6.4: Part II, Inhibition of catechol oxidation

Pre-Lab Assignment for Part II

Pre-lab assignment for Part II

Files to support this experiment are here (click). 

Please submit this assignment prior to the second scheduled lab period for this module. Submit it along with the general pre-lab assignment.

Although different varieties of apples (and other fruits) brown at different rates, they all turn brown eventually. To preserve fruit, a chef may choose a specific apple variety and add something (an inhibitor) to slow down or inhibit the rate of the browning reaction. Inhibitors are often listed as preservatives in recipes or on labels of fruits sold in cans or jars. 

1. Suggest at least two possible inhibitors for this system. Begin by looking for the preservatives used in recipes or on labels. Also, consider the results obtained in the first cycle of this experiment. Prepare to discuss your choice of inhibitors with your instructors.
2. Select a substrate concentration that falls in the (approximately) linear portion of your Michaelis-Menton plot, and write in your lab notebook the volume of substrate in the reaction mixture that resulted in this concentration.
3. The optimal concentration of inhibitor must be determined experimentally; prepare to record the concentration of the stock inhibitor solution in your lab notebook. Create and complete a table similar to the one below (an example of an electronic version of a table is here (click)). The volume of substrate should be the same in each trial and should correspond to the volume you chose above (in pre-lab question 2). Try using concentrations of inhibitor in each mixture that vary by factors of 10. Get approval from your instructor before you actually use these for experiments. Depending on the selection of inhibitor, you may need to run the reactions without buffer.

   Trial	Volume of Enzyme	Volume of Substrate	Volume of Inhibitor	Volume of Buffer or Water
   1	0.50 mL
   2	0.50 mL
   3	0.50 mL

4. Each trial will be carried out in a cuvette. What is the best order in which to combine the enzyme, substrate, inhibitor, and buffer (if needed)? Explain your reasoning.

Experiment Part IIA: What can inhibit the browning of fruit?

In this phase of the experiment, you will answer the following two questions:

• What substance do you choose to inhibit the rate of the browning reaction?
• What concentration of inhibitor will slow but not completely shut down the reaction?

Follow the same general protocol as before to measure the rate of oxidation of catechol using the volumes of enzyme, substrate, inhibitor, and buffer or water determined in pre-lab question 3 above. 

Thinking About the Data: Part IIA

1. Determine the initial rate of the reaction for each mixture and tabulate the results in a spreadsheet that may be shared with team members (or other class members). 

2. Based on the results, decide which concentration of inhibitor slows the enzyme-catalyzed reaction without stopping the reaction completely.

3. Based on your results, how would you prepare apples for a fruit salad?

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Experiment Part IIB: What type of inhibition is at play?

Inhibitors generally work according to one of the following mechanisms:

Equilibrium binding of an inhibitor, \(I\), to the enzyme, \(E\), to form an adduct, \(EI\), as in the following equation:
\[ \mathrm{EI} \rightleftharpoons \mathrm{E}+\mathrm{I} \quad K_1=\frac{[\mathrm{E}][\mathrm{I}]}{[\mathrm{EI}]} \label{i1}\]

Equilibrium binding of an inhibitor, \(I\), to the substrate-enzyme complex, \(ES\), to form an adduct, \(ESI\), as in the following equation:
\[\mathrm{ESI} \rightleftharpoons \mathrm{ES}+\mathrm{I} \quad K_2=\frac{[\mathrm{ES}][\mathrm{I}]}{[\mathrm{ESI}]} \label{i2}\]

Competitive inhibition occurs when a substance similar in structure to the substrate competes with the substrate for the active site on the enzyme, thus blocking the enzyme from forming a complex with the substrate. Uncompetitive inhibition occurs when a substance binds to the enzyme only after it is bound to the substrate. Non-competitive inhibition occurs when a substance binds to the enzyme away from the active site, but the enzyme may still bind the substrate. However, the enzyme’s changed structure means that the enzyme is no longer able to catalyze the browning reaction. In general, inhibitors are selected because they may structurally resemble the reaction substrate (in this case, either molecular oxygen or catechol).

