Baxter 2
Introduction
Phosphatases are a general class of enzymes which remove phosphate groups
from other proteins, a process called dephosphorylation (Berridge, M.J.). Although the
tyrosine phosphatase superfamily has widely divergent structures in a variety of
organisms, all carry a signature motif (H-C-X-X-G-X-X-R) which is responsible for their
catalytic activity (Berridge, M.J.). The mechanism begins by transferring the substrates
phosphate group to the cysteine residue in the signature motif. This is followed by the
phosphoryl residue being hydrolyzed by water coordinated with a glycine, located in the
signature motif, to release the phosphate anion (Berridge, M.J.). The phosphatase is
now reverted back to a non-bound conformation and can catalyze another removal of a
phosphate from a different substrate.
A specific group of phosphatases, known as alkaline phosphatases (APs), are
found in many organisms ranging from bacteria to humans (Milln, 2006). As the name
suggests, this group of phosphatases, regardless of organism, has a more alkaline ideal
pH (Milln). As previously mentioned, the catalytic site is highly conserved. When
comparing mammalian and bacterial APs, the mammalian counterparts are more
frequently membrane bound and more readily inhibited by some L-amino acids and
peptides through uncompetitive inhibition (Milln, 2006).
The goal of this current study is to investigate the proposed inhibitory effects of Lamino acids on the activity of bovine intestinal AP (Ghosh, 1965, Fernley and Walker,
1970). We expect to see inhibition of the breakdown of the enzyme-substrate complex
in a non-competitive manner when introducing L-phenylalanine, as others have reported
before (Ghosh, 1965). This means that we expect the double reciprocal plots of the
Baxter 3
Methods
Materials and reaction protocols
The bovine alkaline phosphatase was provided by Dr. Schramp and kept on ice
until it was needed. The substrate used was p-nitrophenylphosphate (PNPP) which
was also provided by Dr. Schramp and kept on ice until needed. Tris-Hcl was used to
buffer all reactions. Sodium hydroxide (1M) was used to stop the reactions, inactivate
the enzyme, and convert all accumulated product into the p-nitrophenolate ion
(Schramp, 2014). Ten total reactions were run in separate test tubes in a 37 oC water
bath. These included 6 different concentrations of L-Phenylalanine ranging from 2mM
to 7.5mM.
Tyrosine as a structurally similar amino acid control, minus enzyme to correct for the
absorbance due to slight product formation due to pH, minus substrate to blank the
spectrophotometer, and a normal control reaction ran without inhibitor.
The test tubes were then brought to temp in a 37 oC water bath for
Bovine
Baxter 4
alkaline phosphatase was added in 60 second increments in each test tube, with the
exception of one to be used to correct for absorbance.
hydroxide was added to all test tubes to halt each reaction after 3.5 minutes of
respective reaction time. Figure 1, listed in results, shows all relevant reactants and
their perspectives volumes or final concentrations added for each reaction.
Data collection
Absorbance at 400nm was measured from each reaction in a spectrophotometer.
The 400nm wavelength correlates to p-nitrophenolate ion which is what the product, pnitrophenol, dissociates to at an alkaline pH (Schramp, 2014). One milliliter of each
sample was placed in a cuvette and its absorbance was read. Raw collected data is
shown in Table 1 and Figure 2 in results. Reaction velocity was calculated using the
Millimolar Extinction Coefficient, corrected absorbance, and path length in Table 2.
Results
Listed below are all relevant figures and tables for the reactions.
