UNIVERSITI TUNKU ABDUL RAHMAN

FACULTY OF SCIENCE
KAMPAR CAMPUS
PERAK
UDBB 2244 Enzymology
YEAR 2 SEMESTER 2


Name : Wong Chin Keong
ID : 1104311
Tutors name : Dr. Chang Ying Ping
Title : Kinetic Studies with Alkaline Phosphatase: Enzyme
Inhibition I and Enzyme Inhibition II

Experiment 3: Kinetic Studies with Alkaline Phosphatase: Enzyme Inhibition I and
Enzyme Inhibition II.
Objectives:
Learn to investigate the effect of inhibitor on the enzymatic activity of alkaline phosphatase.
Introduction:
Enzyme is essential in the human body to act as a biological catalyst for various reaction to occur.
Enzyme activity refers to the catalytic effect provided by the enzyme. The enzyme activity is
express in how fast a product is form. However, enzyme activity can be affected by various
factors, such as temperature, pH, concentration of enzyme and substrate, and most importantly
the presence of inhibitors.
An inhibitor will bind to enzyme and altering his effectiveness. Inhibitor binding is separate into
two types: reversible an irreversible. A reversible inhibitors will not form bonds covalently with
enzyme, therefor the inhibition can be overcome, and however, irreversible inhibitors form
covalent bond with the enzyme which is very difficult to overcome.
Under reversible inhibition, there are 3 types of inhibitors: competitive inhibitors, non-
competitive inhibitor and uncompetitive inhibitor. Competitive inhibitors are inhibitors that
compete with substrates for the active site and eventually change the value of K
m
. Non-
competitive inhibitors bind to other parts of the enzyme which is not the active site of an enzyme.
By this, the conformation of the active site will be changed. This type of inhibitors will change
the V
max
value. Uncompetitive inhibitors will target enzyme-substrate (ES) complex. This causes
ES complex cannot convert into products.
In this experiment, alkaline phosphatase is used as the enzyme to study the effect of inhibitor on
enzyme activity. Alkaline phosphatase is a membrane-bounded enzyme which functions as a
catalyst in hydrolyzing phosphate monoesters. Alkaline phosphatase works best in an alkaline
condition and it is sometimes term as basic phosphatase.



Procedure:
A. Enzyme inhibition
1. First, we prepared two sets of six tubes based on the table below:
Tube 1 2 3 4 5 6
2.70 mM subsrate (ml) 0.06 0.09 0.12 0.18 0.30 0.60
50
mM Tris-Cl pH 8.0 (ml)
2.84 2.81 2.78 2.72 2.6 2.3
Set 1: 30 mM Sodium phosphate
(monobasic)(ml)
0.1 0.1 0.1 0.1 0.1 0.1
Set 2: Control (no inhibitor,
distilled water which containing
5 mM MgCl
2
)
0.1 0.1 0.1 0.1 0.1 0.1
2. The reaction was started by adding 20 l of enzyme solution into each tube and
then staggered the addition of the enzyme.
3. The tube was mixed gently by inversion and then it was incubate for exactly 5 minute
at room temperature.
4. The reaction was stopped by adding 1.0 ml of 0.5 M NaOH and then we read the
absorbance of each tube at 400 nm by using spectrometer.
5. A graph of enzyme activity (mol/min) against substrate concentration for both of the
sets was set.
B. Enzyme inhibition II
1. The steps in this section was repeated as the section of enzyme inhibition I. the different
was substituted the monobasic sodium phosphate with 150 mM L-phenylalanine which
had 5 mM final concentration.

Result:
Set 1: 30mM sodium phosphate (monobasic)
Tube 1 2 3 4 5 6
Absorbance 0.099 0.105 0.093 0.111 0.126 0.179

Set 2: Distilled water
Tube 1 2 3 4 5 6
Absorbance 0.124 0.126 0.122 0.145 0.162 0.176

Set 3: 150mM L-phenylalanine
Tube 1 2 3 4 5 6
Absorbance 0.135 0.146 0.139 0.155 0.176 0.195

