Non-competitive inhibitors attach themselves to the enzyme at a site other than the active site. However, in doing so they alter the shape of the active site in such a way that the substrate cannot fit into it and the enzyme cannot function. As the substrate and inhibitor are not competing for the same site, an increase in substrate concentration does not diminish the effect of the inhibitor.
Digestive enzymes include amylases, which digest starch, lipases, which digest fats, and proteases, which digest protein. Other enzymes play a part in the conversion of food energy into ATP², the manufacture of all the molecular components of the body, the replication of DNA³ when a cell divides, the production of hormones and the control of substances in and out of cells.
²ATP is an abbreviation for adenosine triphosphate, a nucleotide molecule found in all cells. It can yield large amounts of energy, and is used to drive the thousands of biological processes needed to sustain life, growth, movement and reproduction. Although ATP serves as the energy current for all cells, its quantity is limited. About three ounces of ATP are stored in the body at any one time, and this provides only enough energy to sustain strenuous activity, such as spriting, for 5 to 8 seconds. Therefore, ATP must be constantly synthesized to provide a continuous supply of energy.
³DNA is an abbreviation for deoxyribonucleic acid, a complex molecule that contains, in chemically coded form, the information needed for a cell to make proteins.
Enzymes consist of two parts: the protein portion and the cofactor portion. The protein portion is determined by the genetic code. Either minerals (such as calcium, magnesium or zinc), vitamins, or both in some instances, make up the cofactor portion of the complete enzyme.
Every cell contains a part called the mitochondria which acts like an engine. The enzyme that makes this engine work is called coenzyme Q10. The cells do not use the nutrients consumed in the diet for their immediate supply of energy. Instead, they create and use the energy-rich compound, ATP.
Ubiquinone, another name for coenzyme Q10, was derived from the word ‘ubiquitous’ because the enzyme was found in all cells of the body. It is a naturally occurring molecule, and is the cofactor in the electron transport chain; the biochemical pathway from which ATP and most of the body’s energy is derived.
Enzymes have many medical and industrial uses from washing powders, drug production and as research tools in molecular biology. They can be extracted from bacteria and moulds, and genetic engineering now makes it possible to tailor an enzyme for a specific purpose.
Most chemical reactions require an initial input of energy, called activation energy, to enable them to occur. Enzymes reduce the amount activation energy needed and thus allow reactions to take place faster and at lower temperatures than would otherwise be inaccessible. This can be seen in these graphs:
Lipase is an organic enzyme responsible for breaking down fats into fatty acids and glycerol. It is produced by the pancreas and requires a slightly alkaline environment to work efficiently. The intestinal wall then absorbs the fatty acids and glycerol, and they are converted into a readily available source of energy. Since it is found in the body, we can assume that lipase works best at body temperature, around 37ºC.
Preliminary Work: To prepare for this investigation, our class performed some preliminary experiments to familiarise ourselves with lipase and its properties. We found out that when mixed with sodium carbonate solution (Na²CO³), Lipase slightly slows down. We also found that the weaker sodium carbonate solution is, the faster the lipase works. We also discovered that bile salts increase the rate of reaction. In the body, bile salts assist in the breakdown and absorption of fats into the body.
Predictions: I predict that the higher the concentration of Lipase, the faster the reaction will occur. I believe this is because there will be more particles of reactant in the solution which makes collision more likely. This is linked to the Collision Theory, which explains how chemical reactions take place and why rates of reaction alter.
Method: Equipment needed:
Test tube racks
Test tubes
Thermometers
Beakers
Source of hot water
Electronically controlled water bath or simply a normal water bath
10cm³ Syringes
1cm³ Syringes
Marker pens
Stopwatch accurate to a hundredth of a second
Pipettes
Sodium carbonate solution*
Phenolphthalein**
Full fat milk (3.6% fat)***
Bile salts
Lipase solution
Goggles
*Sodium carbonate is used to neutralize acids.
**Phenolphthalein is an acid-based indicator that is clear below pH8 and red above pH9.6.
***Milk is usually around pH6.8, slightly lower that neutral (pH7).
Step-by-Step Procedure: 1. Start by taking any safety precautions that are needed, bearing in mind that you are working in a laboratory with many alkaline and acidic solutions. Id Est: Put on goggles, tie back long hair and store all bags under tables.
2. Assemble a test tube rack with six test tubes in it. Use the marker pens to number the test tubes from 0 through to 5. This will indicate the percentage of Lipase that will be added later.
3. In all six test tubes add:
3cm³ full fat milk (3.6% fat)
3cm³ sodium carbonate solution
1cm³ bile salts
6 drops of phenolphthalein using a pipette
4. Prepare a water bath at 40ºC and place two 100ml beakers in the water. Each beaker needs to contain 50ml of water. Wait until the beakers and there contents reach 40ºCand progress onto the next step.
5. In test tube 5, using the 1cm³ syringe, add 1cm³ of lipase solution and start the timer immediately. Place the test tube in the first beaker of water in the water bath. Record the time it takes to turn red in seconds and put the results into a suitable table. Your results should be to two decimal places.
