ENZYME KINETICS

DEPARTMENT OF BIOLOGY

FACULTY OF SCIENCE & MATHEMATICS

UNIVERSITI PENDIDIKAN SULTAN IDRIS

SBK3013

PRINCIPLE IN BIOCHEMISTRY

EXPERIMENT 2: ENZYME

NAME	MATRIC NO.
MUHAMMAD FARIS BIN ISMAIL SAZEMI	D20141067089
MAYURIE PHUTHARANT A/P SURIN	D20141067078
NUR AFIQAH SYAHMINA BT MOHD KAMAL	D20141067091

GROUP: A

LECTURER’S NAME: DR. ROSMILAH MISNAN

INSTRUCTOR NAME: NUR ATIEKAH BT AZAHARI

INTRODUCTION

The approach of studying the mechanism of an enzyme-catalyzed reaction is to determine the changes in response and the rate of the reaction with the changes of parameters for example substrate concentration, enzyme concentration, pH, temperature and it is known as enzyme kinetics. Enzymes are protein molecule that will act as a biological catalyst. Enzyme is functioning to increase the rate of reactions without changing the overall process.

Enzymes are chains of amino acids that are bounded together by peptide bonds. In addition, they can be seen in all living cells and they will help in controlling the metabolic processes in which they functioned as converter to convert nutrients into energy and new cells. Furthermore, enzymes also help in the breakdown of food materials into its simplest form.

The reactants of enzyme catalysed reactions are termed substrates and each enzyme is quite specific in character, acting on a particular substrate to produce a particular products.

MATERIAL AND PROCEDURE

1. Preparation of standard reference

1. Starch solutions were 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. Mix the starch solution with distilled water and Iodine Solution.

The mixture for a standard curve were prepared.

The absorbance were measured at 590nm.

5. The following table is used as a guide.

The Standard Reference

Test tube	8 ml starch of x mg/ml	Water (ml)	Iodine (ml)	Absorbance at 590nm
1	0	9	1
2	0.01	1	1
3	0.025	1	1
4	0.05	1	1
5	0.1	1	1
6	0.3	1	1
7	0.5	1	1
8	0.7	1	1
9	1.0	1	1

6. A standard curve of Absorbance (@ 590 nm) vs. concentration of starch/iodine mixture are plotted. 

2. Determination the effect of substrate concentration, temperature and pH on Enzyme velocity 

a. The effect of substrate concentration

 Experiment of starch hydrolysis in different substrate concentration had been 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	8	1		1
2	0.01	0	1		1
3	0.025	0	1		1
4	0.05	0	1		1
5	0.1	0	1		1
6	0.3	0	1		1
7	0.5	0	1		1
8	0.7	0	1		1
9	1.0	0	1		1

b. The effect of temperature

Prepare as the following 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	8	1		1
2	0.01	0	1		1
3	0.025	0	1		1
4	0.05	0	1		1
5	0.1	0	1		1
6	0.3	0	1		1
7	0.5	0	1		1
8	0.7	0	1		1
9	1.0	0	1		1

c. The effect of pH

The Effect of pH

Prepare the following 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

RESULT

1.      Standard reference

1.      Determination the effect of substrate concentration, temperature and pH on Enzyme velocity

a.      The effect of substrate concentration

S0	Sf		S	V= S/t	1/S0	1/V
	Absorbance	Standard curve
0.00	0.182	0.010	0.010	0.0010	0.00	1000.00
0.01	0.147	0.005	0.005	0.0005	100.00	2000.00
0.025	0.208	0.010	0.015	0.0015	40.00	666.67
0.05	0.239	0.015	0.035	0.0035	20.00	285.71
0.10	0.222	0.010	0.090	0.0090	10.00	111.11
0.30	0.231	0.015	0.285	0.0285	3.33	35.09
0.50	0.155	0.005	0.495	0.0495	2.00	20.20
0.70	0.229	0.015	0.885	0.0885	1.43	11.30
1.00	0.251	0.020	0.980	0.0980	1.00	10.20

Michaelis-Menten Plot:

V_max = 0.099 mg/ml min

V_max/2 = 0.0495 mg/ml min

K_m = 0.24 mg/ml

Lineweaver-Burke Plot:

