Updated: Oct 23, 2019
Investigation on the effect of different substrate concentrations, which in this case are the manipulated hydrogen
peroxide concentrations on the rate of enzyme activity of catalase in liver on the decomposition of the hydrogen peroxide.
Enzymes are biological catalysts that catalase in biochemical reactions in living cells. In enzyme reactions, a substrate binds to the active site on the enzyme, forming an enzyme-substrate complex where the enzyme breaks down the bonds in the substrate, creating a whole new bond and formation which will lead to the product and during and after the whole process the enzyme will remain unchanged, thus being able to react in other chemical reactions again.
In this case, the enzyme is catalase which is produced by the liver to break down hydrogen peroxide. This is a very common end product of a metabolism, but very toxic as well if accumulated in the body.
2H2O2 (l) ==> O2 (g) + 2H20 (l)
In this experiment, we will put liver pieces in distilled water and different hydrogen peroxide concentrated solutions for mixed with 2 drops of soap for 60s and measure the foam obtaining gas on top of the liver to measure the rate of enzyme activity. The reason to why the soap is used in this practical, it to be able to measure the gas made from the reaction, which can otherwise not be seen without the foam obtaining it. The manipulation of different substrate concentrations show us a variation of enzymes rate of reaction to see where the enzyme will have full reaction capacity and efficiency as well as in which cases the enzyme will react the least.
As mentioned above, enzymes are proteins and biochemical catalysts that are involved in chemical reactions and metabolism in living cells and organisms. They are globular proteins, with specific shapes and are usually specific for a type of reaction. (1) The part of the enzyme that acts as a catalyst is called the active site and the rest of the enzyme's role is to maintain its specific shape. A reaction occurs from random movements of the enzymes and substrates, and when a substrate/substrates attach to the active site of the enzyme, the reaction is triggered and the result is a product with new bonds and chemical structure.
Some examples of Enzymes are (2) :
ATPase, breaks down ATP ⇒ ADP, producing energy
Glycogen synthase catalyses the formation of glycosidic bonds between glucose molecules
Lactase, breaks down lactose ⇒ glucose and galactose
Catalase, breaks down hydrogen peroxide ⇒ water and oxygen
The reason to why enzymes are very important biological catalysts, is because most reactions inside the cell requires a very high temperature to get going, which would inevitably destroy the cell. However, when enzymes are used a lot lower activation energy for the reaction to start. How enzymes lower the activation energy, is by holding the molecules packet together increasing the chance of a reaction to occur and thus lowering the activation energy.
There are 2 hypothetic models on the enzyme reaction one being the “Lock and key model” and “The induced fit hypothesis”. (3) The lock and key model as the name implies sees the substrate as the key and the enzyme's active site as the lock being specifically modified and complementary to one another. It further explains that when in random movements the complementary substrate to an enzyme collide, the substrate attached to the active site creating an enzyme-substrate complex and producing a product with the enzyme remaining unchanged and ready to react again.
The induced fit model is a more recent and more accepted model with wide evidence and acceptance around the world. The hypothesis explains how the shape of the active site of the enzyme is not in fact complementary to the substrate and when it is near a substrate is changes its shape to be complementary to the substrate. (4) When the composition is specifically correct, an enzyme substrate complex is formed producing a product with the enzyme remaining unchanged, thus able to react again.
Factors affecting the rate of enzyme activity are the competitive and noncompetitive inhibitors, temperature, pH and substrate concentration.
Enzymes have a specific optimum rate of reaction at a certain temperature, pH, substrate concentration and inhibitors presence. In this case the enzyme catalase has an optimum temperature at 37 degrees Celsius and if the temperature would get too high the shape of the active site will be permanently altered, leading to the enzyme no longer working and getting denatured.
The enzyme's optimum pH is at 7 and if it would be too low or high the enzyme will generally lose its complete ability to be active in reactions. When comes to rate of reaction if substrate concentrations, at a certain level the rate of reaction will reach a plateau and increase in substrate concentration will thus no longer affect or increase the rate of reaction considering all the enzyme molecules would be saturated with substrates.
Last but not least the presence of inhibitors affect the rate of reaction of which depends on if the inhibitors would be competitive or noncompetitive. A competitive inhibitor, as the name suggests the active site of the enzyme would be uninvited to attach to any other substrate thus no longer reacting with any other substrate. (5) However, increase in substrate concentration will lead to the competitive inhibitors to instead compete with one another and thus many enzymes will be free on the active site to react with substrates. Non competitive inhibitors inhibit the enzyme by attaching to the allosteric site of the enzyme, which is no the active site leading to the active site changing shape permanently and no longer being able to react and in this case increase in substrate concentration will not let the enzyme activity reach the usual plateau as it would if it would be a competitive inhibitor.
