Bachelor of Biomedical Science (Hons)

Programme

Metabolic Biochemistry
BBS2121

Practical 1: Enzyme Kinetic of Acid Phosphatase

Name: Lim wei-jet

Student ID: 00000031232
Cohort: BM1/19
Introduction

There are two varieties of phosphatase enzymes which are acid phosphatase and alkaline
phosphatase1. Acid phosphatase works more effective in acidic medium whereby alkaline
phosphatase works more effective in an alkaline medium1. Acid phosphatase is an enzyme
that plays a vital position in catalyzing the body’s chemical reactions1.

The highest acid phosphatase (ACP) production rate resides in the liver, spleen, blood,
erythrocytes, platelets, bone marrow, and prostate gland5. The prostate gland contain the highest
source, and a limited proportion of the enzyme found in sera from healthy malescontributes5.1
Increasing amounts of ACP are associated with prostate cancer5.

Phosphatases are enzymes catalyzing the hydrolysis of phosphoric acid esters2. Those enzymes are
located predominantly in the prostate glands of men1. These enzymes have the physiologic
function of liquefying the semen produced1. Increased amount of phosphatase enzymes can be
identified in individuals suffering from prostate cancers2. It is processed in lysosomes, so these
lysosomes combine with endosomes, releasing phosphatase acid2. Acid phosphatase works
effectively in acidic media by separating the phosphate group from its substratum 2. This reaction
is referred to as hydrolysis2.

Calculation with graph

Beer-Lambert equation
C= A/ 𝜀 t
A= absorbance at 400 nm
𝜀 = Molar absorptivity = 18, 120 l.mol -1 .cm- 1

t = Distance taken for the light travel through the solution: 1 cm

[C] = Concentration of the absorbing species, mol.l -1

Example:
A = 0.021nm
𝜀 = 18,120 l.mol -1 .cm- 1

t = 1 cm
Therefore, C = 0.021/(18,120x1) = 1.11x10-6 mol.l -1
Velocity of reaction = (1.15x10-6x0.5x1000)/0 = 0.0 μmol min-1
Result
Part 1: Time linearity of hydrolysis
Test tube at time 0 minutes 7.5 minutes 15 minutes
Absorbance at 0.021 0.165 0.286
400nm
Concentration of 4- 0.021 0.165 0.286
nitrophenol (mol L-1) (18120x1) (18120x1) (18120x1)

= 1.15x10-6 = 9.10x10-6 = 15.78x10-6

Velocity of reaction 1.15x10-6x0.5x1000 9.10x10-6x0.5x1000 15.78x10-6x0.5x1000
(μmol min-1) 0.0 7.5 15

= 0.0 = 6.06x10-4 = 5.26x10-4

Table 1: Absorbances and concentrations of 4-nitrophenol obtained at different time
intervals.

Velocity of enzymatic reaction against time

7.00E+00

6.06 E-04
6.00E+00
5.26 E-04
Velocity of reaction (µmol min-1)

5.00E+00

4.00E+00

3.00E+00

2.00E+00

1.00E+00

0.0 E-04
0.00E+00
0 2 4 6 8 10 12 14 16
Time (minute)

Figure 1: Velocity of enzymatic reaction v time taken graph

Part 2: inhibition of hydrolysis
Tube Concentration of 4- Absorbance
Velocity
nitrophenol in 5mL (μmol min-1)
(400nm)
of reacting solution
(mol L-1)
F0 0.204 0.204 11.25x10-6x0.5x1000
18120x1 7.5
= 11.25x10-6 =7.5x10-4
F0.5 0.108 0.108 5.96x10-6x0.5x1000
18120x1 7.5
= 5.96x10-6 =3.97x10-4
F1 0.095 0.095 5.24x10-6x0.5x1000
18120x1 7.5
-6
= 5.24x10 =3.50x10-4
Table 2: Absorbance, concentration and velocity of three tubes labeled F0, F0.5 and F1.

