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-451485-2216150031508700College of Arts

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-451485-2216150031508700College of Arts & Sciences
Department of Chemistry & Earth Sciences
Principles of Environmental Chemistry CHEM 242
00College of Arts & Sciences
Department of Chemistry & Earth Sciences
Principles of Environmental Chemistry CHEM 242

Experiment (9): Study of Enzyme kinetics using Alkaline Phosphatase Enzyme.

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Name: Areej Ali Alhams
QUID: 201702829
Partner: Aseel yousif
QUID: 201604075
Instructor: Dr.Hanan Rizk and Dr. Olfat Mokhtar.

Due Date: 21st/11/2018
Study of Enzyme kinetics using Alkaline Phosphatase Enzyme
Objectives:
Enzyme assays and determination of kinetic parameters Km and Vmax for alkaline phosphatase (Part 1).
Product inhibition of alkaline phosphatase by phosphate ions and determination of inhibition constant “Ki.” (Part II).

Abstract:
Enzymes are biocatalysts that speeds up the rate of a chemical reaction by decreasing the activation energy of these reactions without undergoing the reaction themselves. The aim of this lab is to determine the activity of alkaline phosphatase enzyme kinetics. This experiment is consisting of two parts, the first part is testing the activity of the alkaline phosphatase enzyme by using 7 different concentrations of the substrates PNPP substrate (para-nitrophenyl-phosphate). The enzyme catalyzes the hydrolysis of PNPP to para-nitro phenol PNP and the absorbance of this canalization is recorded at 210 nm after collecting the data, Michaelis-Menten and linearweaver-burk graphs are illustrated in order to calculate the Vmax and Km which are found to be 2.7 and 4.01 respectively. The second part focuses on how the activity of these enzymes could be controlled using inhibitors. Absorbance values taken and have been used to plot enzyme progression curve and Michaelis-Menten plot to deter mine Vmax and Km values. a linear graph of linearweaver-burk was plotted to study the effect of different concentrations of inhibitors and determine the type of inhibitor used. (0, 0.067, 0.133 and 0.201 mM). According to the results, the type of inhibitor used is a competitive inhibitor. By illustrating Dixon’s plot the Ki is determined and found to be 0.035.
Introduction:
Enzymes are biological catalysts proteins that help in deriving different metabolic reactions in living organisms. Each enzyme binds to a substrate, and this binding is characterized with high specificity in the binding site. To Measure the activity of the enzyme in a reaction the amount of substrate or the amount of the product produced under the optimum condition must be known. Substrates binds to enzymes in a lock and key module. Which means that the binding site on the enzyme which is known as the active site only fits the right substrate. (figure1).
15499852540000
Figure.1
the equation below shows the reaction of enzyme rate constants:
E + S ? ES ? ES* ? EP ? E + P
760195346392500By plotting the initial reaction rate (V0), a rectangular hyperbola is resulted, in which the enzyme concentration is fixed, and the substrates concentrations are different as they are present in excess. When the concentration of the substrate is low, the reaction is considered to be first order reaction, as the substrate concentration gets higher the reaction becomes Zero order reaction. In Michaelis-Menten equation (V0), the Vmax is the maximum velocity of the reaction, Km (Michael’s constant) is substrate concentration at half of the maximal velocity. In order to determine the Vmax and Km values in an accurate way, Lineweaver-Burk graph is used, which is the reciprocal of Vmax and Km. Enzymes can be influenced by several factors such as, Inhibitors, the concentration of the substrate, temperature and pH. Inhibitors are molecules or ions that slow that down or stop the enzymes catalytic activity in reversible and irreversible ways. Reversible Inhibitions have three types: 1. competitive inhibition, 2.non-competitive 3. uncompetitive inhibition. The competitive inhibition inhibitors compete, and the substrate competes over binding to the active site on the enzyme. In non-competitive inhibition both the substrate and the inhibitors bind equally well to the enzyme whether. Moreover, in the uncompetitive inhibitions, the inhibitor binds only to the enzyme-substrate complex. (Figure 3).
Figure.3
In this experiment, the Km and Vmax of Alkaline Phosphatase enzyme are going to be determined by measuring the absorbance of PNPP catalyzation hydrolysis to PNP. PNPP is going to be used as the substrate. In the second part of the experiment, the effect of the inorganic inhibitor phosphate on the catalytic activity of Alkaline phosphatase will be studied by monitoring the change in absorbance, as in part I, but with different inhibitors concentrations.

