Lab Report 2: Enzyme

INTRODUCTION

Enzyme is any of a group of complex proteins or conjugated proteins that are produced by living cells that act as catalyst in specific biochemical reactions. The molecules at the beginning of the process upon which enzymes may act are called substrates and the enzyme converts these into different molecules, called products. Almost all metabolic processes in the cell need enzymes in order to occur at rates fast enough to sustain life. The set of enzymes made in a cell determines which metabolic pathways occur in that cell.

Like all catalysts, enzymes increase the reaction rate by lowering its activation energy. Some enzymes can make their conversion of substrate to product occur many millions of times faster. Chemically, enzymes are like any catalyst and are not consumed in chemical reactions, nor do they alter the equilibrium of a reaction. Enzymes differ from most other catalysts by being much more specific. Enzyme activity can be affected by other molecules: inhibitors are molecules that decrease enzyme activity, and activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. An enzyme's activity decreases markedly outside its optimal temperature and pH.

Enzymes are generally globular proteins, acting alone or in larger complexes. Like all proteins, enzymes are linear chains of amino acids that fold to produce a three-dimensional structure. The sequence of the amino acids specifies the structure which in turn determines the catalytic activity of the enzyme. Enzymes must bind their substrates before they can catalyze any chemical reaction. Enzymes are usually very specific as to what substrates they bind and then the chemical reaction catalysed. Specificity is achieved by binding pockets with complementary shape, charge and hydrophilic/hydrophobic characteristics to the substrates.

Some enzymes are used commercially, for example, in the synthesis of antibiotics. Some household products use enzymes to speed up chemical reactions: enzymes in biological washing powders break down protein, starch or fat stains on clothes, and enzymes in meat tenderizer break down proteins into smaller molecules, making the meat easier to chew.

PROCEDURE

Part A: Preparation of standard reference

1. A series of dilution for starch solution were prepared by using 1.0 mg/ml stock solution.
2. The starch solution that was prepared were mixed with distilled water and iodine solution.
3. The following table was used as a guide.


Test tube
8 ml starch of x (mg/ml)
Water (ml)
Iodine (ml)
Absorbance at 590 nm
1
0.00
9
1
2
0.01
1
1
3
0.025
1
1
4
0.05
1
1
5
0.10
1
1
6
0.30
1
1
7
0.50
1
1
8
0.70
1
1
9
1.00
1
1

4. Graph of standard curve of absorbance (@ 590 nm) vs. concentration of starch/iodine mixture were plotted. 

Part B: Determination Effect Of Substrate Concentration, Temperature And pH On Enzyme Velocity

a.       The Effect Of Substrate Concentration 

Experiment of starch hydrolysis in different substrate concentration was prepared as the following table:

Test tube
8 ml starch of x mg/ml
Water
(ml)
Amylase (ml)
Incubate each sample at 370C for 10 minutes
Iodine
(ml)
Place all test tubes in an ice bath. Measure the absorbance at 590nm
1
0.00
8
1
1
2
0.01
0
1
1
3
0.025
0
1
1
4
0.05
0
1
1
5
0.10
0
1
1
6
0.30
0
1
1
7
0.50
0
1
1
8
0.70
0
1
1
9
1.00
0
1
1

b.       The Effect Of Temperature
The solution was prepared for the experiment of different temperature:


Test tube
8 ml starch of x mg/ml
Water
(ml)
Amylase (ml)
Incubate each sample at 8, 28, 60, 1000C for 10 minutes
Iodine
(ml)
Place all test tubes in an ice bath. Measure the absorbance at 590nm
1
0.00
8
1
1
2
0.01
0
1
1
3
0.025
0
1
1
4
0.05
0
1
1
5
0.10
0
1
1
6
0.30
0
1
1
7
0.50
0
1
1
8
0.70
0
1
1
9
1.00
0
1
1

b.       The Effect Of pH
The following solution was prepared for the experiment using different pH:
Test tube
Starch of 0.5 mg/ml
2 ml buffer of pH x
Amylase (ml)
Incubate each sample at 370C for 10 minutes
Iodine
(ml)
Place all test tubes in an ice bath. Measure the absorbance at 590nm
1
5
4
1
1
2
5
5
1
1
3
5
6
1
1
4
5
7
1
1
5
5
8
1
1
6
5
9
1
1
7
5
10
1
1
Blank
5
8 ml of dH2O
1

