Enzymes Catalysts In Biochemical Reactions Biology Essay

Introduction

Enzymes are resourceful catalysts for biochemical reactions, like all catalysts enzymes tend to speed up reactions. Enzymes use alternative reaction pathway of lower activation energy. They take part in the reaction, and as a result their able to provide alternate pathways. Throughout the reaction enzymes remain unchanged because they cannot experience any permanent changes. Enzymes only have the ability to change the rate of the overall reaction; they can’t affect the reactions position of the equilibrium (Rsc).

In most cases a chemical catalyst will catalyze any sort of reaction, enzymes differ in this sort. Enzymes tend to be specific, and this is due to the shape of enzymes molecules (Rsc).

Enzymes are made up of several proteins in a tertiary structure; these proteins tend to be globular. Many enzymes consist of a protein and a non-protein, called a cofactors and coenzymes. Cofactors are inorganic molecules that bind to enzymes to help them function examples maybe be zinc/magnesium ions (Zn2+, Mn2+), and coenzymes are organic molecules that bind to enzymes to help them function. An example of one of the most important coenzymes is nicotinamide adenine dinucleotide (NAD+), this substrate acts as an electron carrier in cellular respiration (Nelson Biology 12).

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Enzymes consist of active sites, which are parts of the enzyme molecule that have the ideal shape and functional groups to bind to one of the reacting molecules. The reacting molecule that binds to the enzyme is called the substrate. An enzyme-catalyzed reaction takes a different direction than a reaction without catalyst. When the substrate binds to the enzyme a reaction intermediate is produced. This intermediate has lower activation energy than the reaction without the enzyme catalyst (Rsc). There are two kinds of enzyme reactions, catabolic and anabolic. In a catabolic reaction the interactions between the substrate and enzyme causes stress and distorts the bonds in the substrate, allowing bonds to break. In an anabolic reaction the enzyme allows two substrates to have proper orientation to allow bonds to form between them. As a result the activation energy is lowered in both the catabolic and anabolic reaction (Nelson Biology 12).

Catalase is a common enzyme found in most plant and animal cells that functions as an oxidative catalyst, it decomposes hydrogen peroxide into oxygen and water. It’s structure is made of 4 main polypeptide chains, which can each be over 500 amino acids long. Catalase optimum temperature can vary depending on the species; similarly the optimum pH also varies from approximately 4-11. In humans however the optimum pH for catalase tends to be neutral. One molecule of Catalase can break down 40 million molecules of hydrogen peroxide each second (Catalase). The overall reaction is:

2 H2O2 → 2 H2O + O2

Many factors such as temperature, pH, inhibition of enzyme activity, substrate and enzyme concentrations can influence the affect the enzyme has on the reaction.

As the temperature rises, reacting molecules gain more kinetic energy, as a result the chances of a successful collision increase and thus the rate increases. There is a specific temperature when an enzyme’s catalytic activity is at its maximum. This optimal temperature is usually around human body temperature (37.5 oC) for the enzymes in human cells (Figure 1). When the temperature increases past the optimal temperature the enzyme becomes agitated, it begins to denature and ultimately lose its overall affect on the reaction (Nelson Biology 12). This occurs because the increase in temperature achieves higher kinetic energy and as a result the intra- and intermolecular bonds are broken in the enzyme molecule (Rsc).

Each enzyme works within a fairly small range of pH levels. Similar to temperature there is a pH at which its activity is at its maximum, the optimal pH (Figure 2). This is because changes in pH can create and break intra- and intermolecular bonds, changing the shape of the enzyme and ultimately the rate at which it will react.

The rate of an enzyme-catalyzed reaction depends on the concentrations of enzyme and substrate. As the concentration of either is increased the rate of reaction increases (Figure 3). When substrate concentrations are increased the overall reactions proceeds to increase up to a certain point, at this point the active sites have become saturated by the substrate and there are no further significant changes in the rate of reaction (Figure 4) (Rsc).