4. Considering the equations \ref{i1} and \ref{i2} above, explain in words how a substance, acting as an inhibitor, can slow the rate of the browning reaction. 

5. Which equilibrium above corresponds to 

   1. competitive inhibition? 

   2. uncompetitive inhibition? 

   3. Justify your answer.

6. Explain why non-competitive inhibition involves both equilibrium expressions.

7. If a substance slows an enzyme reaction via competitive inhibition, the remains the same and is larger; if the reaction slows via uncompetitive inhibition, both and are smaller; and if the reaction slows via non-competitive inhibition, remains the same but is smaller. Examine your graph of initial rate versus substrate concentration. In your notebook draw three similar graphs (initial rate versus substrate concentration) showing the effect of

   1. competitive inhibition,

   2. uncompetitive inhibition, and

   3. non-competitive inhibition.

8. Complete three tables similar to the one below. It is only necessary to carry out six total trials (six different concentrations of substrate) rather than the eight used in Experiment Part I. In the first table the volume of inhibitor is that determined in #2 above (Thinking About the Data Part IIA). In the second table the volume of inhibitor is about half of that in the first table. In the third table the volume of inhibitor is about double that in the first table. Depending on your selected inhibitor, either water or buffer will be added to bring the reaction mixtures to a volume of 2.5 ml. Get approval from your instructor or teaching assistant before continuing.

Trial	Volume of Enzyme	Volume of Substrate	Volume of Inhibitor	Volume of Water or Buffer
1	0.50 mL
…	0.50 mL
6	0.50 mL
7	0.50 mL	0 mL
8	0 mL	2.00 mL

This cycle requires the most data collection. It is hoped that students, having done the experiment four times, will be efficient.

Experiment IIB: 

What is the most likely inhibition mechanism for your selected inhibitor? Carry out the same protocol as previously, but using the solution mixtures from the three tables in #8 above. 

Thinking About the Data: Part IIB

9. Determine the initial rate of the reaction for each mixture and tabulate in a spreadsheet.  

10. Construct graphs of initial rate versus substrate concentration for each concentration of inhibitor. Compare to your predicted curves for each type of inhibition.

11. Incorporation of the inhibitor processes into the Michaelis-Menten mechanism yields the following equation:
    \[v_0=\frac{v_{\text {max }}\left[\mathrm{S}_0\right]}{\left(1+\frac{[\mathrm{I}]}{K_1}\right) K_m+\left(1+\frac{[\mathrm{I}]}{K_2}\right)\left[\mathrm{S}_0\right]}\]
    where \([I]\) is the concentration of inhibitor. Invert this equation to find the inhibitor version of the Lineweaver-Burk equation. 

12. Show that \(1+\dfrac{[I]}{K_1} >1\)  and  \(1+\dfrac{[I]}{K_2} =1\)  for competitive inhibition. Go on to determine the slope and intercept of a Lineweaver-Burk plot for competitive inhibition.

13. Show that \(1+\dfrac{[I]}{K_1} =1\)  and \(1+\dfrac{[I]}{K_2} >1\)  for uncompetitive inhibition. Go on to determine the slope and intercept of a Lineweaver-Burk plot for competitive inhibition.

14. For non-competitive inhibition, \(1+\dfrac{[I]}{K_1} =1\)  and  \(1+\dfrac{[I]}{K_2} =1\) . What are the slope and intercept of the resulting Lineweaver-Burk plot?

15. Construct and overlay the Lineweaver-Burk plots for the three inhibitor concentrations and tabulate the slopes and intercepts.

16. Decide the mechanism of inhibition for your inhibitor and explain your decision.

 

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Adapted from Cole, Renée S., Marc Muniz, Erica Harvey, Robert Sweeney, and Sally Hunnicutt. “How Should Apples Be Prepared for a Fruit Salad? A Guided Inquiry Physical Chemistry Experiment.” Journal of Chemical Education 97, no. 12 (December 8, 2020): 4475–81. https://doi.org/10.1021/acs.jchemed.0c00517.