DATA ANALYSIS

 1. Preparation of standard reference
( REFER STANDARD CURVE GRAPH)

2. Determination The Effect Of Substrate Concentration, Temperature And Ph On Enzyme Velocity

a. The effect of substrate concentration

So
Absorbance
s.curve
∆S
V= ∆S/t
1/ SO
1/V
0.00
0.182
0.01
0.01
0.001
0.00
1000.0
0.01
0.147
0.005
0.005
0.0005
100
2000.0
0.025
0.208
0.013
0.012
0.0012
40.00
833.3
0.05
0.239
0.015
0.035
0.0035
20.00
285.7
0.10
0.222
0.012
0.088
0.0088
10.00
113.6
0.30
0.231
0.013
0.287
0.0287
3.33
34.8
0.50
0.155
0.009
0.491
0.0491
2.00
20.4
0.70
0.229
0.012
0.688
0.0688
1.43
14.5
1.00
0.251
0.02
0.98
0.098
1.00
10.2

From Michaelis-Menten graph,
Vmax=  0.1
Km  = 0.32 mg/ml
From Lineweaver-burk graph,
1/ Vmax = 40
Vmax= 1 / 40
 = 0.025 mg/mlmin
-1/ Km= -2
Km       = 0.5 mg/ml


b. The effect of temperature          



for 8  ͦC:

SO
Absorbance
s.curve
∆S
V= ∆S/t
1/ SO
1/V
0.00
1.058
0.10
0.10
0.01
0.00
100.00
0.01
0.980
0.09
0.08
0.008
100.00
125.00
0.025
1.060
0.015
0.01
0.001
40.00
1000.00
0.05
0.991
0.095
0.045
0.0045
20.00
222.22
0.10
1.490
0.14
0.04
0.004
10.00
250.00
0.30
2.231
0.24
0.06
0.006
3.33
166.67
0.50
3.235
0.35
0.15
0.015
2.00
66.67
0.70
3.580
0.39
0.31
0.031
1.43
32.26
1.00
3.732
0.41
0.59
0.059
1.00
16.95


for 28  ͦC:


So
Absorbance
s.curve
∆S
V= ∆S/t
1/ SO
1/V
0.00
0.812
0.0700
0.0700
0.0070
0
142.9
0.01
0.317
0.0200
0.0100
0.0010
100.00
1000.0
0.025
0.246
0.0055
0.0195
0.00195
40.00
512.8
0.05
0.293
0.0053
0.0447
0.00447
20.00
223.7
0.10
0.239
0.0050
0.0950
0.0095
10.00
105.3
0.30
0.297
0.0057
0.2943
0.02943
3.33
34.0
0.50
0.348
0.0250
0.4750
0.0475
2.00
21.1
0.70
0.327
0.0230
0.6770
0.0677
1.43
14.8
1.00
0.374
0.0300
0.9700
0.0970
1.00
10.3

for 60  ͦC:

S0
Absorbance
s.curve
∆S
V= ∆S/t
1/ SO
1/V
0.00
1.189
0.11
0.110
0.0110
0.00
90.00
0.01
1.324
0.13
0.120
0.0120
100.00
83.30
0.025
1.100
0.015
0.010
0.0010
40.00
1000.00
0.05
1.141
0.013
0.037
0.0037
20.00
270.30
0.10
1.100
0.015
0.085
0.0085
10.00
117.60
0.30
1.070
0.100
0.200
0.0200
3.33
50.00
0.50
1.336
0.135
0.365
0.0365
2.00
27.40
0.70
1.360
0.138
0.562
0.0562
1.43
17.80
1.00
1.444
0.140
0.86
0.0860
1.00
11.60

for 100  ͦC:

S0
Absorbance
s.curve
∆S
V= ∆S/t
1/ SO
1/V
0.00
1.014
0.09
0.09
0.009
0.00
111.10
0.01
1.181
0.11
0.1
0.01
100.00
100.00
0.025
1.171
0.015
0.01
0.001
40.00
1000.00
0.05
1.417
0.14
0.09
0.009
20.00
111.10
0.10
1.252
0.12
0.02
0.002
10.00
500.00
0.30
1.197
0.115
0.185
0.0185
3.33
54.00
0.50
1.384
0.13
0.37
0.037
2.00
27.00
0.70
1.723
0.171
0.529
0.0529
1.43
19.00
1.00
1.612
0.161
0.839
0.0839
1.00
12.00

Vmax and Km values for each temperature are shown below:

