What Factors affect the rate of enzyme reaction

Investigating which factors affect the rate of enzyme reaction

Introduction to practical:

Enzymes are proteins that are able to catalyse the rate of reaction in a living organism. Each enzyme controls a particular reaction and is consistently re-used. In addition, they are affected by several factors, such as temperature, pH, concentration of enzyme and concentration of substrate.

An enzyme that is common in living organisms is Catalase, which works to catalyse the decomposition of the mixture hydrogen peroxide to water and oxygen. This enzyme is known to have one of the highest reactions out of all; every second, one molecule of catalase is able to transfer 83,000 molecules of hydrogen peroxide into water and oxygen. When hydrogen peroxide is broken down by catalase, oxygen and water are released.

In a catalysed enzyme reaction, molecules named substrates fit into the active site and form an enzyme-substrate complex, a concept which can be called the lock and key theory. As a result of this, while still attached to the enzyme, the substrate breaks down into sub-units (smaller molecules) and the product is released. Below is a diagram to illustrate clearly:

In this practical, pH will be investigated to see how it affects the rate of enzyme reaction. We will use different levels of pH of acid and alkali to break down hydrogen peroxide, thus concluding which level of pH caused the rate of reaction to increase or decrease by calculating the amount of oxygen released, and justifying the reason for our results.

Aim:

To investigate how and why different levels of pH affect the rate of enzyme reaction, when using a combination of Catalase with Hydrogen Peroxide, and modifying the pH of an acid-alkali solution.

Hypothesis:

I predict that for each of the different levels of pH, there will be different results. pH 7 is the level in which the acid and base substances are neutral, and is the physiological pH of most cells in the human body. For these reasons, I predict that the catalase will react best at pH 7, and the rate of reaction will be highest.

For pH level 7.2 and 6.8, I think that the rate of reaction will increase slightly, as there is not much of a difference from these levels of pH to pH 7. Due to this fact, it is less likely for the enzyme to de-nature.

However, for pH level 7.4 and 6.0, I think that the enzyme will not affect the rate of reaction as the acid and alkali levels are too high and also too low, which will cause the active site to denature, not allowing the lock and key theory to take place.

Apparatus:

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Apparatus:

1 side-arm flask
1 rubber tube
2 measuring cylinders (100cm³)
5 small beakers
50cm³ of Hydrochloric Acid
50cm³ of Sodium Hydroxide
50cm³ of Hydrogen Peroxide (20% vol.)
Screw driver
1 beehive stand
1 water-trap
50cm³ of celery cells
Stopwatch
pH meter
buffer (25cm³)

Variables:

Independent Variables:

pH of solution

Dependent Variables:

Amount of Oxygen released (speed of reaction)

Controlled Variables:

Amount of substrate = H202 (10cm³)
Amount of Catalase (10cm³)
Amount of pH solution (10cm³)

Uncontrolled Variables:

Error of accuracy +- (errors in the pH levels, amount of hydrogen peroxide used, and in the amount of catalase used); air pressure; room temperature. These would have an effect as they could interfere in our results. Room temperature because as temperature is another factor which affects the rate of enzyme reaction, so it may make a difference in the accuracy of the results.

Method:

First, set up the apparatus as shown in the diagram:

Fill the water trap with water, and place a beehive stand inside making sure the water is 5cm above the height of the beehive stand.
Get a measuring cylinder and fill completely with water. Once this is completed, place your hand upon the opening of the measuring cylinder so no water escapes while turning it around and putting it on the beehive stand.
Connect the rubber tube to the side-arm flask and place on the side hole of the beehive stand, and up into the measuring cylinder, so the solution of acid-alkali & catalase + H2O2 can cause a reaction for oxygen to be released.

Once the apparatus has been set up as indicated, follow the instructions below:

Take a beaker and fill it with 10cm³ of Hydrochloric acid using a measuring cylinder.
With a pH meter, measure the pH of the acid. If the pH isn’t as required, then add more acid to decrease, or more alkali (sodium hydroxide) to increase the pH of the solution.
Next, measure 10cm³ of catalase, and pour into the beaker with the solution. Mix, and pour into a new measuring cylinder to fill 10cm³ of the new solution.
Then, pour the mixture of catalase and the acid-alkali solution into the side-arm flask.
Once this is done, clean the measuring cylinder and use it to measure 10cm³ of hydrogen peroxide. Pour into the side-arm flask, quickly close with the bung and start timing for the reaction to start. Write down in a table the amount of oxygen released by counting the bubbles formed inside the measuring cylinder every 30 seconds for 2 minutes.
Use this methodology for all pH levels you have decided to investigate to see which pH affects the rate of enzyme reaction. Please note that each level of pH should be repeated twice in order to achieve more accurate results and calculating an average.

