Experiment 3 : Enzyme

 Introduction:

Thousands of enzymes are found in living cells where they act as catalysts for the thousands of chemical reactions which occur. Enzymes are proteins that help to speed up chemical reactions in the body. Enzymes also have their own scientific functions and not changed when they perform their function. It also can be reused. Enzymes are long, linear chains of amino acids that fold to produce a three-dimensional product. Each unique amino acid sequence produces a specific structure, which has unique properties. In addition to making life possible, many enzymes have numerous applications that affect our daily lives in other ways such as food processing, clinical diagnoses, sewage treatment, and the textile industry. 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.

The region that contains the catalytic residues, binds the substrate, and then carries out the reaction is known as the active site. Enzymes can also contain sites that bind cofactors, which are needed for catalysis. Like all catalysts, enzymes work by lowering the activation energy for a reaction, thus dramatically increasing the rate of the reaction. Enzyme activity is also affected by temperature, pressure, chemical environment (pH), and the concentration of substrate. Most enzymes can be denatured and unfolded and inactivated by heating or chemical denaturants, which disrupt the three-dimensional structure of the protein. Depending on the enzyme, denaturation may be reversible or irreversible.

METHODS:

1. Preparation of Standard reference

Starch solution from the stock solution (1.0 mg/ml)  was prepared into dilution 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.

1. Iodine solution was prepared by adding 5g potassium iodide to 100ml water. The dissolved potassium iodide was added with 1 g of iodine and was allowed to dissolved.
2. A standard curve of absorbance (590 nm) vs concentration of starch/iodine mixture was prepared. The data was recorded in Table 1.

 2.   The effect of substrate concentration

1. Experiment of starch hydrolysis in different substrate concentration experiment must be prepared as shown in Table 2.
2. Starch concentration for each sample was calculated after hydrolysis (S_F) through use of the standard curve. The initial starch concentration (S₀) is already known.[S]=(S₀)-(S_F)
3. The velocity (rate of digestion) of the reaction for each sample can be calculated as
4. A table showing rate of hydrolysis (V) was prepared at different the starch concentrations.                V=Δ S/ Δt = (S₀-S_F)/10 minutes
5. Michaelis-Menten graph was plotted.
6. A graph of 1/starch concentration (x-axis) versus 1/rate of digestion(y-axis) was prepared. This type of reciprocal graph displaying enzyme kinetics is a Lineweaver-Burke plot.
7. The value of V_max and Michaelis constant K_m from the graph.
8. The y-intercept of the Line-weaver Burke plot is the reciprocal of the maximum velocity of the reaction (V_max). The x-intercept is the negative reciprocal of the Michaelis constant. (K_{m )}

3.    The effect of temperature

1. Data collected in Table 3
2. The Lineweaver-Burke line for the result of 20,28,35 and 40⁰C. All three plots was compared

4.  The effect of pH

1. Table 4 was prepared using different pH
2. What are the value of V for all pH?
3. The velocity for each of the pH test was compared.

Results :

Part A : Standard Curve determination

Concentration, mg/mL	Absorbance,nm
0.000	0.018
0.010	0.060
0.025	0.148
0.050	0.279
0.100	0.472
0.300	1.245
0.500	1.912
0.700	2.471
1.000	3.324

Part B : The effect of substract concentration

Velocity	[S]
-0.0015	-0.030
-0.0012	-0.023
-0.0002	-0.004
0.0065	0.013
0.0036	0.072
0.0143	0.285
0.0238	0.476
0.0335	0.670
0.0480	0.960

Part C : The effect of temperature on enzyme

8^oC

So	Sf	[S] = So-Sf	1/ [S]	v = [S]/20 min	1/v
0.000	0.050	-0.050	-20.000	-0.003	-400.000
0.010	0.059	-0.049	-20.408	-0.002	-408.163
0.025	0.072	-0.047	-21.277	-0.002	-425.532
0.050	0.069	-0.019	-52.632	-0.001	-1052.632
0.100	0.067	0.033	30.303	0.002	606.061
0.300	0.306	-0.006	-166.667	0.000	-3333.333
0.500	0.062	0.438	2.283	0.022	45.662
0.700	0.061	0.639	1.565	0.032	31.299
1.000	0.030	0.970	1.031	0.049	20.619

1/[S]	1/v
-20.000	-400.000
-20.408	-408.163
-21.277	-425.532
-52.632	-1052.632
30.303	606.061
-166.667	-3333.333
2.283	45.662
1.565	31.299
1.031	20.619

28 ^oC

So	Sf	[S] = So-Sf	1/ [S]	v = [S]/20 min	1/v
0.000	0.073	-0.073	-13.699	-0.004	-273.973
0.010	0.072	-0.062	-16.129	-0.003	-322.581
0.025	0.158	-0.133	-7.519	-0.007	-150.376
0.050	0.100	-0.050	-20.000	-0.003	-400.000
0.100	0.088	0.012	83.333	0.001	1666.667
0.300	0.074	0.226	4.425	0.011	88.496
0.500	0.072	0.428	2.336	0.021	46.729
0.700	0.071	0.629	1.590	0.031	31.797
1.000	0.097	0.903	1.107	0.045	22.148