1/V_max = 60 (mg/ml min)­­^-1

V_max = 0.017 mg/ml min

-1/ K_m =- 4 (mg/ml)^-1

K_m =  0.25 mg/ml

a.      The effect of temperature

8ºC

S0 (mg/ml)	Sf  (mg/ml)		ΔS (mg/ml)	V=ΔS/t (mg/ml)/m	1/S0 (mg/ml)-1	1/V (mg/ml min)­­-1
	Standard curve	Absorbance at 560 nm
0	0.140	1.180	-0.140	-0.0140	0	-71.43
0.010	0.145	1.200	-0.135	-0.0135	100.0	-74.07
0.025	0.146	1.203	-0.121	0.0121	40.0	-82.64
0.050	0.230	1.921	-0.180	-0.018	20.0	-55.56
0.10	0.147	1.227	-0.047	-0.0047	10.0	-212.77
0.30	0.150	1.244	0.150	0.015	3.33	66.67
0.50	0.151	1.253	0.349	0.0349	2.00	28.65
0.70	0.145	1.214	0.555	0.0555	1.43	18.02
01.0	0.133	1.142	0.867	0.0867	1.00	11.53

28ºC

S0 (mg/ml)	Sf  (mg/ml)		ΔS (mg/ml)	V=ΔS/t (mg/ml)/m	1/S0 (mg/ml)-1	1/V (mg/ml min)­­-1
	Standard curve	Absorbance at 560 nm
0	0.145	1.208	-0.145	-0.0145	0	-68.97
0.010	0.130	1.070	-0.120	-0.012	100.0	-83.33
0.025	0.116	0.971	-0.091	-0.0091	40.0	-109.90
0.050	0.115	0.964	-0.065	-0.0065	20.0	-153.85
0.10	0.120	1.036	-0.020	-0.0020	10.0	-500.00
0.30	0.147	1.234	0.153	0.0153	3.33	65.36
0.50	0.161	1.382	0.339	0.0339	2.00	29.50
0.70	0.128	1.068	0.572	0.0572	1.43	17.48
01.0	0.135	1.156	0.865	0.0865	1.00	11.56

60ºC

S0	Sf		ΔS	V=ΔS/t	1/S0	1/V
	Absorbance	Standard curve
0	1.385	0.165	0.165	0.0165	0	60.61
0.01	0.623	0.072	0.062	6.2x10-3	100	161.3
0.025	1.019	0.063	0.038	3.8x10-3	40	263.2
0.05	0.844	0.1	0.500	0.05	20	20.0
0.1	1.215	0.145	0.045	4.5x10-3	10	222.2
0.3	1.570	0.183	0.117	0.0117	3.33	85.47
0.5	1.943	0.23	0.270	0.027	2	37.03
0.7	2.050	0.243	0.457	0.0457	1.43	21.88
1.0	0.627	0.077	0.923	0.0923	1	10.83

100ºC

S0	Sf		ΔS	V=ΔS/t	1/S0	1/V
	Absorbance	Standard curve
0	0.359	0.030	0.030	3.3X10-3	0	303.03
0.01	0.470	0.050	0.04	4.0X10-3	100	250
0.025	2.085	0.25	0.225	0.0225	40	44.44
0.05	2.675	0.32	0.27	0.0270	20	37.04
0.1	0.440	0.043	0.057	5.7X10-3	10	175.44
0.3	0.450	0.047	0.253	0.0253	3.33	39.53
0.5	0.520	0.055	0.445	0.0445	2	22.47
0.7	0.563	0.062	0.638	0.0638	1.43	15.67
1.0	0.504	0.058	0.942	0.0942	1	10.62

Calculation for Km and Vmax for each temperature

Temperature (*C)	Km (mg/mol)	Vmax (mg/mol.min)
8	0.250	0.057
28	0.125	0.057
60	0.083	0.057
100	0.0417	0.057

a. The effect of pH 

pH	So	Sf		∆s = So - Sf	V= ∆s/t
		absorbance	Standard curve
4	0.5	2.099	0.45	0.050	0.005
5	0.5	0.362	0.04	0.460	0.046
6	0.5	0.256	0.01	0.490	0.049
7	0.5	0.254	0.005	0.495	0.0495
8	0.5	0.223	0	0.5	0.05
9	0.5	0.241	0	0.5	0.05
10	0.5	0.278	0.015	0.485	0.0485
Blank	0.5	0.085	0	0.5	0.05

DISCUSSION

The effect of substrate concentration

As the concentration of substrate increases, the rate of reaction also increases until the point saturation occurs. It means as you increase the concentration, rate keeps increasing and then one point comes when the maximum rate is achieved and there is no free enzyme to bind with substrate and all the active sites of enzyme are bound to the substrate. So after that point, increasing the concentration won’t have any effect. What is the maximum for each enzyme is usually given by K_m value (Michealis Menten graph or the other one called something like Lineweaver burke plot). However, at some point, the graph shows that increasing the amount of substrate does not increase the reaction rate. We call this V_max or the maximum velocity of reaction.