Figure.4: Alkaline Phosphatase catalyzing PNPP to PNP
Materials:
For the materials Refer to the lab manual: part I page (65), part II page (67).
Part I: Seven tubes were prepared with the following solutions, by adding a unique amount of 0.4mM PNPP and 0.1 Tris-HCl which used as a buffer. Follow the table in page 65 to prepare the 7 tubes.

Set the spectrophotometer dial to 410nm and be sure to put it on ‘absorbance’ mode.

Mix the (blank) tube content, tube #1, and use it to calibrate the spectrophotometer.

Add 0.1 ml of alkaline phosphate (0.5U/ml) enzyme to the second tube after placing it is contents in the cuvette and mix the content quickly.
Measure the absorbance at 410 nm every 30 seconds for 3 min.

Repeat the previous step for the rest of the tubes in table 1 above.

Plot time Vs. Absorbance in order to make calculations on.

After plotting, determine the slopes for the seven tubes by make 7 trend lines on the graph. Graph 1/S vs. 1/V in order to determine and calculate Vmax and Km.
Part II: In part two, inhibitor is added. There are three groups, A( no inhibitor) , B ( 0.067 mM inhibitor) , and C (0.133 mM inhibitor) , and prepare group A, group B and group C as instructed in pages 68 – 69.

Select absorbance mode in the spectrophotometer and put it on 410 nm.
Calibrate the instrument (adjust the absorbance to zero).

Same as part 1, add 0.1 alkaline phosphate enzymes for each tube (see tables 2, 3, and 4 above). Record the change of absorbance every 30 sec and for 3 minutes.

Plot the graph time vs. absorbance for the three groups.

Plot the graph I against 1/Vmax in order to determine the type of inhibition.
Results:
Table.1: Time and absorbance of the solutions prepared with different concentration of PNPP and alkaline phosphatase in 7 tubes.

Time(min) Absorption of Tubes Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7
0 0 0.003 0.005 0.006 0.012 0.027 0.032
0.5 0 0.021 0.026 0.032 0.034 0.038 0.056
1 0 0.05 0.055 0.057 0.058 0.06 0.071
1.5 0 0.063 0.078 0.084 0.086 0.095 0.1
2 0 0.087 0.099 0.107 0.11 0.112 0.115
2.5 0 0.11 0.119 0.129 0.131 0.139 0.14
3 0 0.134 0.137 0.148 0.15 0.163 0.164
-16484421438700
Graph.1: Alkaline phosphatase activity of different PNPP concentration
Calculations:
The velocity = (slope x 106)/ molar absorptivity (18000).
The Slope= ?A / ?t (A for absorbance and t for time)
The reciprocals of V0 and s is simply done by 1/V0 and 1/s
The calculations are shown in Table 2.

Table.2: Para-Nitrophenylphosphate (PNPP) concentration, slope, and velocity and their reciprocals table.

Substrate s, mMSubstrate s, uM?A/min (Slope) V0, umol PNP / min 1/s, uM1/v0 , (umol/ min)
Tube 2 0.020 20 0.0403 (0.0403×106/18000) = 2.23 1/20= 0.05 1/2.23=0.45
Tube 3 0.025 25 0.0411 (0.0411×106/18000) = 2.28 1/25=0.04 1/2.28=0.44
Tube 4 0.033 33 0.0453 (0.0453×106/18000) = 2.52 1/33= 0.03 1/2.52=0.39
Tube 5 0.050 50 0.0451 (0.0451×106/18000) =2.50 1/50= 0.02 1/2.50= 0.4
Tube 6 0.100 100 0.0478 (0.0478×106/18000) = 2.65 1/100= 0.02 1/2.65= 0.38
Tube 7 0.200 200 0.0453 (0.0453×106/18000) = 2.52 1/200=
0.005 1/2.52=0.39
678094-20548Michaelis-Menten Plot of PNPP Alkaline Phosphatase
00Michaelis-Menten Plot of PNPP Alkaline Phosphatase
6159592075

0
0.5

1

1.5
2

2.5

3
3.5

0

50

100

150

200

250

V0
Linear(V0)
Vmax = 2.7 umol/min
Km = 2.7/ 2 = 1.35 umol/min
00

0
0.5

1

1.5
2

2.5

3
3.5

0

50

100

150

200

250

V0
Linear(V0)
Vmax = 2.7 umol/min
Km = 2.7/ 2 = 1.35 umol/min
5675109-163830-130978-164388-130979-164387
9668956114 Vo(umol/min)
Vo(umol/min)

607838521189y = 0.0063x + 1.7132
00y = 0.0063x + 1.7132
599554441325
2575362192569R2 = 0.21585
00R2 = 0.21585

