LAB REPORT 2: ENZYME

BIOCHEMISTRY SBK3013

LAB REPORT 2: ENZYME

TITLE: ENZYME KINETICS EXPERIMENT

MAUREEN SANTIH ANAK AMBANG	D20141067070
SHARIFAH AWANIS BINTI SYED MOHD ASWAD	D20141067053
HEIDI AMELDA ANAK LAGAT	D20141067086

DATE OF EXPERIMENT: 10 MARCH 2017

LECTURER: ASSOCIATE PROFESSOR ROSMILAH BINTI MISNAN

INSTRUCTOR: NUR ATIEKAH BINTI AZAHARI

Experiment 3: Enzyme Kinetics Experiment

OBJECTIVES

To determine the effects of substrate concentration, pH , and temperature on enzyme activity.

INTRODUCTION

Enzyme kinetics is the study of the rate at which an enzyme works. Enzymes are protein catalysts that speed up the rate of a chemical reaction without being used up during the process. They lowered the activation energy needed to convert it to a product. They also achieve their effect by temporarily binding to the substrate. The activity of an Enzyme is affected by its environmental conditions. Changing these alter the rate of reaction caused by the enzyme.The rate at which an enzyme work is determined by several factors such as substrate concentration , temperatures and pH. Cells control the activity of their enzymes through enzyme activity. Enzyme activity is the changes in affinity or catalytic efficiency such as inhibitors,allosteric or ionic signals (e.g. change pH). The rate of reaction when the enzyme is saturated with substrate is the maximum rate of reaction is called Vmax. This is usually expressed as the Km (Michaelis constant) of the enzyme, an inverse measure of affinity. For practical purposes, Km is the concentration of substrate which permits the enzyme to achieve half Vmax. In nature, organisms adjust the conditions of their enzymes to produce an Optimum rate of reaction, where necessary, or they may have enzymes which are adapted to function well in extreme conditions where they live.

PROCEDURE

1. PREPARATION OF STANDARD SOLUTION

1. Starch solutions is prepared  from the stock solution (1.0 mg/ml) into dilutions of 0.01, 0.025, 0.05, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/ml from the starch stock solution.

2. The starch solution is mixed with distilled water and Iodine Solution. Iodine solution is prepared by adding 5 g potassium iodide to 100 ml water.

3. A standard curve of Absorbance (@ 590 nm) vs. Concentration of a starch/iodine mixture is plotted. The following table is used as a guide.

2. DETERMINATION THE EFFECT OF SUBSTRATE CONCENTRATION,TEMPERATURE AND PH ON ENZYME VELOCITY (PROJECT)

A) THE EFFECT OF SUBSTRATE CONCENTRATION

1. Experiment of starch hydrolysis in different substrate concentration experiment is prepared as the following table.

2.A graph of Michaelis-Menten and Lineweaver-Burke is plotted.

B) THE EFFECT OF TEMPERATURE

1.The experiment of different temperature is prepared as followed.

2. Lineweaver-Burke graph is plotted.

C) THE EFFECT OF pH

1. The following is prepared for the experiment using different pH

RESULTS

1.PREPARATION OF STANDARD REFERENCE

Concentration of starch solutions	Absorbance(@590nm)
0	0.000
0.01	0.262
0.025	0.315
0.05	0.647
0.1	1.055
0.3	2.633
0.5	4.568
0.7	5.358
1.0	5.358