Analysis and Calculation
Calculation for Michaelis-Menten Graph
The linear equation, y =1.2487x + 0.0262 that obtained from graph of absorbance against
concentration of p-nitrophenol, where y is absorbance and x is concentration of p-nitrophenol
which we done it last session of practical report.
The enzyme activity (mol /min) of each tube was obtained by dividing concentration of p-
nitrophenol with the time taken which is 5 minutes.
The substrate concentration of each tube can be calculated by using the formula below:
M
1
V
1
=M
2
V
2,
where:
M
1
=2.7mM V
1
= amount of substrate added (ml),
M
2
=substrate concentration (mM) V
2
= 3ml
The unit of substrate concentration was converted from mM to mol by using the formula:
number of mole = MV / 1000 x 1000.
Set 1: 30mM sodium phosphate (monobasic)
Tubes
Absorbance
1 2 3 4 5 6
Enzyme activity (mol/min) 0.01166 0.01262 0.01070 0.01358 0.01598 0.02447
Substrate concentration (mol) 0.054 0.081 0.108 0.162 0.27 0.54

Set 2: Distilled water
Tubes
Absorbance
1 2 3 4 5 6
Enzyme activity (mol/min) 0.01566 0.01598 0.01534 0.01902 0.02175 0.02399
Substrate concentration (mol) 0.054 0.081 0.108 0.162 0.27 0.54

Set 3: 5mM of L-phenylalanine
Tubes
Absorbance
1 2 3 4 5 6
Enzyme activity (mol/min) 0.01742 0.01918 0.01807 0.02063 0.02399 0.02704
Substrate concentration (mol) 0.054 0.081 0.108 0.162 0.27 0.54



Calculation for Lineweaver-Burk graph
For the calculation of the data that need in the Lineweaver-Burk graph:
Set 1:
Tube 1 2 3 4 5 6
1/V (min/mol) 85.763 79.239 93.458 73.638 62.578 40.866
1/[S] (mol
-1
) 18.519 12.346 9.259 6.173 3.704 1.852

1/V= (K
m
/V
max
)(1/[S]) + 1/V
max,
y = mx + c

, y = 1/V m = (km/Vmax) c = 1/Vmax
Based on the graph,
1/V
max
= 52.855
V
max
= 0.0189
0
0.005
0.01
0.015
0.02
0.025
0.03
0 0.1 0.2 0.3 0.4 0.5 0.6
E
n
z
y
m
e

a
c
t
i
v
i
t
y
,

m
o
l
/
m
i
n

Substrate concentration, mM/min
Graph of enzyme activity (mol/min) against substrate concentration
(mM/min):

Sodium Phosphate
Distilled water
L-phenylalanine
According to Y= mX + C equation,
m= gradient of the equation = 2.2836
K
m
/V
max
= m = 2.2836
K
m
= 2.2836x0.0189 = 0.04316
Set 2:
Tube 1 2 3 4 5 6
1/V (min/mol) 63.857 62.578 65.189 52.576 45.977 41.684
1/[S] (mol
-1
) 18.519 12.346 9.259 6.173 3.704 1.852

1/V= (K
m
/V
max
)(1/[S]) + 1/V
max,
y = mx + c

, y = 1/V m = (km/Vmax) c = 1/Vmax
Based on the graph,
1/V
max
= 43.245
V
max
= 0.0231
According to Y= mX + C equation,
m= gradient of the equation = 1.3961
K
m
/V
max
= m = 1.3961
K
m
= 1.3961 x 0.0231= 0.03225
Set 3:
Tube 1 2 3 4 5 6
1/V (min/mol) 57.405 52.138 55.340 48.473 41.684 36.982
1/[S] (mol
-1
) 18.519 12.346 9.259 6.173 3.704 1.852

1/V= (K
m
/V
max
)(1/[S]) + 1/V
max,
y = mx + c

, y = 1/V m = (km/Vmax) c = 1/Vmax

Based on the graph,
1/V
max
= 38.681
V
max
= 0.02585
According to Y= mX + C equation,
m= gradient of the equation = 1.1559
K
m
/V
max
= m = 1.1559
K
m
= 1.1559x 0.02585= 0.02988

Graph of Lineweaver-Burk plot of 1/enzyme activity against 1/substrate concentration

y = 2.2836x + 52.855
R = 0.5575
y = 1.3961x + 43.245
R = 0.7283
y = 1.1559x + 38.681
R = 0.7902
-40
-20
0
20
40
60
80
100
120
-40 -30 -20 -10 0 10 20 30
1
/
V