1851118380811Substrate S (uM)
Substrate S (uM)

584509249326 Graph.2: Michaels-Menton plot of Alkaline phosphatase
0 Graph.2: Michaels-Menton plot of Alkaline phosphatase
-130979192483

-36004530734000
Graph.3: Lineweaver-Burk plot of alkaline phosphatase
The calculation of Vmax and Km:
Vmax:
1/Vmax=c
equation of the line: y=1.4849x+0.37 (From Graph3.) c= 0.37
Vmax=1/0.37= 2.7 umole/PNP/min
Km:
Km= slope (m) x Vmaxequation of the line: y=1.4849x+0.37 (From Graph.3) m= 1.4849Vmax= 2.7
1.4849 x 2.7= 4.01 uM
II: Part A:
Table3.: PartIIA: Inhibitor (Phosphate) concentration: 0 mM.Time(min) Absorption of Tubes Tube 1 Tube 2 Tube 3 Tube 4 Tube 5
0 0.012 0.024 0.055 0.062 0.075
0.5 0.048 0.059 0.087 0.107 0.117
1 0.086 0.095 0.131 0.147 0.15
1.5 0.114 0.125 0.163 0.181 0.188
2 0.142 0.158 0.194 0.211 0.213
2.5 0.175 0.182 0.211 0.250 0.261
3 0.192 0.206 0.234 0.253 0.288

Graph.4: Absorbance at 410 nm of reaction mixture (alkaline Phosphatase) at different times
Table.4: for Part IIA: This table shows the slop, Vo, 1/s, and 1/v values when inhibitor conc. is (0.0 mM):
Substrate s, mMSubstrate s, uM?A/min (Slope) V0, umol PNP / min 1/s, uM1/v0 , (umol/ min)
Tube 1 0.020 20 0.0607 (0.0607×106/18000) = 3.37 1/20= 0.05 1/3.37=0.30
Tube 2 0.025 25 0.0611 (0.0611×106/18000) = 3.39 1/25=0.04 1/3.39=0.29
Tube 3 0.033 33 0.0606 (0.0606×106/18000) = 3.37 1/33= 0.03 1/3.37=0.30
Tube 0.050 50 0.0659 (0.0659×106/18000) =3.66 1/50= 0.02 1/3.66= 0.27
Tube 5 0.100 100 0.0706 (0.0706×106/18000) = 3.92 1/100= 0.01 1/3.92= 0.26
Part B:
Table.5: PartIIB: Inhibitor (Phosphate) concentration: 0.067 mM.Time(min) -599440-13335Absorbance of tubes
00Absorbance of tubes
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5
0 0.006 0.008 0.010 0.006 0.010
0.5 0.022 0.027 0.031 0.032 0.045
1 0.036 0.039 0.046 0.057 0.066
1.5 0.049 0.055 0.062 0.084 0.091
2 0.062 0.071 0.078 0.107 0.113
2.5 0.074 0.084 0.093 0.129 0.134
3 0.086 0.098 0.113 0.156 0.160

61595190500Graph5: Absorbance at 410 nm of reaction mixture (alkaline Phosphatase) with 0.067 mM phosphate inhibitor at different times
Table.6: for Part IIB: This table shows the slop, Vo, 1/s, and 1/v values when inhibitor conc. is (0.067 mM):
Substrate s, mMSubstrate s, uM?A/min (Slope) V0, umol PNP / min 1/s, uM1/v0 , (umol/ min)
Tube 1 0.020 20 0.0264 (0.0264×106/18000) = 1.47 1/20= 0.05 1/1.47=0.68
Tube 2 0.025 25 0.0297 (0.0297×106/18000) = 1.65 1/25=0.04 1/1.65= 0.61
Tube 3 0.033 33 0.0332 (0.0332×106/18000) = 1.84 1/33= 0.03 1/1.84=0.54
Tube 0.050 50 0.0469 (0.0469×106/18000) =2.61 1/50= 0.02 1/2.61=0.38
Tube 5 0.100 100 0.482 (0.482×106/18000) = 2.68 1/100= 0.01 1/2.68= 0.37
-194688374650Table.7: PartIIC: Inhibitor (Phosphate) concentration: 0.133 Mm.

0Table.7: PartIIC: Inhibitor (Phosphate) concentration: 0.133 Mm.