2.Determination the effect of substrate concentration, temperature and pH on enzyme velocity (PROJECT)

a) The effect of substrate concentration

S0	ABSORBENT	SF	∆S	V = ∆S/T	1/SO	1/V
0	0.990	0.10	-0.100	-0.0100	0	-100.00
0.01	1.193	0.12	-0.110	-0.0110	100	-90.90
0.025	0.948	0.09	-0.065	-0.0065	40	-153.80
0.05	1.183	0.12	-0.070	-0.0070	20	-14.30
0.1	1.172	0.12	0.020	0.0020	10	500.00
0.3	0.987	0.10	0.200	0.0200	3.3	50.00
0.5	1.125	0.11	0.390	0.0390	2.0	25.60
0.7	1.231	0.12	0.580	0.0580	1.4	17.20
1.0	1.248	0.13	0.870	0.0870	1.0	11.50

b) The effect of temperature

i) Temperature=8⁰C

S0	ABSORBENT	SF	∆S	V = ∆S/T	1/SO	1/V
0	1.058	0.100	-0.100	-0.0100	0	100.00
0.01	0.980	0.090	-0.080	-0.0080	100	-125.00
0.025	1.060	0.015	0.010	0.0010	40	-1000.00
0.05	0.991	0.095	-0.045	-0.0045	20	-222.22
0.1	1.490	0.140	-0.040	-0.0040	10	-250.00
0.3	2.231	0.240	0.060	0.0060	3.3	166.67
0.5	3.235	0.350	0.150	0.0150	2.0	66.67
0.7	3.580	0.390	0.310	0.0310	1.4	32.36
1.0	3.732	0.410	0.590	0.0590	1.0	16.95

ii) Temperature = 28⁰C

S0	ABSORBENT	SF	∆S	V = ∆S/T	1/SO	1/V
0	0.812	0.0700	-0.0700	-0.0070	0	-142.90
0.01	0.317	0.0200	-0.0100	-0.0010	100	-1000.00
0.025	0.323	0.0055	0.0195	0.0019	40	512.80
0.05	0.293	0.0053	0.0447	0.0047	20	212.80
0.1	0.239	0.0056	0.0950	0.0095	10	105.30
0.3	0.297	0.0057	0.2943	0.0294	3.3	34.00
0.5	0.348	0.0250	0.4750	0.0475	2.0	21.11
0.7	0.327	0.0230	0.6770	0.0677	1.4	14.80
1.0	0.347	0.0300	0.9700	0.0970	1.0	10.30

iii) Temperature = 60⁰C

S0	ABSORBENT	SF	∆S	V = ∆S/T	1/SO	1/V
0	1.189	0.110	-0.110	-0.0110	0	-90.9
0.01	1.324	0.130	-0.120	-0.0120	100	-83.3
0.025	1.100	0.015	0.010	0.0010	40	1000.0
0.05	1.141	0.013	0.037	0.0037	20	270.3
0.1	1.100	0.015	0.085	0.0085	10	117.6
0.3	1.070	0.100	0.200	0.0200	3.3	50.0
0.5	1.336	0.135	0.365	0.0365	2.0	27.4
0.7	1.360	0.138	0.562	0.0562	1.4	17.8
1.0	1.444	0.140	0.860	0.0860	1.0	11.6

iv) Temperature = 100⁰C

S0	ABSORBENT	SF	∆S	V = ∆S/T	1/SO	1/V
0	1.014	0.090	-0.090	-0.0090	0	-111.10
0.01	1.181	0.110	-0.100	-0.0100	100	-100.00
0.025	1.171	0.015	-0.010	-0.0010	40	-1000.00
0.05	1.417	0.140	-0.090	-0.0090	20	-111.10
0.1	1.252	0.120	-0.020	-0.0020	10	-500.00
0.3	1.197	0.115	0.185	0.0185	3.3	54.00
0.5	1.384	0.130	0.370	0.0370	2.0	27.00
0.7	1.723	0.171	0.529	0.0529	1.4	19.00
1.0	1.612	0.161	0.839	0.0839	1.0	12.00

c) The effect of pH

pH	S0	ABSORBENT	SF	∆S	V = ∆S/T
4	0.5	1.470	0.145	0.355	0.0355
5	0.5	1.230	0.120	0.380	0.0380
6	0.5	1.395	0.135	0.365	0.0365
7	0.5	1.390	0.135	0.365	0.0365
8	0.5	1.240	0.125	0.375	0.0375
9	0.5	1.390	0.135	0.365	0.0365
10	0.5	1.500	0.150	0.350	0.0350
Blank	0.0	1.189	0.110	-0.110	-0.011

