-BIOLOGY LABORATORY REPORT-

NAME : MOHAMMAD HAZZIQ BIN SELAMAT NRIC : 930712-01-6563 GROUP : 12M6 STUDENT ID : 2011629718 TITLE : ENZYME CONCENTRATION AND RATE OF REACTION DATE: 15 SEPTEMBER 2011 LECTURER : MR. MOHD HAFIZ BIN MOHD ROTHI
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TITLE: THE EFFECT OF ENZYME CONCENTRATION ON THE RATE OF REACTION. OBJECTIVES: 1) To investigate how enzyme concentration can affect the rate of reaction. 2) To develop experimental and investigative skills.

INTRODUCTION Enzymes are proteins that participate in cellular metabolic processes with the ability to enhance the rate of reaction between bimolecular. Some enzymes can even reverse a reaction from the direction it would normally take, by reducing the activation energy (Ea) to the extent that the reaction favours the reverse direction. Similarly, enzymes can catalyze reactions that might not otherwise occur, by lowering the Ea to a more "affordable" level for the cell. Enzymes are classified according to the reactions they catalyze. The six classes are: 1. Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases

Like all catalysts, enzymes work by lowering the activation energy (Ea) for a reaction, thus dramatically increasing the rate of the reaction. As a result, products are formed faster and reactions reach their equilibrium state more rapidly. Most enzyme reaction rates are millions of times faster than those of comparable un-catalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts in that they are highly specific for their substrates. Enzymes are known to catalyze about 4,000 biochemical

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reactions.[1] A few RNA molecules called ribozymes also catalyze reactions, with an important example being some parts of the ribosome.[2][3] Synthetic molecules called artificial enzymes also display enzyme-like catalysis.[4] Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity; activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, chemical environment (e.g., pH), and the concentration of substrate. Some enzymes are used commercially, for example, in the synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes; enzymes in meat tenderizers break down proteins into smaller molecules, making the meat easier to chew). Enzymes are generally globular proteins and range from just 62 amino acid residues in size, for the monomer of 4-oxalocrotonate tautomerase, to over 2,500 residues in the animal fatty acid synthase. A small number of RNA-based biological catalysts exist, with the most common being the ribosome; these are referred to as either RNA-enzymes or ribozymes. The activities of enzymes are determined by their three-dimensional structure. However, although structure does determine function, predicting a novel enzyme's activity just from its structure is a very difficult problem that has not yet been solved. Like all proteins, enzymes are long, linear chains of amino acids that fold to produce a threedimensional product. Each unique amino acid sequence produces a specific structure, which has unique properties. Individual protein chains may sometimes group together to form a protein complex. Most enzymes can be denaturedthat is, unfolded and inactivatedby heating or chemical denaturants, which disrupt the three-dimensional structure of the protein. Depending on the enzyme, denaturation may be reversible or irreversible. Structures of enzymes in complex with substrates or substrate analogs during a reaction may be obtained using Time resolved crystallography methods. Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions. Complementary shape, charge and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible for this specificity. Enzymes can also show impressive levels of stereospecificity, regioselectivity and chemoselectivity.

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Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. These enzymes have "proof-reading" mechanisms. Here, an enzyme such as DNA polymerase catalyzes a reaction in a first step and then checks that the product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases. Similar proofreading mechanisms are also found in RNA polymerase, aminoacyl tRNA synthetases and ribosomes. Some enzymes that produce secondary metabolites are described as promiscuous, as they can act on a relatively broad range of different substrates. It has been suggested that this broad substrate specificity is important for the evolution of new biosynthetic pathways. Enzymes are very specific, and it was suggested by the Nobel laureate organic chemist Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model. However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve.

Figure 1: Lock and key model.

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PROBLEM STATEMENT:

HYPOTHESIS: The higher the concentration of enzyme, the higher the rate of reaction.

NULL HYPOTHESIS: The higher concentration of enzyme, the rate of reaction is remaining the same.

VARIABLES

Manipulated variable: The number of spatula of blended potato. Responding variable : Volume of oxygen released. Fixed variable: Volume of hydrogen peroxide, H2O2 solution.

APPARATUS Test tubes Conical flask Spatula Stopwatch Beaker Delivery tube with stopper Syringe Graduated tube Small measuring cylinder

MATERIALS Blended potato Hydrogen peroxide solution, H2O2 Distilled water Buffer solution

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PROCEDURE 1. 1 spatula of blended potato was placed into the conical flask. 5cm3 of buffer solution was measured using a syringe. 2. Then, 5cm3 of buffer solution (pH6.3) was added into the conical flask. 3. The conical flask was swirled to mix up all of the solution. 4. The mouth of the conical flask then was closed temporarily with a stopper. 5. The graduated tube was filled with distilled water and it was inverted carefully into the beaker. 6. 25 cm3 of hydrogen peroxide, H2O2 was measured into a syringe and the conical flask was connected. 7. Then, the graduated tube was placed immediately over tube end of the delivery tube and the volume of oxygen gas, O2 was measured every 10 seconds for 5 minutes. 8. The experiment then was repeated by used 2 and 3 spatula of blended potato added into the conical flask. 9. The results then was recorded in a table showing the times and volume of oxygen gas, O2 collected. 10. A several graph was plotted and the initial rate of reaction was calculated by finding the gradient.