Some substances reduce or even stop the activity of enzymes in biochemical reactions. They do this by blocking or distorting active sites of enzymes. These substances are referred to as inhibitors. Inhibitors that occupy the active site and prevent a substrate molecule from binding to the enzyme are said to be competitive, as they compete with the substrate for the active site. Inhibitors that attach to other parts of the enzyme molecule, perhaps distorting its shape, are said to be non competitive (Nelson Biology 12).

Figure 1: Table 1Analysis

Amount of H2O2 (mL)

Amount of Distilled Water (mL)

Amount of pH Buffer (mL)

pH Level

Vertical Distance Travelled by Filter Paper Towards Meniscus

Time taken by filter paper disc to move to meniscus (s)

Upward velocity of Filter Paper Disc (cm/s)

10 mL

5 mL

–

7 (Control)

8.15

6.6

1.23

10 mL

–

5 mL

4

8.15

7.05

1.16

10 mL

–

5 mL

9

8.1

10.4

0.78

10 mL

–

5 mL

12

7.85

8.14

0.96

Figure 2: Graph 1

Test Tube

Temperature (°C)

Distance (cm)

Time (s)

Rate of Reaction (cm/s)

A

10.0

8.00

5.85

1.38

B

21.0

8.00

4.83

1.66

C

35.0

8.00

2.99

2.68

D

50.0

8.00

4.21

1.90

E

80.0

8.00

5.52

1.45

Figure 3: Table 2As the pH increased from 2-7 so did the velocity of the reaction (refer to figure 1: table 1). The reaction had an optimal pH of 7, and as the pH increased after the velocity of the reaction rapidly decreased. Notice the velocity for pH 12 is higher then the velocity of pH 9 (refer to figure 2: graph 1).

Figure 4: Graph 2 As the temperature increased from 10oC-30oC so did the rate of the reaction (refer to figure 3: table 2). The reaction had an optimal temperature of 35oC, and as the temperature increased after the rate of the reaction began to rapidly decrease (refer to figure 4: graph 2).

Enzyme concentration

Distance (cm)

Time (s)

Rate of Change (cm/s)

Other observations

100 % concentration

8 cm

3.02 s

2.65 cm/s

– bubbles appeared

80 % concentration

8 cm

5.06 s

1.58 cm/s

– fewer bubbles than previous composition

60 % concentration

8 cm

6.28 s

1.27 cm/s

– fewer bubbles than previous composition

40% concentration

8 cm

7.5 s

1.07 cm/s

– fewer bubbles than previous composition

Figure 5: Table 320% concentration

8 cm

19.65 s

0.41 cm/s

– no bubbles appeared

Figure 6: Graph 3

Figure 7: Table 4

Figure 6: Graph 3Increasing the concentration of the enzyme catalase (potato juice) rapidly increased enzyme activity (refer to figure 6: graph 3).

Concentration of

H202 of Distilled Water

Trial

Time of catalase to travel from the bottom of the test tube to the top (s)

Distance of bottom of test tube to substrate(cm)

Rate of change of the catalyzed reaction (cm/s)

15 mL of H202

3%

1

5.89

8.0

1.36

2

6.86

8.0

1.17

Total

6.38

8.0

1.27

13 mL of H202 2.6%

1

8.13

8.0

0.98

2

7.11

8.0

1.13

Total

7.62

8.0

1.01

10 mL of H202 2%

1

8.65

8.0

0.87

2

12.8

8.0

0.63

Total

10.73

8.0

0.75

7.5 mL of H202 1.5%

1

9.43

8.0

0.84

2

12.53

8.0

0.64

Total

10.98

8.0

0.74

5 mL of H202 1%

1

10.37

8.0

0.77

2

12.88

8.0

0.62

Total

12.63

8.0

0.70

Figure 9: Table 5

Figure 8: Graph 4Increasing concentrations of the substrate slowly increased from 1% to 2% (refer to figure 8: table 4), then as substrate concentrations increased more the rate of change became more rapid (refer to figure 9: graph 4).