6. In test tube 4 repeat the procedure but add a solution made up of 0.8cm³ lipase and 0.2cm³ of water from the second beaker of water from the water bath. In test tube 3 repeat the procedure but add a solution made up of 0.6cm³ lipase and 0.4cm³ of water. In test tube 2 repeat the procedure but add a solution made up of 0.4cm³ lipase and 0.6cm³ of water. In test tube 1 repeat the procedure but add a solution made up of 0.2cm³ lipase and 0.8cm³ of water. In test tube 0 repeat the procedure but add 1cm³ of water from the second beaker of 40ºC water. If the reaction in test tube 0 isn’t complete within about half an hour, simply record that in your results table.
7. Repeat this experiment (steps 1 though to 6) twice more to ensure accuracy. Make sure to use clean apparatus and equipment each time. When you have completed your three results tables, make another one containing the average results from the three. Your averages should be calculated to three decimal places.
There are many accuracy issues to take into consideration while performing this experiment. It is paramount that all measurements are as accurate as can be and that the water bath it maintained at 40ºC. This would be easier with an electronically controlled water bath but using a thermometer and a source of hot water, you should be able to control the temperature of the water. I plan to repeat the experiment three times to ensure that I will be able to see a pattern if one occurs and with three different results, I should be able to spot an anomalous result, if one should arise.
There are also many safety issues to take into consideration while performing this experiment. Standard lab safety rules should be followed and extra things need to be taken into account; id est: take the whole test tube rack to the solution that you require instead of a single test tube. Be careful when dealing with any kind of corrosive liquid or irritant and always have a clean & clear working space.
Variables: Variables are the things that can be changed in an experiment. For it to be a fair test, only one variable may be changed at any one time. These are variables that could affect the outcome of the experiment:
Amount of phenolphthalein (propose to keep the same)
Amount of milk (propose to keep the same)
Fat content of milk (propose to keep the same)
Amount of bile salts (propose to keep the same)
Amount of sodium carbonate solution (propose to keep the same)
Concentration of sodium carbonate solution (propose to keep the same)
Lipase solution to water ratio (this is the variable that I plan to change and investigate)
Temperature of water bath (propose to keep the same)
Analysing Evidence and Concluding: From my results, I can observe that as I predicted, the highest concentration of lipase solution broke down the lipids in the milk and thus neutralised the sodium carbonate solution in shortest amount of time. Therefore I can conclude that the higher the concentration of lipase, the faster the rate of reaction.
My results show no sign of the presence of a limiting factor, meaning that there was still enough substrate for the enzyme to act upon with the maximum concentration I tested.
As the concentration decreases so does the rate of reaction, as I predicted, for it takes more time for the fewer numbers of lipase molecules to breakdown the lipids and there are less collisions. The lipase did not completely stop reacting in the experiment apart from in the experiment with 0% concentration because enzymes are not affected by the reactions they catalyse, and can be used over and over again. I think that because my experiment was performed at 40ºC, the enzyme worked much better than it would have at, for example room temperature.
Evaluating Evidence: I feel that the results I have collected were pretty accurate but because this is such a complex experiment with many different variables and accuracy issues, I concede that my results may not be as accurate as they could have been. I am sure that if I repeated the results more times my results would become more accurate. Also, in a school laboratory with thirty other people in, it is extremely hard to attain perfect accuracy. From the graph I produced, I don’t think there were any anomalous results. There were a few results that were not entirely similar to the other two, but were not more than a few seconds different. I believe the evidence I assembled is strong enough to support a conclusion. On the graph we can see a correlation between the concentration of the lipase solution and rate of reaction. This means that as the lipase concentration is increased, the rate of reaction falls.
To improve this experiment, I think it would be useful to have access to data-loggers and other electronically controlled equipment rather than doing things manually. This is because whenever there is human involvement; there are chances for human err. Using an electronically controlled water bath would reduce the margin of error and would maintain a constant temperature to perform the experiment in. Another limitation with this particular experiment is that the volume of liquid in each pipette-drop can vary slightly, so the results may not be truly accurate. I think that the primary limitation with this experiment was the judgement by eye of when the solution had turned colour, as this is not going to be completely accurate and the same for each experiment. I considered conducting the experiment in another way, but as no gas was given off that I could collect, I could not measure this.
To extend the enquiry, I would devise more experiments to discover the exact pH that lipase works best at and following on from that, the precise optimum temperature that it works at. In a further experiment I could increase the ratio of lipase to water until I can tell that all the substrate is occupying an enzyme molecule, and the addition of more lipase would have no effect. I could also investigate the affect that lipase has on different types of milk. For example, skimmed milk, semi-skimmed milk and condensed milk, which all have different fat contents. From this you can build up a perfect picture of how lipase behaves in a variety of different situations.
Bibliography:
1. The Hutchinson Encyclopaedia 2001 CD-Rom
2. Schott’s Original Miscellany – Ben Schott
3. Rennin Investigation – Tamsin Chipperfield
4. Coenzyme Q10 – LifeLink
5. Edexcel Modular Science – Coordination Group Publications