1/[S]
Sodium Phosphate Distilled water
L-phenylalanine Linear (Sodium Phosphate)
Discussion:
Alkaline phosphatase is the enzyme used in this experiment. It can bind to substrate to form a
product called p-nitrophenol. In this experiment, 3 types of substances are used as inhibitors:
Sodium phosphate, distilled water and L-phenylalanine. Besides the enzyme, substrate and
inhibitors, we also add in NaOH into the mixture. NaOH is added right before bringing the
mixture into the spectrophotometer to read its absorbance. The purpose of doing this is to stop
the enzymatic activity of alkaline phosphatase on p-nitrophenyl phosphate. By doing this, we can
standardize the absorbance value obtained, as no more product will form to alter the result.
Enzyme is sensitive to pH, therefore adding NaOH will increase the pH of the mixture causing
the enzyme to denature. Furthermore, NaOH is also responsible to deprotonate the p-nitrophenyl
phosphate to give the yellow coloured p-nitrophenol.
In the first set, sodium phosphate was used as the inhibitor. Based on lineweaver-burk graph
constructed, the sodium phosphatase containing mixture showed the steepest gradient compared
to the other two sets. However, the Vmax reading is the same as set 2, which is the control. This
meant that sodium phosphatase is a competitive inhibitor. Sodium phosphatase competes for
active site against the substrate, maximum enzyme velocity will still be reached.
In the second set, distilled water was used as the inhibitor. The purpose of using distilled water is
to create a control for the experiment. A controlled group is like any other experiment group,
however it does not go through the experimental changes, therefore it is important to eliminate
alternate explanations of experimental results by comparing the control to other experimental
samples. In this experiment, the experimental changes will be the inhibitors. Thus, a control can
be used to be compared to study the effect of inhibitors.
In the third set, L-phenylalanine was used as the inhibitor. Hypothetically, the Km and Vmax of
this set is supposed to be lower compare to the controlled set 2. So, from this it can be said that
L-phenylalanine is an uncompetitive inhibitor. An uncompetitive inhibitor will interacts with the
ES complex to form an EIS complex, changing the rate-limiting step. But in the actual
experiment, the activity of set 3 is higher than the control, denying the hypothesis. This error
might be caused by unskilled handling of apparatus while adding the substances into the mixture.
The mistake can be added too much enzyme or too less inhibitor.
Fixed time assay or discontinuous assay is an assay used to measure enzyme concentration in a
fixed periods of time. The benefits of this assay is that it enable the researchers to measure many
assays simultaneously. However, the weakness of this assay is that it required a series of reaction
time courses, thus more time will be needed compared to continuous assay. Therefore in this
experiment, continuous assay is used instead. A spectrophotometer is used to measure the
forming and disappearance of product by reading its absorbance in real-time. Continuous assay is
suitable to measure rate reactions in the condition that the optimum pH must be determined
beforehand. But the weakness of this assay is that it can only measure one reaction at a time.
There are some precaution that should be taken to ensure the experiment completes with positive
results. Firstly, apparatus handling skill basics such as micropipettes using, 1 stop and 2 stops.
Then avoid shaking the mixture when enzyme is added, because shaking will cause formation of
bubbles that can lead to enzyme denaturation. When using the spectrophotometer, blank must be
set by using distilled water. When inserting the cuvette into spectrophotometer ensure it is in the
correct position, with the clear sides facing the beam of light.
Conclusion:
Both set 1 and set 2 had the same V
max
value. The K
m
values for set 1 is lower than set 2. The K
m

and V
max
values for set 3 supposed to be lower, but appeared to be higher due to errors. Set 1 is a
competitive inhibitor and set 3 is an uncompetitive inhibitor. Set 2 is the control in this
experiment.

References:
Anon., n.d. Enzyme Activity. [Online]
Available at: http://www.rpi.edu/dept/chem-eng/Biotech-Environ/IMMOB/enzymeac.htm
[Accessed 9 July 2014].
Hair, S., 2009. The purpose of using a control in an experiment. [Online]
Available at: http://www.thestudentroom.co.uk/showthread.php?t=879371
[Accessed 8 July 2014].
Kimball, J. W., 2011. Enzyme Kinetics. [Online]
Available at: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html
[Accessed 8 July 2014].
University, N., 2007. 4 Enzyme assays. [Online]
Available at: http://learning.covcollege.ac.uk/content/Jorum/CHB_Intro-to-basic-prac-
skills_LM-1.2/page29.htm
[Accessed 9 July 2014].