Experiment Number

Amount of Inhibitor (copper (II) sulphate) (drops)

Time taken by enzyme disc to float to top of test tube (s)

Distance travelled by enzyme disc to top of test tube(cm)

Rate of Change of Enzyme Activity(cm/s)

1

0

4.13

8.0

1.94

2

1

4.68

8.0

1.71

3

5

5.57

8.0

1.44

4

10

6.66

8.0

1.20

5

15

8.57

8.0

0.93

Figure 10: Graph 5

As the amount of copper (II) sulphate increases the overall reactions begins to slow down, and the rate of reaction decreases (refer to figure 10: graph 5).

Evaluation

Part One: Affects of pH Enzymes are very sensitive to changes in pH, and significant changes in pH can affect enzymes in numerous ways. The effects of pH on enzyme activity are due to changes in the ionic state of the amino acid deposits of the enzyme and the substrate molecules. These variations in charge will affect the binding of the enzyme and as a result, enzyme activity will increase or decrease. Over a tapered pH range these effects will be reversible however high acid levels often cause permanent denaturation of the enzyme (Users.rcn). Before conducting this experiment one can anticipate that pH levels too high or too low would cause the enzyme to denature and thus it would no longer have an affect on the overall reaction. In this experiment 5 pH levels were used 2, 4, 7(control), 9, and 12. When the buffer solution affected the pH levels of the H2O2 from 2 to 4 there was a slight increase in enzyme activity (from 0.47 m/s to 1.16 m/s). There was one control test tube containing H2O2 with a neutral pH of 7. This test tube conducted the highest velocity of 1.23 m/s. As a result the optimal pH for the H2O2 was at a neutral pH of 7. When the pH level of the H2O2 increased to 9 the velocity seemed to decrease, which illustrated the loss of the effect of the enzyme. However this trend did not seem to remain consistent because when the pH level was increased to 12 the velocity of the enzyme also increased. As a result, it can be stated that enzymes work best in the region of neutral pH levels, and when pH levels become too high or to low enzyme activity decreases thus the hypothesis proved to be partly correct.

Part Two: Affects of Temperature The temperature of the H2O2 can severely affect the overall outcome of a reaction. Like most chemical reactions, enzyme-catalyzed reactions also increase in speed with an increase in temperature. As the temperature of the enzyme increases past a critical point thermal agitation begins to disrupt the protein structure resulting in the denaturation and loss of enzyme function (Nelson Biology 12). The hypothesis for this experiment was similar to that of pH, temperatures too high or too low would cause denaturation of the enzyme and thus it would no longer have an affect on the overall reaction. In this experiment 5 different temperatures were used 10oC, 21oC, 35oC (control), 50oC, and 80oC. When the temperature was decreased to 10oC the rate of the reaction was at it lowest of 1.38 m/s. At 21oC the rate slightly increased to 1.66 m/s. Thus there is a trend of lower temperatures causing the enzyme to lose its overall affect. There was one control test tube containing H2O2 that was at room temperature which was 35oC. This test tube conducted the highest rate of reactions of 2.68 m/s. As a result the control test tube achieved the optimal temperature. When the temperature of the H2O2 began to increase from 50oC to 80oC there was a trend of the enzyme losing its affect, and having an overall lower rate of reaction. As the temperature increased before the optimal temperature the rate of the reaction increased, and when the temperature continued to increase past the optimal point there was a rapid decrease in the rate of the reaction thus it is evident the hypothesis was correct.

Part Three: Affects of Changes in Concentrations The rates of enzyme-catalyzed reactions severely depend on the concentrations of enzymes and substrates. If one person is pushing a car it likely that car will take longer to get to and end point, however if 10 people are pushing that same car it will obviously get to the end point a lot quicker. It is the same with enzyme and substrate concentrations, the higher the concentrations the faster the reaction works. As the enzyme concentration increases so does the number of enzyme molecules, thus more substrate molecules can be acted upon at the same time which means they breakdown a lot faster. As the substrate concentrations increase, the reaction also proceeds to increase however with high levels of substrate concentrations the active sites become saturated and the enzyme no longer has an effect of the reaction (Worthington-biochem). The hypothesis for this experiment was simple, as enzyme and substrate concentrations increase so will the speed of the reactions. When changing the substrate concentrations, the five H2O2 concentrations where 3% (control), 2.6%, 2%, 1.5%, and 1%. The main trend in this experiment was the higher the concentration of the substrate the higher the rate of change. There was a significant and rapid increase in the rate of change from concentrations of 2% to 3%. When changing the enzyme concentrations, the five potato juice concentrations where 20%, 40%, 60%, 80%, and 100%. Changing the concentration of the enzyme had a similar affect to when the substrate concentrations were changed. The more concentrated the enzyme was the higher the rate of the reaction. The rate of the reaction rapidly increased from 20% to 40%, however it became a bit constant from 40% to 80%, and from about 80% to 100% it began to promptly increase again. As a result, it is evident the hypothesis was correct as the concentrations increased so did the reactions.