The
Baxter 5
[Phe]
mM
[Tyr]
mM
Enzyme
(mL)
PNPP
(L)
Tris
(mL)
H20
(mL)
Baxter 6
7.5
6.5
5
4
3
2
-
7.5
1
1
1
1
1
1
1
1
150
150
150
150
150
150
150
150
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.1
0.6
1.1
1.6
2.1
2.6
3.85
0.1
7.5
7.5
150
-
0.5
0.5
1.1
0.25
normal reaction
Tyr control
Correct
absorbance
Blank spec
Baxter 7
0.91
0.9
0.89
Absorbance
0.88
0.87
0.86
0.85
0
Abs
7.5mM
Phe
6.5mM
Phe
5mM
Phe
4mM
Phe
3mM
Phe
2mM
Phe
Normal
rxn
7.5mM
Tyr
0.85
8
0.89
8
0.88
5
0.89
6
0.90
1
0.88
8
0.88
1
0.84
3
Correcte
mM
d
extinction
Absorba
coefficient
nce
product
formed
(mM)
Reaction Velocity
(mM
product/3.5
minutes)
0.848
18.4
4.61E-02
1.32E-02
0.898
18.4
4.88E-02
1.39E-02
0.885
18.4
4.81E-02
1.37E-02
0.896
18.4
4.87E-02
1.39E-02
0.901
18.4
4.90E-02
1.40E-02
0.888
18.4
4.83E-02
1.38E-02
0.881
18.4
4.79E-02
1.37E-02
0.843
18.4
4.58E-02
1.31E-02
Baxter 8
Correct
abs
Blank
spec
0.01
0.01
18.4
5.43E-04
1.55E-04
18.4
0.00E+00
0.00E+00
Normal
reaction
1.38E-02
1.36E-02
1.34E-02
1.32E-02
L-Tyr
control
1.30E-02
1.28E-02
1.26E-02
Baxter 9
Figure 4 Double reciprocal plot of 1/v as a function of 1/[s] for L-Phe data only
0.6
0.5
0.4
0.3
0.2
1/v
0.1
0
1/[s]
Discussion
We hypothesized that L-Phenylalanine would function as a non-competitive
inhibitor of bovine intestinal alkaline phosphatase.
Baxter 10
absorbance, resulting from decreased product formation, and reaction rate as the
concentration of L-Phenylalanine rose due to less product being formed. Less product
was hypothesized to be formed since it was proposed that L-Phenylalanine binds to the
enzyme-substrate complex and disallows their dissociation.
Table 1 and Figure 2 show no clear trend in absorbance as a function of LPhenylalanine concentration. We would have expected to see a linear decrease in
absorbance with increasing concentrations of L-Phenylalanine added in solution. The
absorbance at 400nm is proportional to the amount of free product in solution. If LPhenylalanines concentration was high enough in solution, it should have theoretically
bound up most or all enzyme-substrate complexes and disallowed them from degrading
into enzyme, dephosphorylated substrate, and free phosphate.
Figure 3 shows no clear trend in decreased reaction rate by increasing LPhenylalanine concentrations. We would have expected to see a linear decrease in
reaction velocity with increasing L-Phenylalanine concentrations. This was expected
based on the fact that if the enzyme-substrate complex cannot degrade, very little or no
product would be formed.
amount would be very limited if L-Phenylalanine can prevent the release of substrate.
Since the reaction velocity is measured by the substrate production over a period of
time, we would have seen a decrease in reaction velocity if L-Phenylalanine would have
functioned as an inhibitor. Our data do not support the hypothesis. We do not have
evidence in support of L-Phenylalanine acting as an inhibitor of bovine intestinal alkaline
phosphatase.
Baxter 11
Baxter 12
Milln, Jos Luis. "Alkaline Phosphatases." Purinergic Signal 2.2 (2006): 335-41. Print.
Ghosh, Nemai K., Fishman, William H. On the Mechanism of Inhibition of Intestinal
Alkaline Phosphatase by L-Phenylalanine. Biological Chemistry (1965): 26162522. Print
Fernley, H. N., Walker, P. G., Inhibition of Alkaline Phosphatase by L-Phenylalanine
Biochem (1970): 543-544. Print
Schramp, Mark. Alkaline phosphatase enzyme kinetics, structure, and activity (2014).
Lab handout, print.