DISCUSSION

1)PREPARATION OF STANDARD REFERENCE

   In this experiment, we used the equation of M₁V₁=M₂V₂ to determine the amount of starch that we need. First, we put small amount of distilled water into the measuring cylinder. Then, from the calculation, we add the amount of starch needed into the measuring cylinder. After that, we add distilled water until it reach the point of 30ml. Next, we take about 8 ml from the 30ml of the solutions into the test tube. This is followed by adding water and iodine respectively according to the amount that is provided in the table. This process is continued with other starch concentration such as 0.01,0.025,0.05,0.1,0.3,0.5,0.7 and 1.0.

M_IV₁=M₂V₂

(1.0)(V₁)= (0.01,0.025,0.05….)(30)

According to the equation above, the amount of starch solutions that we need is:

8ml of starch of x mg/ml	Amount of starch solution needed(ml)
0	0.00
0.01	0.30
0.025	0.75
0.05	1.50
0.1	3.00
0.3	9.00
0.5	15.00
0.7	21.00
1.0	30.00

From the standard curve of Absorbance (@590nm) vs concentration of starch mixture that we plotted, the absorbance increase, as the concentration of starch increase.

2.Determination the effect of substrate concentration, temperature and pH on enzyme velocity (PROJECT)

a) The effect of substrate concentration

The enzyme velocity will be affected if the enzyme and substrate concentration is changed. In this experiment, we changed the  substrate concentration. If we increase the substrate concentration, the rate of reaction will increase as there will be more substrate molecules available to collide with enzyme molecules. As a result, more product will be formed. Isf the substrate concentration is a limiting factor, then increasing concentration will increase the rate of reaction until at one point where there will be no any increase of the rate of reaction. This is because the substrate concentration is no longer be the limiting factor and the enzymes will effectively become saturated and will be working at their maximum possible rate. Controlling enzymes and substrate concentration in a cell is one way that an organism regulates its enzyme activity and so its Metabolism

Michaelis constants,Km have been determined for many of the commonly used enzymes. The size of Km tells us several things about a particular enzyme. From Michealis-Menten graph(MM), the value of Km is 0.45mmol while for LB graph is 0.25mmol. Vmax is the maximum point reached when there are enough substrate molecules to completely fill (saturate) the enzyme's active sites. At this point, the rate of enzyme activity will be not increase any longer. The value of Vmax for MM graph is 0.089mg/mol while for LB graph is 0.083 mg/mol. From our results, we found that we get negative for several value of rate of hydrolysis (V) due to several errors that occurs during the experiment.

Michaelis constants have been determined for many of the commonly used enzymes. A small Km indicates that the enzyme requires only a small amount of substrate to become saturated. Large Km indicates that high substrate concentrations is needed to achieve maximum reaction velocity.

B) THE EFFECT OF TEMPERATURE

When the temperature increases, the rate of reaction of the enzyme also increase. This has to do with the kinetic energy. This is because when the temperature increase, there are more random collisions between molecules per unit time, thus forming more product. The increase in the temperature will also break  more bonds especially hydrogen and ionic bonds.  When the bond is broken, the active site of the enzyme will also change shape. As a result, the active site becoming less complementary to the shape of substrate. This will decrease the rate of reaction as the enzyme will become denatured and will no longer function.  As a conclusion, as temperature increases, the rate of reaction will increase as the kinetic energy increase. However, as the temperature increase, the breaking of bond will become greater and greater so the rate of reaction  become decrease.The temperature at which the maximum rate of reaction occurs is called the enzyme's optimum temperature. Different enzymes has their own optimum temperature. Most enzymes in the human body have an optimum temperature of around 37.0 °C.

