Enzymes are complex protein molecules that reduce the activation energy of biochemical reactions. They catalyse and accelerate the reactions to turn the substrate into the needed product. For a long time the “lock and key” theory was used to explain how enzymes work. It suggested that each enzyme has an active site that only the specific substrate would fit into (like a key fits into a lock). However, the theory was modernised and now the induced fit model is recognised mostly. It states that an enzyme can change its shape to bound with the substrate. Enzyme activity can be affected by such factors as temperature, pH, concentration of inhibitors, and concentration of substrate. Enzymes denature at high temperature or very acidic or basic solutions. Enzymes are involved in a number of biochemical processes, including the synthesis of starch in a potato tuber. Glucose that is produced during photosynthesis is stored in the form of starch. Phosphorylase is an enzyme which catalyzes this reaction. The aim of this experiment was to observe the activity of this enzyme and find out which polysaccharide will be synthesised into starch. The results will show if this is actually the enzyme found in the potato extract and will prove that this enzyme is specific and will catalyze only the specific reaction with the specific substrate.
Method and Materials
The potato was grinded in a pestle and mortar. Fine sand and water was added to ease the process. After becoming more or less homogenised, the liquid was moved to a centrifuge tube. It was centrifuged for five minutes at 4000 rpm in a bench centrifuge. A centrifuge role is to separate the mixture by rotating the sample at a high speed. By this, the more dense parts of the mixture will stay at the bottom of the container and the less dense would be at the top. The supernatant was them removed and transferred into a separate test tube. At this point, the supernatant contained the potato enzymes that are involved in the starch synthesis, and not the already formed starch or the remainings of sand. This is due to the fact that the enzyme solution is less dense than the starch or the sand. The extract was then tested to check if any starch using iodine on a white tile. If the result was positive, the mixture was centrifuged again. The extract was stored in an ice bath.
Glucose, glucose-1-phosphate, maltose and water were placed in four different test tubes, 3 ml each. The test tubes were marked – G for glucose, GP for glucose-1-phosphate, \m for maltose and C for control (water). The control sample was important because obviously no reaction was expected there, so this would allow to fully realise if there were any changes on colour in other samples. 12 drops of iodine was added to a spotting tile. 1 ml of the potato extract supernatant was added to the glucose test tube. It was mixed well and the clock was started at that time. Immediately, a few drops of the mixture was added to the first iodine spot. Every one minute drops of the mixture was added to the next iodine tile. After 12 minutes the result was recorded and the spotting tile cleaned. The experiment was repeated with glucose-1-phosphate, maltose and water.
numbers resemble minutes (0 – zero time)
Positive reaction of the mixture to the iodine test indicated that starch was produced after the addition of the enzyme. When starch is present, iodine turns from yellow to dark brown or black, the darker the substance the more starch is present. The results showed no visible color change after the test for glucose or maltose after they were exposed to the supernatant, so no starch was produced from those carbohydrates. The results with the control substance, water, was similar to that of maltose and glucose, which proves that the results were indeed negative. Glucose-1-phosphate was the only carbohydrate that showed a positive result, so it was the only one from which the starch was produced. This happened due to the fact that the supernatant contained the enzyme phosphorylase. This means that in the potato starch production glucose-1-phosphate is the polysaccharide on which phosphorylase acts. This is the specific substrate for phosphorylase and this enzyme only works when added to this polysaccharide.
Starch phosphorylase is responsible for phosphorolytic degradation of starch in plants. It catalyzes the transfer of glucosyl units from glucose-1-phosphate to the end of shortened starch chains to build them up with the release of phosphate. This is a reversible reaction.
image source: http://www1.biologie.uni-hamburg.de/b-online/e19/19j.htm Accessed 10/11/2018
Enzymes are very specific to ensure that they will maximize their impact on the reaction and turn as much substrate into product as possible. Their active site is build specifically to achieve the closest and the most tight bond with the substrate to be effective. This practical proved that the active site of phosphorylase is build that way so it won’t bond with other molecules, such as glucose or maltose, apart from glucose-1-phosphate. This ensures the effectiveness and the highest possible rate of reaction.
Another aspect of the results that can be discussed is the rate of reaction.The color change after performing the iodine test was not seen immediately. After 4 minutes of reacting the test results were positive, and the amount of starch increased with time. The rate of the reaction can be shown on an indicative graph:
With time, the rate of reaction slows down, because less substrate is available to produce more product.
Overall, the results met the predictions made. The enzyme found in the potato extract was phosphorylase and it was proven to be specific to act on glucose-1-phosphate.