Analysis:

From my results, I can see that the catalase reacted exactly as I predicted. The catalase worked best at pH 7.0, and so the reaction rate was highest. This is because this neutral level of pH is the optimum pH, which is where the enzymes work fastest, causing more oxygen to be released.

The pH levels surrounding the optimum pH, 7.2 and 6.8, did not have as great of an effect as the pH 7, but they still increase the rate of reaction considerably high, as they were near the optimum pH. In addition, even though there is a difference of 0.4 pH from pH 7.2 and 6.8, our results show that the effect of the pH on the catalase reaction is oscillating around the same level: from 60 to 90 seconds, the amount of oxygen released was 22cm³ for pH 7.2, and 21.5cm³ for pH 6.8 – giving a difference of 0.5cm³. All of this proves that the rate of reaction is not necessarily affected by whether there is more or less acidity, but is affected by how far away the pH level is from its optimum pH level (example of pH 7.2 (more basic) and pH 6.8 (more acidic) giving almost same results).

Aside from that, pH’s 7.4 and 6.0 did not give any increase at all in the rate of reaction as the solution was either too acidic or too basic. This is related with how many H+ or OH- ions are present. Ions are charged molecules which tend to pull on other molecules, and if too many molecules are present, then the enzyme will become denatured, causing the active site to change its shape and properties, which prohibits the substrate from binding to it to undergo catalysis.

In addition, my findings indicate that even though the levels of pH were changing from 0.2 to 0.2, it makes such a big difference to the final reaction, proving how important it is for us to maintain our pH levels as neutral as possible for fast digestion. However, this does not mean that all enzymes work best when pH is at a neutral level: some enzymes work even better in acidic or basic environments, depending on the characteristics of the enzyme itself. For example, protease enzymes in animal stomachs work best at pH 1, which is extremely acidic.

Conclusion:

In conclusion, to summarise my findings in my analysis, it can be said that my hypothesis was entirely correct « I predict that for each of the different levels of pH, there will be different results. pH 7 is the level in which the acid and base substances are neutral, and is the physiological pH of most cells in the human body. For these reasons, I predict that the catalase will react best at pH 7, and the rate of reaction will be highest. » The other levels of pH used in this experiment gave results which showed that the rate of reaction increased slightly, or did not increase at all. This is because when the level of pH is not neutral, the active site modifies into another shape, inhibiting the substrate from breaking down into sub-units.

Evaluation:

Overall, throughout our entire practical, we encountered several problems, but all were resolved. Initially, we were having problems obtaining results as when we poured all the solutions and mixtures inside the side arm flask, there were no oxygen bubbles released. We tried repeatedly, and after several tries, we managed to get a set of accurate results. These problems may have occurred due to the loss of oxygen before placing the bung quickly enough onto the side arm flask. Because since the moment all the substances are inserted inside the flask, the reaction has commenced; and from that time to the time the bung is put on, oxygen will have been lost, causing less bubbles to arise in the measuring cylinder.

To take this experiment further, we could look at other factors which affect the rate of enzyme reaction, such as temperature, concentration of enzyme or concentration of substrate. Temperature is known to have around the same affects on the rate of reaction as pH does, with the optimum pH being 37ºC (body temperature). And so, we could compare results between pH and temperature, to see the differences and similarities.

Or, we could develop this practical with pH by focusing on the level of pH that gave us best results: from 6.8 to 7.2. Even though we are aware that 7.0 is the optimum pH for increasing the rate of reaction, we can also see that pH’s 6.8 and 7.2 gave increasing results, and so we could focus on this area by performing the same experiment but looking at pHs 6.8, 6.9, 7.0, 7.1 and 7.2, in order to a greater accuracy in our results, and see the transition from 1 pH level to another.

Bibliography: