Plan: The effect of the end product, phosphate, on the enzyme phosphatase

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The effect of the end product, phosphate, on the enzyme phosphatase

1. Plan

Phosphatase enzymes release phosphates from a variety of substrates for synthesis of nucleotides, phospholipids, etc. They are found in both plant and animal tissues and can be classified as acid or alkaline depending on their optimum pH. In this experiment an acid phosphatase from potato was used.

Hypothesis

Since phosphate is a product of phosphatase activity, it may act as an end product inhibitor of the phosphatase enzyme, therefore slowing down the enzyme-catalyzed reaction.

Background knowledge

Enzymes are biological catalysts which speed up the metabolic reaction inside the cells. Enzymes are proteins and therefore can function because of their specific 3D shape. The active site is part of the molecule which allows the substrate to enter and form enzyme-substrate complex. If the shape of the active site changed, the substrate will not fit in, therefore the enzyme cannot function properly.

The activity of enzyme is not only affected by temperature, pH, but also by enzyme inhibitors. These are the substances which can reduce the activity of enzymes. There are two types of enzyme inhibitors which refer to competitive and non-competitive inhibitor.

Competitive inhibitor is a molecule which has a similar shape to the substrate, competing with the substrate for the active site of the enzyme and the inhibition can be either reversible or irreversible. The reversible inhibition means the inhibitors form a loose association with the enzyme and may become detached to the enzyme later, thus enabling the enzymes to function again. Irreversible inhibition means that the inhibitors combine permanently with the enzymes, making it impossible for substrates to react. Non-competitive inhibitor is a molecule which does not enter the active site of the enzyme but changes its shape by entering the other part of the enzyme, leaving the enzyme unable to work.

However, Cells always contain many natural inhibitors which control the rate of metabolism and known as the reversible non-competitive inhibitor. Supposing a metabolic pathway becomes overactive and too much end products are produced, the end products will inhibit one of the enzymes in the metabolic pathway and therefore lower the whole series of reactions. By this means, the further formation of end products is slowed down. This is called an end product inhibition.

e.g

           enzyme a                enzyme b                   enzyme c

Substrate A          Substrate B             substrate C          substrate D

In this experiment, sodium phosphate can work as a natural inhibitor as mentioned above. The enzyme phosphatase in the potatoes breaks phenolphthalein phosphate (PPP) phenolphthalein and phosphate:

                          phosphatase

Phenolphthalein phosphate                 phenolphthalein + phosphate

The reaction will be carried out in sodium phosphate solutions with different concentrations. As a non-competitive inhibitor to the phosphatase. sodium phosphate may enter the phosphatase other than it’s active site. When the sodium phosphate combines with the phosphatase, the substrate PPP can no longer fit in since the shape of the active site has changed, therefore there will be less product produced. Less phenolphthalein being produced means there will be less color change in the solution when sodium carbonate is added to the solution. Therefore, the more active the phosphatase, the more intense the pink color produced. Since the sodium carbonate is an alkaline, it denatures the acid enzyme whose optimum pH is acid, therefore stops the reaction.

Preliminary Trial

Summary of method

I used an artificial substrate Phenolphthalein Phosphate (PPP) which produces phenolphthalein and phosphate when reacts with phosphotase in a buffer solution. The reaction was carried out in 5 test tubes with different concentrations of sodium phosphate added to the buffer in each beaker and incubated in the same water bath for 20 minutes. Upon addition of sodium carbonate, the reaction was stopped and any phenolphthalein produced was detected by the color change (from colorless to pink). The quantitative intensity of color was then measured using the colorimeter.

Range of measurement

The range of different concentrations of sodium phosphate used is 0M, 0.05M, 0.10M, 0.20M and 0.30M., with 0M used as a control. This range of the concentrations ensures that during the experiment, not all the enzymes will be inhibited but the given conditions are different enough to produce different results as an indication of the degree of inhibition. The trial results are plotted on the graph below:

From the graph I can tell that the last result is an anomaly, which doesn’t fit the pattern of the graph. I think it was because the quality of the enzyme added to the last test tube. Since the enzyme solution had been stored in the fridge for 2 days after being centrifuged during the trial, some precipitates may have dissolved in the liquid again. So the last portion of enzyme suspension I added to the test tube may contain some impurities since. That  I should use the enzyme suspension immediately after it has being made.

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But overall the result is reasonable, and accords with my hypothesis. So I will use the same range of concentrations of sodium phosphate next time.

Repeats

In the preliminary experiment, I only did the experiment once, the results are not very reliable. So I decide to do three repeats next time. Less than three repeats will not give me enough data to plot error bars, but more than three may be too much during the limited time period(since the enzyme can not maintain their activity very long after been extracted).

To make it most efficient, I will ...

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This is a very well written report that demonstrates a good understanding of scientific processes and methods. 1. The background knowledge section is well written and concise but should include references. 2. The variables section is well thought through. 3. The method section should be simplified and restructured. 4. The data is presented well. 5. The conclusion is detailed but again needs references. 6. The evaluation is the strongest section in the report and shows good statistical knowledge. ***** (5 stars)