Enzyme Kinetics Lab Simulation answers PDF

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Enzyme Kinetics Lab Online Simulation answers...

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Enzyme Kinetics Lab Simulation – Data and Solutions for Enzyme 1 Experiment A: pH of incubation In this experiment, we are going to find the pH optimum of the enzyme by varying the pH of the buffer we use to measure enzyme activity. Click on pH of incubation and set the incubation conditions from pH 1 to pH 14, start the experiment by clicking on use these values. Keep all of the other incubation conditions as they appear on this page but select no inhibitor. Now click perform the incubations.

Are you happy with your result? If needed, you can repeat the experiment with smaller pH intervals near the pH optimum to get a better estimate of the pH optimum. Select perform more experiments to optimize your experiment.

It seems that the optimum pH is somewhere between 3.6 and 6.2. I want to narrow this down. Therefore, I will run the experiment again, varying the pH between 3.5 and 6.5, accepting all the default values for the other parameters.

If you are satisfied with your results, you may click the save these results to print button. You will only get a print out when you close the program. Make a note of the optimum pH for your enzyme for use in future experiments. Based on the table with results, the optimum pH is 4.7.

Experiment B: Incubation time In this experiment, we are going to vary the measuring time at a constant enzyme concentration and a constant substrate concentration so we can choose an optimum assay time. If you choose a really long time, you will see how the enzyme behaves when product has started to accumulate. If you choose too short a time, you will end up getting very small amounts of product formed, so any experimental error will contribute more to your results, which makes them more inaccurate. Click perform more experiments followed by the incubation time button. Set the range from 0 – 60 minutes, start the experiment by clicking on use these values. Enter your optimum pH value obtained from the previous experiment by selecting change pH and entering the correct value. Click the no inhibitor button followed by perform the incubations.

Are you happy with your result? If needed, you can repeat the experiment with a shorter time period to get a better estimate of the optimum assay time. Select perform more experiments to optimize your experiment. As you can see, the rate of product formation starts to decrease after a while. So I will check a shorter time, the aim is to get a straight line. Maybe 15 minutes?

If you are satisfied with your results, you may click the save these results to print button. You will only get a print out when you close the program. Make a note of your optimum assay time for use in further experiments. I’m going to pick 10 minutes just to be on the safe side.

Experiment C: Amount of enzyme In this experiment, you are going to vary the enzyme concentration at a constant substrate concentration. The idea is to find a small of volume of enzyme (you do not want to waste precious material!) that still results in a measurable amount of product. You will see that the rate will increase linearly with enzyme at first, but starts to curve ever so slightly. This indicates that at the higher enzyme concentrations the substrate may be running out too quickly over the assay time we have chosen. Click perform more experiments followed by the amount of enzyme button. Set the range from 0 – 250L of enzyme, start the experiment by clicking on use these values. Enter your optimum pH value and assay time obtained from the previous experiments by selecting change pH and change incubation time and entering the correct values. Click the no inhibitor button followed by perform the incubations.

You may click the save these results to print button. You will only get a print out when you close the program. Make a note of your optimum amount of enzyme for use in further experiments. The rate increases linearly with enzyme at first, but then starts to curve ever so slightly. Around 50 to 100 µL of enzyme should be fine. As these are computer simulations and the enzyme does not cost anything, I am opting for 100 µL.

Experiment D: Concentration of substrate In this experiment, you are going to investigate the effect of changing the substrate concentration whilst keeping the enzyme concentration constant. The experiments will look at initial velocities of your enzyme at varying substrate concentrations. The substrate concentration range should produce a graph where most of the results are on the more linear portion (i.e. the first-order region) of the graph. You may have to try a few different ranges before you find an appropriate one for your enzyme. Click the concentration of substrate button and choose a range of substrate concentrations. Click on the use these values button. Enter your optimum pH value, assay time and amount of enzyme obtained from the previous experiments by selecting change pH, change incubation time and change amount of enzyme and entering the correct values. Click the no inhibitor button followed by perform the incubations.

Are you happy with your result? If needed, you can repeat the experiment with a different substrate concentration range to get more data points in the first order region of the direct plot. Select perform more experiments to optimize your experiment. I do not have enough data points in the first-order region of the plot. I am going to run the experiment again, over a smaller range of concentrations, to focus on the more useful part of the graph. I will use 0 to 75mM.

You may click the save these results to print button. You will only get a print out when you close the program. Make a note of your optimum substrate concentration range for use in further experiments. 0 to 75 mM What is the Km and Vmax of your enzyme looking at the direct plot? Based on this direct plot, Vmax is around 30 µmol product formed per 10 minutes or 3 µmol per minute and Km is around 25 mM.

Experiment E: Concentration of substrate, but with inhibitor present These experiments will show the effects of inhibitors A and B on your enzyme. The experiments will look at initial velocities of your enzyme at varying substrate concentrations in the presence of varying concentrations of inhibitor. Click the concentration of substrate button and choose the range of substrate concentrations determined in the previous experiment. Click on the use these values button. Enter your optimum pH value, assay time and amount of enzyme obtained from the previous experiments by selecting change pH, change incubation time and change amount of enzyme and entering the correct values. Click the inhibitor A button, select an inhibitor concentration range and then click perform the incubations.

I selected the 125 – 1000 mmol/l range for both inhibitors to start with:

Are you happy with your result? The effect of inhibition should be such that there is a measurable effect, but that a measurable amount of product is still produced. If needed, you can repeat the experiment with a different inhibitor concentration range. Select perform more experiments to optimize your experiment. I am not happy with my results for inhibitor B, so I will run the experiment again using 12.5 – 100 mmol/l inhibitor.

You may click the save these results to print button. You will only get a print out when you close the program. How do Km,app and Vmax,app (so Km and Vmax of your enzyme in the presence of inhibitor A) compare to Km and Vmax looking at the direct plot? Based on the direct plot, For inhibitor A: Vmax,app seems to be the same as Vmax, which is around 3 µmol per minute (maybe I should run a new enzyme assay with higher substrate concentrations to check this claim!) and Km,app seems to be different then Km, which is around 25 mM. For inhibitor B: Vmax,app seems to be different then Vmax, which is around 3 µmol per minute and Km,app seems to be the same as Km, which is around 25 mM. What type of inhibitor is inhibitor A according to the direct plot? Based on the direct plot, For inhibitor A: probably a competitive inhibitor For inhibitor B: probably a non-competitive inhibitor → To repeat this process for Inhibitor B, click perform more experiments.

Data analysis in Excel You have now finished your experiments. Time to analyse your data! Click end and print the saved results and end. Your data but NOT the graphs will be printed out. You must now draw Lineweaver-Burk plots using Excel or a similar graphical package and determine the kinetic parameters Km and Vmax for your enzyme and find out what type of inhibitors A and B are. You do not need to submit your answers. A document containing the solutions for enzyme 1 is available. It works similar for the other enzymes. I do advice you to finish the Enzyme Kinetics Lab Simulation as analysing enzyme kinetics data will be present on test 4. See the Help video...