Part C:
Time(min) Absorption of Tubes Tube 1 Tube 2 Tube 3 Tube 4 Tube 5
0 0.003 0.017 0.025 0.016 0.031
0.5 0.014 0.028 0.032 0.048 0.056
1 0.024 0.040 0.045 0.077 0.081
1.5 0.032 0.051 0.078 0.096 0.104
2 0.041 0.064 0.090 0.115 0.119
2.5 48 0.074 0.106 0.132 0.140
3 0.058 0.085 0.125 0.144 0.158

Graph.6: Absorbance at 410 nm of reaction mixture (alkaline Phosphatase) with 0.133 mM phosphate inhibitor at different times
Table.8: for Part IIC: This table shows the slop, Vo, 1/s, and 1/v values when inhibitor conc. is (0.113 mM):
Substrate s, mMSubstrate s, uM?A/min (Slope) V0, umol PNP / min 1/s, uM1/v0 , (umol/ min)
Tube 1 0.020 20 0.0179 (0.0179×106/18000) = 0.99 1/20= 0.05 1/0.99=1.01
Tube 2 0.025 25 0.0229 (0.0229×106/18000) = 1.27 1/25=0.04 1/1.27=0.79
Tube 3 0.033 33 0.0352 (0.0352×106/18000) = 1.96 1/33= 0.03 1.196=0.51
Tube 0.050 50 0.0421 (0.0421×106/18000) = 2.34 1/50= 0.02 1/2.34=0.43
Tube 5 0.100 100 0.0419 (0.0419×106/18000) = 2.33 1/100= 0.01 1/2.33=0.43
Part D:
Table9.: PartIID: Inhibitor (Phosphate) concentration: 0.201 Mm.
Time(min) Absorption of Tubes Tube 1 Tube 2 Tube 3 Tube 4 Tube 5
0 0.003 0.007 0.018 0.011 0.025
0.5 0.013 0.014 0.030 0.033 0.041
1 0.020 0.022 0.043 0.049 0.053
1.5 0.29 0.032 0.055 0.064 0.068
2 0.035 0.042 0.070 0.079 0.082
2.5 0.44 0.051 0.081 0.095 0.102
3 0.052 0.059 0.093 0.113 0.117
61595288946004109163760713Graph.7: Absorbance at 410 nm of reaction mixture (alkaline Phosphatase) with
0.201 mM phosphate inhibitor at different times
0Graph.7: Absorbance at 410 nm of reaction mixture (alkaline Phosphatase) with
0.201 mM phosphate inhibitor at different times

Table.10: for Part IID: This table shows the slop, Vo, 1/s, and 1/v values when inhibitor conc. is (0.201mM):
Substrate s, mMSubstrate s, uM?A/min (Slope) V0, umol PNP / min 1/s, uM1/v0 , (umol/ min)
Tube 1 0.020 20 0.0160 (0.0160×106/18000) = 0.89 1/20= 0.05 1/0.89= 1.12
Tube 2 0.025 25 0.0179 (0.0179×106/18000) = 1.00 1/25=0.04 1.00/1.00=
1.00
Tube 3 0.033 33 0.0253 (0.0253×106/18000) = 1.41 1/33= 0.03 1/1.41= 0.71
Tube 0.050 50 0.0329 (0.0329×106/18000) = 1.83 1/50= 0.02 1/1.83= 0.55
Tube 5 0.100 100 0.0305 (0.0305×106/18000) = 1.69 1/100= 0.01 1/1.69= 0.59
4064028112000
Graph.8: Lineweaver Burk plot analysis of enzyme alkaline phosphatase inhibition by inorganic phosphate inhibitor.

-133350-174119The calculation of Vmax and Km for PART A:
0The calculation of Vmax and Km for PART A:

1- Vmax:
1/Vmax=cequation of the line: y = x + 0.254 (From Graph8.) c= 0.254
Vmax= 1/0.254= 3.94 umole/PNP/min
2- Km:
Km= slope (m) x Vmaxequation of the line: y = x + 0.254 (From Graph8.) m= 1
Vmax= 0.254Km= 1 x 3.937 = 3.94 uM
The calculation of Vmax and Km for PART B:
1- Vmax:
1/Vmax=cequation of the line: y = 8.5x + 0.261 (From Graph8.)
c= 0.261Vmax= 1/0.261= 3.83 umole/PNP/min
2- Km:
Km= slope (m) x Vmaxequation of the line: y = 8.5x + 0.261 (From Graph8.) m= 8.5Vmax= 3.831Km= 3.831 x 8.5 = 32.56 uM
The calculation of Vmax and Km for PART C:
1- Vmax:1/Vmax=cequation of the line: y = 15.2x + 0.178 (From Graph8.)
c= 0.178Vmax= 1/0.178= 5.62 umole/PNP/min
2- Km:
Km= slope (m) x Vmaxequation of the line: y = 15.2x + 0.178 (From Graph8.) m= 15.2Vmax= 5.62Km= 15.2 x 5.62= 85.42 uM
The calculation of Vmax and Km for PART D:
1- Vmax:1/Vmax=cequation of the line: y = 15.1x + 0.341 (From Graph8.) c= 0.341
Vmax= 1/0.341= 2.93 umole/PNP/min2- Km:Km= slope (m) x Vmaxequation of the line: y = 15.1x + 0.341 (From Graph8.) m= 15.1
Vmax= 2.93Km= 15.1 x 2.93= 44.24 uM
-400692205069Table 11: This table shows the slopes of the inhibitors and their concentrations to illustrate Dixon’s plot.