1/[S]	1/v
-13.699	-273.973
-16.129	-322.581
-7.519	-150.376
-20.000	-400.000
83.333	1666.667
4.425	88.496
2.336	46.729
1.590	31.797
1.107	22.148

35 ^oC

So	Sf	[S] = So-Sf	1/ [S]	v = [S]/20 min	1/v
0.000	0.048	-0.048	-20.833	-0.002	-416.667
0.010	0.056	-0.046	-21.739	-0.002	-434.783
0.025	0.067	-0.042	-23.810	-0.002	-476.190
0.050	0.066	-0.016	-62.500	-0.001	-1250.000
0.100	0.068	0.032	31.250	0.002	625.000
0.300	0.079	0.221	4.525	0.011	90.498
0.500	0.071	0.429	2.331	0.021	46.620
0.700	0.073	0.627	1.595	0.031	31.898
1.000	0.094	0.906	1.104	0.045	22.075

1/[S]	1/v
-20.833	-416.667
-21.739	-434.783
-23.810	-476.190
-62.500	-1250.000
31.250	525.000
4.525	90.498
2.331	46.620
1.595	31.898
1.104	22.075

^40 oC

So	Sf	[S] = So-Sf	1/ [S]	v = [S]/20 min	1/v
0.000	0.089	-0.089	-11.236	-0.004	-224.719
0.010	0.060	-0.050	-20.000	-0.003	-400.000
0.025	0.073	-0.048	-20.833	-0.002	-416.667
0.050	0.063	-0.013	-76.923	-0.001	-1538.462
0.100	0.086	0.014	71.429	0.001	1428.571
0.300	0.034	0.266	3.759	0.013	75.188
0.500	0.102	0.398	2.513	0.020	50.251
0.700	0.020	0.680	1.471	0.034	29.412
1.000	0.090	0.910	1.099	0.046	21.978

1/ [S]	1/v
-11.236	-224.719
-20.000	-400.000
-20.833	-416.667
-76.923	-1538.462
71.429	1428.571
3.759	75.188
2.513	50.251
1.471	29.412
1.099	21.978

LVineweaver-Burke plot graph

Calculation :

All the line intercept at the same y-intercept which represent 1/Vmax

Therefore the value of 1/ Vmax is 30.0

1/Vmax = 30.0

Vmax    = 1 / 30.0

Vmax    = 0.03 mg/ ml.min

The x-intercept represent the value of 1/Km.

Therefore Km value for temperature 8^oC is 

1/Km = -2

Km    = 1/( -2)

Km    = -0.5

Km value for temperature 28^oC is

1/ Km = -8

Km     = 1/ (-8)

Km     = - 0.125

Km value for temperature 35^oC is

1/Km = -5

Km    = 1/ (-5)

Km    = -0.2

Km value for temperature 40^oC is

1/Km = -1.75

Km    = 1/ (-1.75)

Km    = - 0.57

Part D : The effect of pH 

Discussions :

The reactants of enzyme catalyzed reactions are termed substrates and each enzyme is quite specific in character, acting on a particular substrates to produce a particular products. In Experiment 2, we were studying the effects of substrate concentration in enzyme activities. As we know, the concentration of starch, was changes due to the conversion of substrate to product. It has been shown that if the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum. After this point, increases in substrate concentration will not increase the velocity. It is theorized that when this maximum velocity had been reached, all of the available enzyme has been converted to enzyme substrate complex. This point on the graph is designated as maximum velocity, Vmax.

For experiment 3, we observed the effect of temperature on the enzyme amylase. The experiment was developed to test the enzymes reaction rate of amylase digesting starch at several different temperatures and see how the rate changed. The enzyme reaction was tested on 4 different temperatures which are at 8°C, 28°C, 35°C and finally 40°C.Based on theories, the rate of reaction was found to increase as the temperature of the environment was raised. The result recorded was plotted in linear form by using Lineweaver-Burke line. The graph was drawn by plotting 1/V against 1/[S]. However, based on the result and plotted graph, we cannot identify its 1/V_max and its -1/K_m.

Enzymes are affected by changes in pH. The most favorable pH value or the point where the enzyme is most active is known as the optimum pH. Based on this experiment, the enzyme amylase is preferred to work best at 0.42 mg/ml concentration of starch with an optimum pH which is 7. At this point, it indicates the Vmax of the enzyme rate of reaction. The amylase is really performed well with starch at the range of pH in between 4-7. Outside of its pH range the amylase is denatured. Extremely high pH and low pH values generally result in complete loss of activity for most enzymes. pH is also a factor in the stability of enzymes. As with the activity, for each enzyme there is also a region of pH optimal stability.