Part Four: Effect of the Inhibitors Inhibitors are used to block active sites of enzymes. They are substances used to slow down, or in some cases stop catalysis. Inhibitors either compete with a substance for the enzymes active site (competitive), or they bind to another site on the enzyme changing its shape (non-competitive) (Nelson Biology 12). Before conducting this experiment one can anticipate the more amount of inhibitor present the slower the reactions will proceed. In this experiment copper (II) sulphate was used as the inhibitor. In the five trials 0, 1, 5, 10, and 15 drops of the copper (II) sulphate were used. The obvious trend was the more inhibitor the lower the rate of reaction. Thus, the hypothesis was correct.

Sources of Error

Error #1: Consistency of Filter Paper

When conducting each individual experiment for many groups it seemed the most difficult task was getting the filter paper to arrive at the bottom of the test tube. When the filter paper was placed in the test tube it would go about half way down the test tube, however because the reaction catalyzed quickly the filter paper would begin to rise and travel back up to the top of the hydrogen peroxide liquid. As a result you would have to perform the experiment again, with a new catalyzed filter paper. This became a source of error because it made it difficult to collect consistent data. For every test tube, and trial the filter paper did not reach the bottom of the test tube at the exact same time. In some cases it would reach the bottom without difficulty, and in other situations it became a constant struggle to push it down the test tube. During certain trials the experiment had to be performed again and the hydrogen peroxide had already lost its affect from the previous catalyzed reaction. As a result, it is evident that the consistency and rate at which the filter paper travelled down the test tube is a significant source of error. To improve this source of error, heavier and more durable filter paper should be used. One can purchase “wet strength” filter paper which will make its way down the test tube on its own without any human force.

Error # 2: Accuracy of Inhibitor

During this experiment it became difficult to get exactly 15 mL of hydrogen peroxide after the inhibitor has been added. Copper (II) Sulphate is a severely small solvent so when added to the hydrogen peroxide one cannot control the amount of liquid present. This occurs because before adding the copper (II) sulphate it is uncertain how much hydrogen peroxide needs to be reduced in order to have exactly 15 mL. This creates a source of error because now the data collected is inconsistent because of the different volumes of hydrogen peroxide. To prevent this source of error one can use a different inhibitor that will dissolve in the hydrogen peroxide and not change its volume.

Error # 3: Catalase in Potatoes

During the experiment potato juice was constantly being pumped and used as the enzyme to catalyze the reactions. However it was not considered that each potato is harvested in a different way and one potato may have several nutrients, while the other may be completely dead. This results in the difference of concentrations of catalase that was taken from each specific potato. Once again this source of error causes a inconsistency in the collection of data because one cannot be certain they used the same potato, that pumped a constant concentration of catalase throughout the whole experiment. For the purpose of this experiment if only one potato was ground and made into potato juice then catalase concentrations would be consistent and it would eliminate this source of error.

Next Steps

A similar experiment that could be performed is Saturation Points of Substrate Concentrations. In the current lab saturation was not tested when changing around substrate concentrations. One can test the amount of substrate it would take to saturate the active site on the enzyme, and proceed to evaluate how much more of the enzyme concentration is needed to unsaturate and dissociate the substrates from the active site of the enzyme.

Another experiment that could be performed is Affects on Various Enzymes. Instead of just observing the affects of change of pH, temperature, concentrations, and inhibitors on Catalase it can be tested on other enzymes. For example Cellulase, Lactase, and Pepsin.