End product inhibition
The non competitive inhibitor can act as a end product to control the reaction and the amount of substrate produced e.g (5) Threonine reacting with enzymes to produce Isoleucine and in this case the product will act as a non competitive inhibitor attaching to the allosteric site of the first enzyme in the reaction chain Threonine Deaminase to stop more production of Isoleucine which otherwise could be toxic if accumulated for too long.
(6) There is also something called an activator, which if present increases the velocity of the enzymatic reaction. Enzyme activators can appear sometimes in order to eliminate the inhibitors and to help the enzymes to get back to reacting. Many enzymes are activated by inorganic molecules, mainly Ca2+ and the enzyme even has a specific binding site for this particular molecules, which changes the conformation of the enzyme and increases the rate of reaction.
Activators can also bind to the substrates when the substrate is trying to bind to the active site, thus increasing the rate of reaction in this way also. An example would be the magnesium ions that interact with ATP or other negatively charged nucleotides, thus decreasing their overall charge making the binding of them to the active site more efficient and higher velocity in the enzymatic reaction.
The hypothesis of this practical is that the rate of reaction will indeed increase with the increase of hydrogen peroxide concentrations, only if the other crucial factors of enzyme activity are kept constant, such as pH, enzyme concentration, inhibition and temperature. We believe that the rate of reaction in the distilled water will be zero, considering there will be no substrates inside of the liquid and that the rate of reaction will reach a limit called the plateau in which case the enzyme will be a limiting factor at one point in the highest concentration of hydrogen peroxide, which is in our case 3.0% concentration. When the enzyme activity has reached the plateau it can no longer be manipulated by adding more substrate concentration in the solution because all the enzyme molecules will be saturated at any given time, thus being unable to react with other substrates.
Material and apparatus
The conclusion is that the results were the same as expected and just like the hypothesis, no reaction occurred at distilled water and as the substrate concentration increased, so did the rate of reaction. However we thought that at 3.0% hydrogen peroxide we would reach the plateau, which was not the case and it would have been a better data if we would have continued with higher substrate concentrations to see where the plateau would be and analyze upon that. The graph 1 and 2’s linear aggression and high R2 value show a positive correlation between the rate of reaction and hydrogen peroxide concentration, however it cannot be the final answer that this will always be valid and proportional considering no plateau was reached.
At 0.0% substrate concentration, being the distilled water, as said above no reaction occurred proving that the hydrogen peroxide must be the sole factor to the enzyme rate of reaction to occur. As you can see from the data table the biggest volume change occurred between 0.5% and 1.0% hydrogen peroxide concentration with the change of 11.2 cm3 volume and the biggest rate of reaction change was between 2.0% and 3.0% hydrogen peroxide concentration, the plateau was never reached.
Something to pinpoint from the collected raw-data, is that two volumes came off very wrong and far from the other of its trials, thus us believing they were errors and mistakes that should have been redone. However we had short of liver cubes as well as time to do so, thus the conclusion is that those mistakes must have been systematic errors of which impurities/enzyme residues must have been existing in the measuring cylinders, causing extra foam to build up from the start before even putting any liver in. The two wrong volume values in the table are from the substrate concentration 2.0% in trial 4 with the volume 39.0 cm3 as well as substrate concentration 3.0% in trial 2 with the volume 47.0 cm3 . These values definitely affected our standard deviation, giving it a high value as well as became a limitation in our average reaction rate, so a great improvement for next time would probably be to wash the measuring cylinders more carefully and under less stress (which we were in considering the low time we had making us have to hurry up with the cleaning). Or, we could use 5 different measuring cylinders for all the different substrate concentration, thus eliminating any further systematic errors.
Another improvement would be to do less, but more precise steps in measuring 4 ml of the wanted substrate concentration, because even though human errors are unnecessary to mention by now in any lab report, a lot of uncertainty was built up from the first step with the pipette and measuring with sight 4 ml in the 10 ml measuring cylinder as well as with sight measure the foam created in the 100 ml measuring cylinder.
Specially in the section of which we had to by sight measure the gas produced with the help of the foam was to a high extent problematic considering bits of big bubbles were up and down the surface of the measuring cylinder with a complete uneven surface, (7) so maybe a different technique in measuring the gas produced would have left us with less random errors, such as a circular enzyme-soaked disc in the substrate solution would have given a better, more flat surface on the foam to measure by sight. We would have also measured the pressure change as well in the sealing and instead would have been left with a less uncertainty from the digital value or put a syringe cap in the solution and measured the displacement of it with the increase in volume of the gas.
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