Velocity of enzymatic reaction against the concentration of NaF

8.00E-04
7.50E-04

7.00E-04
Velocity of reaction (µmol min-1)

6.00E-04

5.00E-04
3.97E-04
4.00E-04 3.50E-04

3.00E-04

2.00E-04

1.00E-04

0.00E+00
0 1 2 3 4 5 6
Concentration of NaF (mM)

Figure 2: Velocity of enzymatic reaction against the concentration of sodium fluoride, NaF
Discussion
Part 1: Time linearity of hydrolysis
Based on the graph for Part 1, there is a linear relationship between the concentration of the drug
and the reaction rate. This means that the enzymatic reaction increases as the time increases too.
The substratum (NPP) are served as a limiting factor for this enzyme activity, thus the activity of
the enzyme continues to increase until it reaches a certain and ends, which can be observed as a
plateau is formed in the graph, showing the reaction has reached the max velocity.

Part 2: inhibition of hydrolysis

From Table 2 the sodium fluoride concentration increases, the enzymatic reaction reduces can be
observed. The test tube labelled with F0.5 and F1 provides a lower absorbance reading than F0,
based on the tests obtained4. This is due to the involvement of NaF in the F0.5 and F1 test tubes.
Therefore, it can be concluded that Sodium fluoride, NaF is an inhibitor of the enzymes4. NaF is a
chemical or molecules product that changes, slows down and even prevents enzyme activity. As
NaF concentration is increasing, the enzyme absorbance decreases4. The inhibitor we used in this
experiment is sodium fluoride which is dependent on concentration4.

Questions
Part 1
During catalysis, is the product (4-nitrophenol) liberated from the substrate (NPP) linearly with
time?
Answer:
The product (4-nitrophenol) liberated from the substrate (NPP) is linear with the time3. Based on
the graph, the absorbance is proportional to the concentration of the phenolate ion formed3. The
diagram shows a straight positive axis. With the advantage of acid phosphatase, NPP is hydrolyzed
to4-nitrophenol and then it is4-nitrophenolate by adding an OH-which can absorb lights at 400
nm3. The stronger the absorbance the higher the concentration of 4-nitrophenolate. -As time pass
by, the absorbance of the product increases, this shows that more and more 4- nitrophenolate is
produced3.
Part 2
What is the modulatory effect of sodium fluoride that you could observe on the reactions catalysed
by acid phosphatase? Is this effect concentration dependent?
Answer:
Sodium fluoride (NaF) is used as an enzyme inhibitor; therefore, the acid phosphatase catalysed
reaction will be reduce by sodium fluoride4. From the graph, the velocity of reaction showed
decreases as the concentration of NaF increases4. Therefore, it can be concluded that the inhibition
effect of NaF is concentration dependent4.

References:
1. Lab 4_Enzyme Kinetics Report.docx - Enzyme Kinetics Analysis of Alkaline Phosphatase
Introduction The experimental goal of this lab was to examine the | Course Hero [Internet].
Coursehero.com. 2020 [cited 16 March 2020]. Available from:
https://www.coursehero.com/file/39127336/Lab-4-Enzyme-Kinetics-Reportdocx/
2. Dean R. Kinetic studies with alkaline phosphatase in the presence and absence of inhibitors and
divalent cations. Biochemistry and Molecular Biology Education. 2002;30(6):401-407.
3. 4. Enzyme Kinetics - Structure - Function - Michaelis-Menten Kinetics [Internet].
TeachMePhysiology. 2020 [cited 17 March 2020]. Available from:
https://teachmephysiology.com/basics/enzyme-activity/enzyme-kinetics/
4. Sciences L, Biology P, Center P, Library P, Methods P, Inhibitors P. Protease and Phosphatase
Inhibitors | Thermo Fisher Scientific - UK [Internet]. Thermofisher.com. 2020 [cited 17 March
2020]. Available from: https://www.thermofisher.com/my/en/home/life-science/protein-
biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-
methods/protease-phosphatase-inhibitors.html
5. Acid phosphatase [Internet]. Ilexmedical.com. 2020 [cited 19 March 2020]. Available from:
http://www.ilexmedical.com/files/PDF/ACIDPOHS_ARC_CHEM.pdf