Temperature
Vmax (mg/mlmin1)
Km(mg/ml -1)
8  ͦC
0.01667
0.4
28  ͦC
0.0125
0.125
60  ͦC
0.05
1
100  ͦC
0.025
1

c. The effect of pH

S0
Absorbance
S.curve
∆S
V= ∆S/t
0.5
1.470
0.15
0.35
0.0350
0.5
1.230
0.12
0.38
0.0380
0.5
1.395
0.14
0.36
0.0360
0.5
1.390
0.137
0.363
0.0363
0.5
1.390
0.137
0.363
0.0363
0.5
1.500
0.151
0.349
0.0349
0.5
1.189
0.10
0.10
0.040


Standard Curve
Graph of Substrate Concentration A
Graph of Substrate Concentration B
Graph of Temperature

 DISCUSSION

From this experiment, we can discuss that the activity of enzyme is affected by its environmental conditions. Changing this alter the rate of reaction caused by the enzyme. 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.
                
First factor that affected the activity of enzyme is substrate concentration. Increasing substrate concentration increases the rate of the reaction. This is because more substrate molecules will be colliding with enzyme molecules, so more product will be formed. Based on our result, our Vmax on Michaelis-Menten graph is 0.10 µM/min and Km is 0.32 µM, while Vmax on Lineweaver-Burke graph is 0.025mg/mlmin and Km is 0.50 mg/ml. After a certain concentration, any increase will have no effect on the rate of reaction, since substrate concentration will no longer be the limiting factor. The enzymes will affectively become saturated, and will be working at maximum possible rate.

Second factor that affected the activity of enzyme is temperature. Increasing temperature increase the kinetic energy that molecules possess. In a fluid, this means that there are random collisions between molecules per unit times. In this experiment, we needed to incubate each sample in 4 different temperature which are at 8, 28, 60 and 100 oC for 10 minutes. Based on our result, the Vmax  for 8 oC   is 0.0167mg/mlmin-1and Km is 0.4mg/ml-1,Vmax  for 28 oC   is 0.0125 mg/mlmin-1 and Km is 0.125 mg/ml-1, Vmax  for 60oC   is 0.05 mg/mlmin-1 and Km is 1.0 mg/ml-1 and Vmax  for 100oC   is 0.025 mg/mlmin-1 and Km is 1.0 mg/ml-1. Our result is not accurate because of different hand during making a different concentration of starch.

Since enzymes catalyze reactions by randomly colliding with substrate molecules, increasing temperature increases the rate of reaction, forming more product. However, increasing temperature also increases the vibrational energy that molecules have, specifically in this case enzyme molecules, which puts strain on the bonds that hold them together. As temperature increases, more bonds, especially the weaker hydrogen and ionic bonds, will break as a result of this strain. Breaking bonds within the enzyme will cause the Active Site to change shape. This change in shape means that the active site is less complementary to the shape of the Substrate, so that it is less likely to catalyze the reaction. Eventually, the enzyme will become denatured and will no longer function. As temperature increases, more enzymes' molecules' active sites' shapes will be less complementary to the shape of their substrate, and more enzymes will be denatured. This will decrease the rate of reaction. In summary, as temperature increases, initially the rate of reaction will increase, because of increased Kinetic Energy. However, the effect of bond breaking will become greater and greater, and the rate of reaction will begin to decrease.

Last factor that affected the activity of enzyme is pH.Based on our experiment, the values of velocity every pH are 0.0350 mg/mlmin-1, 0.0380 mg/mlmin-1,0.0360 mg/mlmin-1,0.0363 mg/mlmin-1, 0.0363 mg/mlmin-1,0.0439 mg/mlmin-1 and 0.040 mg/mlmin-1.

CONCLUSION

 Knowledge of basic enzyme kinetic theory is important in enzyme analysis in order both to understand the basic enzymatic mechanism and to select a method for enzyme analysis. The conditions selected to measure the activity of an enzyme would not be the same as those selected to measure the concentration of its substrate. Several factors affect the rate at which enzymatic reactions proceed - temperature, pH, enzyme concentration, substrate concentration, and the presence of any inhibitors or activators.

 REFERENCES
1. Alton Meister (1979), Advances In Enzymology And Related Idea Of Molecular Biology, Interscience ® Publication, Cornell University Medical College, New York.
2. John T. Moore and Richard H. Langley (2011), Biochemistry for Dummies ®, 2nd Edition, Wiley Publishing, Icn. , Indianapolis, Indiana.
3. http://www.chemguide.co.uk/organicprops/aminoacids/enzymes2.html