I HAVE BIO QUESTIONS I NEED HELP ON!!!

Question

1) What is the purpose of using I₂KI when determining amylase activity?

 

2) How do enzymes speed up reactions? Be brief but specific in your description.

 

3) How does temperature and pH affect the 3-D confirmation and activity of enzymes? Which factor has the biggest effect? Address each case and use your analysis of the data provided to support your answer for full credit.

  

4) Protein denaturation can occur in various ways but they all alter the 3-D structure of the protein rendering it unable to take part in reactions. Is it possible for a protein to re-nature and become functional again or is denaturation permanent for all proteins? Briefly explain and support your answer.

Answer 1

1.) Amylase is an enzyme that is present in saliva and helps to break down starch. In order to determine amylase activity, Iodine is used. The reason iodine is used is because it combines with starch to form a violet/purple color. In the presence of amylase (depending on how active the enzyme is), starch gets digested and hence there is only little starch left to react with Iodine, leading to a pale purple color. Thus Iodine is used to test the activity of Amylase enzyme.

2.) Enzymes speed up reaction by decreasing the activation energy needed for a reaction to happen and thus acts as catalyst to increase the rate of a reaction.

3.) Both Temperature and pH affect the activity of an enzyme. If the temperature is high, the activity is faster while at low temperatures, the activity of the enzyme is slow. However, if there are extremes of temperature or pH, the enzyme gets denatured since the folding of the enzyme protein is affected.

Normally, all proteins (as enzymes are proteins) have a primary, secondary, tertiary and a quatenary structure and it refers to the folding of the proteins. When there is too high a temperature or pH, for example, the folding of the enzyme protein is affected and so it loses its functional activity.

The structure and function of the enzyme is affected more by temperature than pH, though both need to be at optimum levels for the enzyme to be active.

4.) Even if proteins are denatured after protein purification, they can be refolded to its proper conformation by dialysis, using dilute buffers with low Urea and low pH. For instance, when you purify recombinant proteins from prokaryotes like E. coli, sometimes the purified protein gets denatured due to misfolding. This protein can be dialysed after elution in dialysis buffer, saline, buffers containing urea, low pH etc that can help protein to refold to its native structure and thereby getting its function.

Answer 2

1. We use I₂KI for understanding Amylase activity as I₂KI changes colors in the presence of starch. If starch is present, there will be a bluish/ purple color present. This is significant as when amylase enzymes are in the presence of starch, the Amylase will begin to break down the starch into maltose. Whenever there is no longer any starch present, the indicator will show yellow. Thus, the change from blue to yellow allows one to asses the time and rate of change in amylase activity in a solution. This demonstrating that we can use I₂KI as an indicator for not only presence of starch, but also to study amylase activity
2. Enzymes speed up reactions as they act as reusable catalysts that lowers the activation energy needed to perform said reaction. Some processes without catalysts/ enzymes rarely would happen for proper bodily function to occur so with enzymes speed this along by inserting the substrate onto the active site where it speeds up the chemical process in either the hydrolysis or condensation reaction mechanism ( just depends on the specific enzyme. Amylase breaks down a larger molecule starch to maltose so it is a hydrolysis reaction).
3. Enzymes are awesome and complex, but they are extremely susceptible to sudden changes in pH and heat. This being the case as enzymes are just complex folds of amino acids with different charges and different number of hydrogen bonds. So things like pH can come in to play as it changes the confirmation of the enzyme due to the level of Hydrogen present (pH= -log₁₀ [H⁺] ). Some Enzymes, like the ones in your stomach, require a low pH/ acidic (1-6.9) so they can function while others prefer a more basic conditions in order to operate. Hemoglobin heavily depends on pH changes as this explains whether hemoglobin is picking up Oxygen and unloading Carbon Dioxide in the lungs or if it is releasing Oxygen and picking up Carbon Dioxide in the muscle tissues. Enzymes are also susceptible to heat in that heat also changes there confirmation, but in a different way. Heat manipulates the hydrogen bonds that the individual amino acids make and causes the enzymes to become unstable. This instability is referred to as enzyme denaturation. And in regards to which factor has the biggest effect, I would say both can be argued as the most important as a enzyme that may have the right pH but is too hot/ too cold = enzyme denaturation and vice versa where an enzyme is at the right temperature but the wrong pH = enzyme denaturation. Denatured Enzymes have a different 3D confirmation and therefore the substrate does not bind to the activation site and the chemical reaction does not get carried out.
4. In regards to denaturation, typically whenever a protein denatures, it tends to be permanent. That being the case, it is not always permanent for all enzymes. Some enzymes can handle slight denaturation in regards to confrontational changes; however, these enzymes are designed to change confirmation depending upon function and usually don't denature entirely ( Again, Hemoglobin is a good example for this).

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