UNIVERSITI TUNKU ABDUL RAHMAN FACULTY OF SCIENCE KAMPAR CAMPUS PERAK UDBB 2244 Enzymology YEAR 2 SEMESTER 2
Name
:
Wong Chin Keong
ID
:
1104311
Tutor’s 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 b y 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 eventuall y change the value of K m. Noncompetitive 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 Vmax value. Uncompetitive inhibitors will target enzyme-substrate (ES) complex. This c auses 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
2.84
2.81
2.78
2.72
2.6
2.3
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
mM Tris-Cl pH 8.0 (ml) Set 1: 30 mM Sodium phosphate (monobasic)(ml) Set 2: Control (no inhibitor, distilled water which containing 5 mM MgCl2) 2. The reaction was started by adding 20 µl 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 (µmol/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
Absorbance 0.099
2
3
4
5
6
0.105
0.093
0.111
0.126
0.179
2
3
4
5
6
0.126
0.122
0.145
0.162
0.176
2
3
4
5
6
0.146
0.139
0.155
0.176
0.195
Set 2: Distilled water Tube
1
Absorbance 0.124
Set 3: 150mM L-phenylalanine Tube
1
Absorbance 0.135
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 (µmol /min) of each tube was obtained by dividing concentration of pnitrophenol with the time taken which is 5 minutes. The substrate concentration of each tube can be calculated by using the formula below: M1V1=M2V2, where: M1=2.7mM
V1= amount of substrate added (ml),
M2=substrate concentration (mM)
V2= 3ml
The unit of substrate concentration was converted from mM to µmol by using the formula: number of mole = MV / 1000 x 1000. Set 1: 30mM sodium phosphate (monobasic) Absorbance Tubes 1 Enzyme activity (µmol/min)
2
0.01166 0.01262
Substrate concentration (µmol) 0.054
0.081
3
4
5
6
0.01070 0.01358
0.01598 0.02447
0.108
0.162
0.27
0.54
3
4
5
6
Set 2: Distilled water Absorbance Tubes 1 Enzyme activity (µmol/min)
2
0.01566 0.01598
Substrate concentration (µmol) 0.054
0.081
0.01534 0.01902
0.02175 0.02399
0.108
0.162
0.27
0.54
3
4
5
6
Set 3: 5mM of L-phenylalanine Absorbance Tubes 1 Enzyme activity (µmol/min)
2
0.01742 0.01918
Substrate concentration (µmol) 0.054
0.081
0.01807 0.02063
0.02399 0.02704
0.108
0.27
0.162
0.54
Graph of enzyme activity (µmol/min) against substrate concentration (mM/min):
0.03 n i m0.025 / l o m 0.02 µ , y t i 0.015 v i t c a e 0.01 m y z n0.005 E
Sodium Phosphate Distilled water L-phenylalanine
0 0
0.1
0.2
0.3
0.4
0.5
0.6
Substrate concentration, mM/min
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/ϻmol)
85.763
79.239
93.458
73.638
62.578
40.866
18.519
12.346
9.259
6.173
3.704
1.852
-
1/[S] (ϻmol )
1/V= (K m/Vmax)(1/[S]) + 1/Vmax, y = mx + c , y = 1/V m = (km/Vmax) Based on the graph, 1/Vmax = 52.855 Vmax = 0.0189
c = 1/Vmax
According to Y= mX + C equation, m= gradient of the equation = 2.2836 K m/Vmax = m = 2.2836 K m= 2.2836x0.0189 = 0.04316 Set 2: Tube
1
2
3
4
5
6
1/V (min/ϻmol)
63.857
62.578
65.189
52.576
45.977
41.684
18.519
12.346
9.259
6.173
3.704
1.852
-
1/[S] (ϻmol )
1/V= (K m/Vmax)(1/[S]) + 1/Vmax, y = mx + c , y = 1/V m = (km/Vmax)
c = 1/Vmax
Based on the graph, 1/Vmax = 43.245 Vmax = 0.0231 According to Y= mX + C equation, m= gradient of the equation = 1.3961 K m/Vmax = m = 1.3961 K m= 1.3961 x 0.0231= 0.03225 Set 3: Tube
1
2
3
4
5
6
1/V (min/ϻmol)
57.405
52.138
55.340
48.473
41.684
36.982
18.519
12.346
9.259
6.173
3.704
1.852
-
1/[S] (ϻmol )
1/V= (K m/Vmax)(1/[S]) + 1/Vmax, y = mx + c , y = 1/V m = (km/Vmax)
c = 1/Vmax
Based on the graph, 1/Vmax = 38.681 Vmax = 0.02585 According to Y= mX + C equation, m= gradient of the equation = 1.1559 K m/Vmax = m = 1.1559 K m= 1.1559x 0.02585= 0.02988
120 100 80
y = 1.3961x + 43.245 R² = 0.7283
60 V / 1
40 y = 1.1559x + 38.681 R² = 0.7902
20 0 -40
-30
-20
-10
y = 2.2836x + 52.855 R² = 0.5575
-20
0
10
20
30
-40 1/[S]
Sodium Phosphate
Distilled water
L-phenylalanine
Linear (Sodium Phosphate)
Graph of Lineweaver-Burk plot of 1/enzyme activity against 1/substrate concentration
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-phen ylalanine. 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 unco mpetitive 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 h ypothesis. 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 b y 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 e nsure it is in the correct position, with the clear sides facing the be am of light. Conclusion:
Both set 1 and set 2 had the same Vmax value. The K m values for set 1 is lower than set 2. The K m and Vmax 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-pracskills_LM-1.2/page29.htm [Accessed 9 July 2014].
