I am working on an enzyme kinetics experiment. I want to compare the enzyme acti

Question

I am working on an enzyme kinetics experiment. I want to compare the enzyme activity and activation energies for the catalytic decomposition of hydrogen peroxide by catalase with and withouth inhibitors. It has gotten very complicated lately, because at first I though increasing temperature would increase enzyme activity linearly. However, enzymes do not behave that way as substrate concentration leads to a maximum velocity given by the Michaelis-Menten equation. However, I'm working with TEMPERATURE, and all I have read on enzyme kinetics leads me to substrate concentration.

My project consists on many parts that lead to, in the end, calculating the Activation energies for the reaction with and without inhibitor. These parts are:

1. Determine the substrate concentration that behaves in a linear way.
What I though is that I don't want to difficult my project with the Michaelis-Menten equation, so I though doing the reaction in the school lab changing substrate concentration to see reaction rate (v), with constant enzyme concentration, repeting this the with the different inhibitors concentrations. Plotting the results I would get the Michaelis-Menten, but my idea was to look in the graph up until which substrate concentration the reaction is linear. That way, I set a constant substrate concentration for the next experiments, so that when calculating the value of k k=[E][S]

2. Enzyme activity v/s Temperature and Inhibitors.
With the substrate and enzyme concentrations defined, it should be possible to test its activity in terms of temperature. (the ...s in the table correspond to the different reaction rates [v=c/t] that I would get in the lab) (Ignore the blank spaces I didn't know how to do the table lol)

3. Calculate the value of k (rate law) for the different reactions (TROUBLE BEGINS)

Calculating the value of k is necessary to calculate the activation energy later. THIS IS WHERE I START HAVING TROUBLE. As I stated before, k would be k=[E][S]. This is valid for the reaction with no inhibitor, BUT IS IT VALID ALSO FOR THE ONES WITH INHIBITOR??????? OR WOULD IT BE k=[E][S][I]. I also want to know if is it valid to do what i do in step one, because otherwise I must change everything.

4. Calculating the activation energies for the reactions (this is doable if part 3 is ok, otherwise it must change because k is on of the variables in the Arrenhius equation for activation energy.

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EDIT: Answering to: "the values of reaction velocity and substrate/enzyme concentrations used initially are required to answer and calculate the desired entities. Please provide the information to answer" --> I understand, but I don't know these values yet, since I need to have this method / procedure approved so that I can proceed with the experiment. When I try to understand it myself, I use replace them using simple numbers representing hypotethical values, but I don't know if that really works, eg.

[S] (hydrogen peroxide): 2 ml
[E] (catalase): 5 grams

The part where I get more confused is in determining the values of k, since I don't know if I should include the concentration of the inhibitor in the formula (k=[E][S][I]) or not, or if there is another way to calculate it. I need k so that I can determine the Activiation Energy as a cuantitative parameter to compare the reactions. Thank you for your time!

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I WOULD APPREACIATE SO MUCH YOUR HELP ORIENTATING ME IF IM GOOD TO GO, OR IF I MUST CHANGE THE METHOD. THANKS!! Greetings from Patagonia, Chile!

Inhibitor - Tem pe ra tu re - concentration 15°C 30°C 45°C 60°C 75°C 0 ... ... ... ... ... 1 ... ... ... ... ... 2 ... ... ... ... ... 3 ... ... ... ... ...

Explanation / Answer

First at all. In you work with temperature you must know that temperature affects the enzyme structure. If the structure is affected the active site will not work as it should and then the enzyme efficiency will be get shorted. To calculate this factors i recomend you to do a test with these next steps.

First we need to know what buffer really works with the enzyme, and with a known volume we can determinate the concentration of the enzyme, for example 2 grams of enzyme in 10mL of buffer. With that the enzyme should work properly.

The next step is calculate the reaction velocity of the enzyme. With a Michaelis-Menten model we can calculate this to know at what substrate concentration the enzyme works better, enzyme saturation, etc. Also i recommend you to work with shorter ranges of temperature, like 15, 18, 21, etc, until you reach the temperature where the enzyme cannot work efficiently. And to determine the k constant you can out inhibitor concentration. It is ok.

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