Experiment 3 : Enzyme Kinetics

TITLE

Experiment 3: Enzyme Kinetics Experiment

OBJECTIVES

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

INTRODUCTION

Enzymes are protein molecule that acts as biological catalysts. Without changing of the overall process, they increase the rate of reactions. Enzymes are long chains of amino acids bound together by peptide bonds. Besides that, they are seen in all living cells and controlling the metabolic processes in which they converted nutrients into energy and new cells. Other than that, enzymes also help in the breakdown of food materials into its simplest form. 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. The central approach for studying the mechanism of an enzyme-catalyzed reaction is to determine the rate of the reaction and its changes in response with the changes in parameters such as substrate concentration, enzyme concentration, pH, temperature and known as enzyme kinetics. The substrate concentration, [S] is one of the important parameter that affecting the rate of a reaction that catalyzed by an enzyme. However, studying the effects of substrate concentration is elaborated by the fact that during the course of an in vitro reaction, [S] changes due to the conversion of substrate to product. One simplest method to study enzyme kinetics is to measure the initial rate of the reaction designated V₀ ( Initial velocity), when [S] is much greater than the concentration of enzyme, [E]. The effect on V₀, when the enzyme concentration becomes constant is shown in Figure 1.

Figure 1

PROCEDURE

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

The Standard Graph

 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 350C 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

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 20, 28, 35, 400C 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

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 350C 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	3 ml of dH2O			1

RESULT
Result for substrate concentration

Test tube	So ( 8 ml starch of x mg/ml)	SF	S (So – SF )	V = S/ 20 minute
1	0	0.015	-0.015	-7.5×10-4
2	0.01	0.045	-0.035	-1.75×10-3
3	0.025	0.040	-0.015	-7.5×10-4
4	0.05	0.050	0	0
5	0.1	0.050	0.050	2.5×10-3
6	0.3	0.030	0.270	1.35×10-2
7	0.5	0.050	0.450	2.25×10-2
8	0.7	0.060	0.640	3.2×10-2
9	1.0	0.100	0.900	4.5×10-2

Graph Rate of Hydrolysis (V) at different Starch Concetration (Substrate)

Result for Temperature 

Graph of 1/V against 1/[S]

Result for pH

Test Tube	pH	Absorbance (mg/ml)
1	4	0.447
2	5	0.391
3	6	0.876
4	7	0.373
5	8	0.301
6	9	0.344
7	10	0.202
8	3ml of H2O	0.186

By using,

ΔS /V = (S₀ – S_f) / 20 min 

Test Tube	pH	S0(mg/ml)	Sf (mg/ml)	ΔS / V (mg/ml min)
1	4	5	0.097	0.2452
2	5	5	0.081	0.2459
3	6	5	0.023	0.2488
4	7	5	0.076	0.2462
5	8	5	0.052	0.2474
6	9	5	0.064	0.2468
7	10	5	0.025	0.2487
8	3ml of H2O	5	0.018	0.2491

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 wont have any effect. What is the maximum for each enzyme is usually given by Km value (michealis menten graph or the other one called sumting like Lineweaver burke plot). The Km value is the rate constant or it can be explained as how much substrate concentration is required by an enzyme to reach to the half of maximum rate or velocity of enzyme. Each enzyme has different Km values. So I hope that answers ur quesiton- wherever the Vmax occurs and it intersects the curve drawn for substrate concentraion and velocity (or rate of reaction), that point is the saturation point or maximum substrate concentration to have maximum rate of the reaction.

Based on our experiment, we got negative value for rate of hydrolysis (V). According to the theory we are not supposed to get negative value for (V). In other words it states that, when the substrate concentration change and while enzyme concentration is keeps constant, the rate of reaction will increase. This is because there are a few error that occurred during we ran the experiment. 

The Effect of Temperature

           Based on the plotted graph, the line for 10°C has the Km value of 0.30. The line for 28°C has the Km value of 0.14. The line for 35°C has Km value of 0.07 while the Km value for 40°C is 0.10. 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 35°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 0.003. 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 amylase reacts the best at temperature of 35°C.

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 increases from pH of 4 up to pH 6. But, starting from pH 7, the velocity decreases from 0.2488 for pH 6 to 0.2462 for pH 7. The velocity increase until pH 8 with value of 0.2474. Starting from pH 9, the velocity decreases again but after pH 9, the velocity keep increased until pH 10 and so with test tube blank test tube.

Based on this result of experiment, the optimum pH that is with highest velocity is sample pH 6 with velocity 0.2488. The lowest velocity is pH 1.

There is fluctuation of value of absorbance that give the fluctuation of value of velocity might because of the sample is contaminated. The other factor might be the time for taking reading of absorbance for every of pH sample is not fixed. Besides that, the process of placing all test tubes in ice bath might affect the result because, while taking reading of absorbance, the water vapour from condensation of the sample is still there, so, it might affect the absorbance reading.

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. However, the obtained result is different 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 6.   

REFERENCE

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

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

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

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

Worthington Biochemical Corporation (2013). Introduction to enzyme. Retrieved on April 28,

            2013 from http://www.worthington-biochem.com/introbiochem/effectsph.html

Anonymous, Effect of pH on enzyme. Retrieved on April 28, 2013

from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm