vmax: 0,00873 µmol/min
Km: 0,08466 mM
Slope: 9,68
A410 values recorded at indicated time intervals of the assay:
Sample no.
Time [s]
0
20
40
60
80
100
120
1
2
3
4
5
6
0,097
0,104
0,116
0,126
0,135
0,145
0,157
0,007
0,02
0,03
0,04
0,053
0,067
0,079
0,005
0,027
0,037
0,045
0,057
0,07
0,083
0,007
0,024
0,039
0,049
0,063
0,081
0,092
0,008
0,026
0,042
0,055
0,072
0,088
0,104
0,011
0,03
0,054
0,066
0,09
0,108
0,128
Table 4
Plot of ΔA410 vs. time [s]
Graph 4
4
Assay conditions:
Sample
no.
C Pnpp
[Mm]
1
0,08
0,108
0,162
0,216
0,270
0,540
2
3
4
5
6
ΔC/min
V0
ΔA410/min [µM/min] [µmol/min]
0,031
0,039
0,040
0,043
0,049
0,062
1,68
2,11
2,16
2,32
2.65
3,35
4,2*10-3
5,28*10-3
5,4*10-3
5,8*10-3
6,6*10-2
8,34*10-3
1/[S]
[1/Mm]
1/V0
[min/µmol]
12,5
9,26
6,17
4,63
3,7
1,85
238
189,39
185,19
172,41
151,52
119,90
Table 5
Plot of V0 vs. [S]:
Graph 5
Plot of 1/V0 vs. 1/[S]:
Graph 6
5
Lineaweaver-Burk plot:
Graph 7
6