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RESULTS Table: Number of spatula of blended potato Time (s) 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 1 4.4 9.6 11.6 14.8 16.4 17.4 18.8 19.6 20.0 20.4 20.6 21.0 21.2 21.4 21.6 21.7 21.8 22.0 22.2 22.3 22.4 22.5 2 9.0 19.2 22.7 25.5 27.8 30.6 32.7 35.5 37.2 40.5 42.4 44.3 44.5 45 45.6 46.2 46.8 47.3 47.9 48.2 48.5 49 3 16.4 26.0 34.1 41.8 47.1 49.6 52.5 57.5 61.4 64 65.6 68.2 70 70.6 71.7 73.4 75.4 77.3 78.2 79.6 80.0 81.3

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230 240 250 260 270 280 290 300

22.6 22.6 22.6 22.6 22.6 22.6 22.6 22.6

49.3 50.1 50.1 50.1 50.1 50.1 50.1 50.1

8106 83.0 83.4 83.6 83.6 83.6 83.6 83.6

DISCUSSION Based on the result obtained, we can see clearly that there are slightly differences between the fresh fruits and the carton fruits. To obtain the result, first of all we must find the standard curve of ascorbic acid using 100mg of vitamin C tablet. The concentration is different between each other which are range from 0.2%-1.0%. Dissolving that particular tablet in 100ml of water is then used to decolourise the DCPIP solution of fixed concentration and volume. The various concentration of ascorbic acid are 0.2%, 0.4% 0.6%, 0.8% and 1.0%, which is had been used to decolourise the DCPIP solution. The volume of ascorbic acid used to decolourise the DCPIP solution is 1.0cm3, 0.8cm3, 0.6cm3, 0.4cm3 and 0.2 cm3 respectively. According to the standard curve drawn, the concentration of vitamin C is higher if small volume of ascorbic acid is used to decolourise the DCPIP solution. Based on the Graph 2, the volume of carton orange juices needed to decolourise the DCPIP solution is 1.0cm. This juice contains 0.2% of vitamin C. When the lemon juice is used, the volume needed to decolourise the DCPIP solution is 0.2 cm3. Its means thats it contain 1.0% 0f vitamin C. the result is same as when using lime juices. The volujme of lemon juices needed to decolourise the DCPIP solution is same as lemon juices which are 0.2cm3 and it shows that its contains 1.0% of concentration of vitamin C. the lowest volume needed to decolourise the DCPIP solution between all three cartons fruits jices are 0.2 cm3. The carton orange juices need 1.0 cm3 to decolourise DCPIP solution. Between the three carton fruit juices, orange contain the lowest concentration of vitamin C compared to lemon and lime.

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If we refer to the Graph 3, there are slightly differences between fresh juices and carton juices. When we used fresh orange juices, its only need 0.5cm3 rather than carton juices which is 1.0 cm3 to decolourise the DCPIP solution. Thus, it contains 0.5% of vitamin C. lemon juices and lime juices share the same result. The volume needed to decolourise the DCPIP solution by both lemon and lime juices are the same. They contain same concentration of vitamin C which is 1.1%. Therefore, lime and lemon juices have the highest concentration of vitamin C compared to orange juices. So, based on the result obtained we can conclude that fresh juices have higher concentration of vitamin C content compared to carton juices. The differences also affected by high rate of oxidation in carton juices because they might be undergo oxidation for several times before it has been processed.c c LIMITATIONS

There are several limitations while we carry out the experiment. First and the foremost is regarding the volume distilled water. Parallax

SAFETY PRECAUTIONS 1. Before beginning the experiment, we need to make sure that we wear a lab coat. This is important so that our clothes would not get stained by the blended potato, buffer solution and hydrogen peroxide solution. 2. We also have to wear a glove when handling the hydrogen peroxide solution, H2O2 and buffer solution as they was an acidic state which might be able to irritate our hand. Besides that, we also have to wear the glove when handling with the blended potato because the potato blended contain enzyme which are potential allergens and we should avoid rubbing our eyes in case we have potato blended solution on our hands. 3. Other than that, we also must wear a goggle when handling the hydrogen peroxide solution to make sure that our eyes are not in contact with the quite acidic hydrogen peroxide solution which might be able to irritate the eyes.

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CONCLUSION

In conclusion, we can say that the higher the concentration of enzyme which is the number of spatula of blended potato, the higher the rate of rate reaction. The hypothesis are accepted while the null hypothesis that says the higher the concentration of enzyme, the rate of reaction remaining the same is rejected by obtaining the result.

REFERENCES 1. Bairoch A. (2000). "The ENZYME database in 2000" (PDF). Nucleic Acids Res 28 (1): 3045. doi:10.1093/nar/28.1.304. PMC 102465. PMID 10592255. http://www.expasy.org/NAR/enz00.pdf. 2. Lilley D (2005). "Structure, folding and mechanisms of ribozymes". Curr Opin Struct Biol 15 (3): 31323. doi:10.1016/j.sbi.2005.05.002. PMID 15919196. 3. Cech T (2000). "Structural biology. The ribosome is a ribozyme". Science 289 (5481): 8789. doi:10.1126/science.289.5481.878. PMID 10960319. 4. Groves JT (1997). "Artificial enzymes. The importance of being selective". Nature 389 (6649): 32930. doi:10.1038/38602. PMID 9311771.

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