Based on the plotted graph, the line for 8°C has the Km value of 0.0625. The line for 28°C has the Km value of 0.10. The line for 60°C has Km value of 0.083 while the Km value for 100°C is 0.25. The lower the Km value, the higher the affinity to the substrate. As a result, the rate of reaction of the ezyme is greater. Based on the result, temperature of 8°C is the optimum temperature for the enzyme to react as its Km value is the lowest among all. All the temperature share the same Vmax value, which is 0.01. The enzymes are competitive inhibitors because the vlue of Km is different but the value of Vmax has no changed.

C) THE EFFECT OF pH

pH is a measure of the hydorgen ion concentration of a solution. Solutions with a high concentration of hydrogen ions have a low pH and solutions with a low concentrations of H+ ions have a high.It ranges from pH1 to pH14. Acid solutions have pH values below 7, and Basic solutions (alkalis are bases) have pH values above 7. Deionised water is pH7, which is termed as neutral. Different enzymes have different optimum pH value. For example, the enzyme Pepsin functions best at around pH2 and is found in the stomach, which contains Hydrochloric Acid (pH2).

As we know, enzymes has their own bonds. The bonds of the enzyme are influenced by H+ and OH- Ions in such a way that the shape of their active site is the most complementary to the shape of their substrate. At the optimum pH, the rate of reaction is at an optimum.Any change in pH above or below the optimum will quickly cause a decrease in the rate of reaction, since more of the enzyme molecules will have active sites whose shapes are not or less complementary to the shape of their substrate.

The velocity increase from pH 4 to pH 5 and slightly decrease and stay the same at pH 6 and pH 7. The velocity increase again at pH 8. The velocity then drop from pH 9, pH 10 and blank test tube. Based on this result of experiment, the optimum pH that is with highest velocity is sample pH 5 with velocity 0.380. The lowest velocity is blank test tube.

There is fluctuation of value of absorbance . This is because while taking reading of absorbance, the water vapour from condensation of the sample is still there, thus  it might affect the absorbance reading.

CONCLUSION

The enzyme activity is affected by the factors such as substrate concentration,temperature and pH.  For substrate concentration,                                 For temperature, every enzymes has its own optimum temperature. As temperature increases, the rate of reaction will increase as the kinetic energy increase. However, as the temperature increase, the breaking of bond will become greater and greater so the rate of reaction  become decrease. For pH, enzyme has its own optimum pH. Any change in pH above or below the optimum will cause a decrease in the rate of reaction because of the active site are not or less complementary to the shape of their substrate.

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REFERENCES

vlab.amrita.edu,. (2011). Effect of Substrate Concentration on Enzyme Kinetics. Retrieved 9 April 2017, from vlab.amrita.edu/?sub=3&brch=64&sim=1090&cnt=1

Worthington Biochemical Corporation. (2017). Introduction to Enzymes.  Retrieved April 09, 2017, from http://www.worthington-biochem.com/introbiochem/substrateconc.html

REFLECTIONS

MAUREEN SANTIH – This experiment is quite confusing for me as we required to prepare starch solutions with different volume. But after explanation by our lab instructor and help from some of my friends, I able to carry out the experiment successfully.  Besides, I learned how to use the spectrophotometry to measure the absorbance of the starch mixture and to study the effect of pH on enzyme activity. During plotting the graph for Absorbance vs starch concentration, I got some error. Luckily, I able to change my mistake as I showed it to the lab instructor first before submitting the report.

HEIDI AMELDA- From this experiment, it needs a lot of teamwork to do it. I have learnt how the difference temperature and ph value affect the enzyme activity. From this experiment also we prepared our own concentration for the for 8ml starch. We handled the solution carefully so that we used the correct solution.

SHARIFAH AWANIS- From this experiment, there are a lot of graph to be plot. So it is wise that the lab instructor devided the task to each group. We can share our results from another group members and discuss about it if there are problems. for our group, we have done the preparation of standard reference and the effect of pH by using different pH value. Although we have some difficulties during plot the graph, luckily we are able to complete our graph and share it with other groups.