0Table 11: This table shows the slopes of the inhibitors and their concentrations to illustrate Dixon’s plot.

Inhibitorconcentra8onmMISlopes
0 1
0.067 8.5
0.133 15.2
0.201 15.1
13271525600Graph.9: Dixon’s plot of competitive inhibition of alkaline phosphatase at different concentrations
Calculating the Ki:
Ki = x-interceptThe line of the equation (graph9.): y = 73.116x + 2.6201 0 = 73.116x+2.6201-2.6201/73.116 = x x= -0.035
Discussion:
Part I:
As can be seen in MM plot graph.2, it is clear that the velocity increases as the substrate concentration increases until it reaches the Vmax, where the enzyme is saturated with substrate. Which means that the reaction is first order regarding enzyme at the very beginning and when it reached the saturation point it became zero order reaction with respect to substrate. After collecting and calculating the 1/V0 and 1/S, Linewaver curve was illustrated by plotting 1/V0 Vs. 1/S graph.3. From the curve, Km and Vmax values are calculated. Vmax was found to be 2.7 umole/PNP/min and Km is 4.01 uM.
Part II:
In the second part of the experiment, four trails with different inhibitor (phosphate) concentrations were performed (0 mM/ 0.067mM/ 0.133mM and 0.201 mM. In the first graph (graph.4), part IIA, no inhibitor was added, the absorbance increases as the substrate concentration increase, just as Graph1. Graph IIB (graph.5) represents the progression of the enzymatic catalyzed reaction with the presence of 0.067 inhibitor concentration. It was clear that increasing the concentration of substrate increases the amount of product produced, which indicates that this type of inhibition is competitive inhibitor. In graph IIC (graph6.) the enzymatic reaction with higher phosphate (inhibitor) concentration (0.133 mM) is monitored. The absorbance value increases with time, but the amount of product in comparison to the Part IIB is lower. In the last graph, Part IID (graph.7), 0.201 mM of the inhibitor was added. The amount of product was also increasing as the concentration of the substrates increasing, but the total amount is lower than the other previous trials. Concluding that if more substrate is added, this will overcome the inhibition. Another indication that the type of inhibition is competitive is by illustrating (Lineweaver-Burk Plot) (graph.8) The y-intercept (1/ Vmax) is approximately were all inhibitor concentrations intersected. Also, the km values are increasing as the inhibitor concentration increases (x-intercept), which indicate that the type of inhibition is competitive inhibition. A Dixon’s plot was then illustrated to calculate the Ki of the competitive inhibition using the line of the equation. The x- intercept which is Ki is found to be 0.035.
Conclusion:
In conclusion, this experiment aims to determine the Km and the Vmax values of alkaline phosphatase and identify the type of inhibition by the inhibitor phosphate. Lineweaver-Burk Plot was illustrated to determine their values. Despite the accuracy of LB plot, it still has its disadvantage which is that the error increases if the amount of substrate used is low. The values of Vmax and Km were calculated for each part, and the type of inhibition was identified as competitive inhibition. There might be some experimental errors that might have occurred due to improper use of the pipette or for not inserting the solution in the spectrophotometer directly after adding the enzyme alkaline phosphatase, which means that the absorbance was measured after the reaction has already preceded without detecting its beginning in the spectrophotometer. All in all, enzyme kinetics have a very important role in biochemical reactions that takes place in human body, thus, it is important to learn about these enzyme activities and know how to monitor and measure them accurately, to understand what effects inhibitors have on them.

References:
1. Experimental biochemistry lab manual page 57-69, Fall 2016.
2. https://en.wikipedia.org/wiki/Enzyme_kinetics3. https://www.chem.wisc.edu/deptfiles/s/enzymes/enzyme4.htm

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