Our experiment is accordance to the theory. This is because, from the test tube 2 until 9 (there are no starch is the first test tube), the value for V= S/t  is increase from 0.0050, 0.0015, 0.0035, 0.0090, 0.0285, 0.0495, 0.0885, 0.0980 respectively. The value for V= S/t indicate velocity (rate of digestion) of the reaction for each sample. When the substrate concentration change and while enzyme concentration is keeps constant, the rate of reaction will increase.

The effect of temperature

           Based on the plotted graph, the line for 8°C has the Km value of 0.250. The line for 28°C has the Km value of 0.125. The line for 60°C has Km value of 0.083 while the Km value for 100°C is 0.0417. Theoretically, the lower the Km value, the higher the affinity to the substrate. As a result, the rate of reaction is greater. According to the three different temperatures applied, temperature of 100°C is the optimum temperature for the enzyme to react as its Km value is the lowest among all. On the other hand, the lines share the same Vmax value, which is 0.057. Although temperatures change, the active site does not change. The substrates can still bind with the enzyme. The only difference is the rate of reaction.

The purpose was to study the effect of temperature of enzyme functioning. The major finding was that at 100°C the amylase broke down the starch the fastest. The other temperatures (8°C, 28°C, 60°C) didn’t break down starch until 10 minutes while 100°C broke down starch in 5 minutes. Our hypothesis, if amylase is used at 37°C, then it would more effectively to break down starch than amylase at any other temperature, was not supported. The unexpected result was that 100°C was more effective to break down starch.

The effect of pH

pH can give several effect on structure and activity of an enzyme. For example, pH can have an effect of the state of ionization of acidic or basic amino acids. Acidic amino acids have carboxyl functional groups in their side chains. Basic amino acids have amine functional groups in their side chains. If the state of ionization of amino acids in a protein is altered then the ionic bonds that help to determine the 3-D shape of the protein can be altered. This can lead to altered protein recognition or an enzyme might become inactive.

Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate cannot bind to the active site or it cannot undergo catalysis.

The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH.

The velocity of pH is increased from pH 4 to 8. The velocity for each pH from pH 4, 5, 6, 7 and 8 is 0.005, 0.046, 0.049, 0.0495 0.05. But starting from pH 8 and 9, the velocity is the same that is 0.05 and the velocity to become lower at pH 10.

It is important to study the effect of pH on enzyme activity so it can be learned when amylase will function with maximun efficiency. In this experiment, the effect of seven different pH’s ( 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) on the efficiency of an amylase reaction is studied. We tested in order to study the general pattern of an enzyme efficiency as a result of the pH environment. Theoretically, the optimal pH will be 7.0 because amylase is generally found in saliva of many animals, and it would therefore make sense for the enzyme to function best at the typical pH of those organisms, which is around 7.0, then the rate of disappearance would be greater than that of the reaction  occuring in the solution with pH 6.0 or pH 10.0. In the experiment that we had done, the result that we obtained is the pH that work best for amylase is pH 8 and it is slightly differences with the theoretical value for pH that work best for amylase that is pH 7. This maybe due to the error that we had done during the experiment such as we put too much of buffer solution in the test tubes.

CONCLUSION

Based on our experiment, some of the results are not the same as the theory. For the substrate concentration, as the concentration increase the rate of enzymatic reaction. The obtained result is same as from the expected. As for the temperature, the rate of reaction increase gradually as the temperature increase. For the effect of pH, the most favorable pH value for amylase is pH 8 and 9.

REFERENCES

Analyzing Enzyme Kinetic Data with a Graphing Calculator. Retrieved on 1 May, 2013 from

http://dwb.unl.edu/calculators/pdf/Enzyme-Calc.pdf

Anonymous, Effect of pH on enzyme. Retrieved on April 28, 2013from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm

Wise Geek (2013). What Is Substrate Concentration? Retrieved on May 1, 2013 from

 http://www.wisegeek.com/what-is-substrate-concentration.htm

Worthington Biochemical Corporation (2013). Introduction to enzyme. Retrieved on April 28 2013 from http://www.worthington-biochem.com/introbiochem/effectsph.html