Baxter 1

Investigating the Inhibitory Effects of L-Phenylalanine on Alkaline Phosphatase Activity

Jared Baxter, Rachel Foguth, Nathan Ptak
March 17th, 2014

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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

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data from reactions in the presence of varying concentrations of L-Phenylalanine to be

straight lines parallel to the control reactions without L-Phenylalanine. (Ghosh, 1965).

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.

The remaining four reactions included one of each of the following: L-

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.

Reaction Setup and Execution

All reactions took place in a standard test tube in a test tube rack suitable for a
hot water bath. The reactions were set up by first adding Tris-HCl followed by the PNPP
substrate and appropriate amino acids (Phe, Tyr, or none). Distilled water was then
added to bring all reactions to the same volume to ensure proper concentration of
reactants.

The test tubes were then brought to temp in a 37 oC water bath for

approximately one minute before adding the bovine alkaline phosphatase.

Bovine

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alkaline phosphatase was added in 60 second increments in each test tube, with the
exception of one to be used to correct for absorbance.

One milliliter of Sodium

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

dephosphorylation reaction was noted by presence of 400nm light-absorbing material in

solution. All solutions started off as clear. All reactions which contained PNPP were
visibly yellow to the naked eye after the reactions were carried out. The color change
indicates that a chemical change took place in the test tube, presumably the conversion
of PNPP to the p-nitrophenolate ion. The reaction used to blank the spectrophotomer
did not contain PNPP and did not have any visible yellow tint.
Figure 1 All reactions and their respective reactants

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[Phe]
mM

[Tyr]
mM

Enzyme
(mL)

PNPP
(L)

Tris
(mL)

H20
(mL)

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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

Table 1 Absorbance of 400nm light by post-reaction solutions

Figure 2Graphical display of absorbance of 400nm light by post-reaction solution

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0.91
0.9
0.89

Absorbance

0.88
0.87
0.86
0.85
0

Concentration of L-Phenylalanine (mM)

Table 2 Calculation of velocity using Millimolar Extinction Coefficient

Reactio
n

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

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Correct
abs
Blank
spec

0.01

0.01

18.4

5.43E-04

1.55E-04

18.4

0.00E+00

0.00E+00

Figure 3 Reaction rate as a function of L-Phenylalanine concentration

1.42E-02
1.40E-02

Normal
reaction

1.38E-02
1.36E-02

Reaction velocity (mM product formed/minute)

1.34E-02
1.32E-02

L-Tyr
control

1.30E-02
1.28E-02
1.26E-02

Conc. L-Phe (mM)

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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.

We expected to see decreased

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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.

Surely some product would have formed, however the

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.

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One possible source of error is contaminated or otherwise imperfect reactants,

specifically L-Phenylalanine. If the stock had degraded based on light, heat, etc it could
no longer function as an inhibitor of AP. Any introduced contaminants could have
reacted with L-Phenylalanine to form a new product which may not be able to inhibit AP.
Another source of error could be in making solutions of proper concentrations and the
addition of the solutions. If the concentrations of solutions were too low, we would not
expect to see much inhibition. If the incorrect amount of the solutions were added,
despite having proper concentrations, the same result could be seen. Another source of
error could be that only one trial of the experiment was run, however this does not seem
as likely as either of the previously mentioned sources.
A curious data point is that of the L-Tyrosine, meant to be a control. The data
point for absorbance is approximately 5% lower absorbance than the average
absorbance for all of the L-Phenylalanine absorbances. This could just simply be by
chance, or it could be a potentially better inhibitor of AP.
With more time we would have liked to do more trials, include more
concentrations of L-Tyrosine, and also obtain the R enantiomers of both amino acids for
a true control. We would cross check more literature to see if we used concentrations
of L-Phenylalanine efficacious enough to elicit a substantial inhibition. We would like to
include more concentrations of L-Tyrosine to see if there is a concentration dependent
inhibition of AP by L-Tyrosine. Another potential for improvement is to test the amino
acids for purity via TLC and purify them if needed via column chromatography.
Works Cited
Berridge, M.J. (2012) Cell Signalling Biology; doi:10.1042/csb0001005

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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.