Chapter 1. Case Study on Enzyme Inhibition: A Likely Story

Instructor's Notes

Topic Pre-requisites: Students should have exposure to the topics of Sections 6.1–6.3 in Lehninger POB, 6th ed. In particular, it is important to discuss mechanisms of reversible inhibition, irreversible inhibition, Lineweaver-Burk plots, and how to calculate k_cat (if the optional k_cat question is assigned).

Case Overview

Students will be given enzyme kinetic data; they will use Microsoft EXCEL (or another graphing program) to graph the data. They will then determine kinetic parameters and type of inhibition and correlate this information to descriptions of molecular mechanisms. Note: This case has two versions that the instructor can choose to assign. These two versions differ only by the inclusion of a question in which students are asked to calculate k_cat from experimental data. Instructors should review this question and assign only one version of the case to students.

Students may work individually or in groups to complete this case study. Students are constantly encouraged to refer to their textbook throughout the case, and internet access is permitted, although it is not necessary for the completion of the case. Students are required to iteratively acquire, analyze, and integrate data as they progress through the case and answer assessment questions found throughout the case. All assessments are automatically scored and a tutorial to guide students through using MS EXCEL is provided.

Learning Objectives

This case is intended for remediating or extending student capabilities in these difficult topics:

1. Real-world applications of the study of enzyme kinetics. Students should be able to:
   • describe the distinctions between kinetic mechanisms of enzyme inhibition.
   • use inhibition kinetics to identify inhibitors by kinetic mechanism.
2. Connections between molecular mechanism and enzyme kinetics. Students should be able to draw connections and distinctions between molecular binding mechanisms and kinetic mechanisms of inhibitors.
3. Practice critical thinking skills involving data. Students should be able to:
   • evaluate data from tables.
   • create graphical representations of data.
   • calculate common kinetic parameters (V_max, K_m and k_cat) from data.

Questions designed to show areas of difficulty for students

1. Students often struggle with the practical calculation of k_cat. This issue will be apparent if students incorrectly answer the optional question about k_cat .
2. Students may mischaracterize the description of an irreversible inhibitor because they do not know what a stable Schiff-base linkage is. The case provides a teachable moment to point out that the student should learn to recognize when they do not know a term and remedy the problem.
3. Students often have difficulty identifying a noncompetitive inhibitor when using real data because real data is not ”perfect”; fit-lines from real data are unlikely to intersect perfectly on the x-axis in a Lineweaver-Burk plot.
4. Similar to the issue above, students will often assign mixed inhibition to an uncompetitive inhibitor.
5. The example of a lab technician neglecting to properly label samples is, of course, a very common issue in teaching and academic research laboratories. This oversight was inserted in the case study as a teachable moment to remind students to be sure to properly label samples throughout their manipulations when they carry out their own laboratory work.

Suggested implementation

Below we describe two options for course implementation. The hybrid Online/In-class approach is recommended. Time required for students to complete the online case will vary greatly depending on students’ prior experience using MS EXCEL or a similar program. The case study can be started and stopped, and so it is recommended to give students a window of 2 to 3 days in which to complete the assignment.

1. Share the case study link with your students to work online outside of class. Assign the case study to be due before your next class meeting. Students should be instructed to bring copies of notes, graphs, and calculations to class.
2. Review the online answers before the following class for difficult areas for students (see expected areas of student difficulty above).
3. Lead students in a discussion in pairs, groups, or as a class (depending upon class size and instructor preference) to address unresolved difficulties (~30 minutes in-class time).
4. We recommend that you select questions from the supplied assessment questions to use on exams oras homework assignments to reinforce the difficult concepts covered. Please see the document “Exam Questions for Case 1: Enzyme Inhibition.”

1. Share the case study link with your students to work online, preferably in pairs or groups of three. Assign the case study to be due before your next class meeting.
2. Review the online answers for difficult areas for students (see expected areas of difficulty above).
3. Mention or remediate tough points during a portion of lecture.
4. We recommend that you select questions from the supplied assessment questions to use on exams oras homework assignments to reinforce the difficult concepts covered. Please see the document “Exam Questions for Case 1: Enzyme Inhibition.”

Suggestions for in-class discussions (these questions may also be used in summative assessments—i.e., exams, scored quizzes, etc.):

• What type of kinetic mechanism would be expected for an irreversible inhibitor and why?

• Have students read/view descriptions of inhibitors in the case: ask what specific information in each description is relevant and how it indicates the expected kinetic mechanism.

• How could an investigator distinguish between a noncompetitive inhibitor and an irreversible inhibitor?

• Why does V_max remain unchanged for a competitive inhibitor?
• When calculating k_cat, when should you use E_total and when should you use [E_total] and why?

• Why does the measured Km value go down in the presence of an uncompetitive inhibitor?

You are missing vital information

You may be missing vital information needed to sufficiently explain this incident. You must complete all investigations before proceeding to the final assessment questions.

You are missing vital information

You may be missing vital information needed to sufficiently explain this incident. You must complete all investigations before proceeding to the final assessment questions.

inv1

true

Results: The compound found in the coffee (Compound X) has yet to be identified, but using the small amount extracted, you have discovered that it is an inhibitor of XYZase. Given this revelation, it seems clear that this compound is the most likely murder weapon. Shown below is the data you have gathered. Note: [S] is given in nM; other values represent observed V₀ values given in units of µmol product formed per minute.

inv6

true

Results: By gathering samples from the labs of Drs. Wight, Greene, and Gray, you hope to discover which of these people may have had access to what is now clearly the murder weapon, compound X. Shown below is the data you have gathered. Note: [S] is given in nM; other values represent observed V₀ values given in units of µmol product formed per minute.

inv9

true

Results: Dr. Gray is studying the effects of a transition-state analog. This molecule, which is a fluorinated lactone called F-lac by the company, mimics the geometry of the theoretical transition state in the XYZase reaction mechanism. Dr. Gray has solved crystal structures that have confirmed that F-lac binds noncovalently in the active site as expected.

inv11

true

Results: Dr. Greene is studying a compound that binds noncovalently to an allosteric site on the protein approximately 20Å away from the active site. She has solved crystal structures, which have revealed that this allosteric site is blocked in the absence of substrate; a tryptophan residue blocks the ligand-binding pocket. When the enzyme binds the substrate, a shift in a helix connecting the active site and the allosteric site displaces the Trp sidechain, clearing the pocket and allowing the inhibitor to bind. When the inhibitor is bound, it prevents a nearby loop from moving to associate with the remainder of the active site. The association of this loop with the active site is necessary for catalysis to occur. Binding studies have confirmed predictions made by the crystal structures: in the absence of substrate, the inhibitor binds with a K_d of 10 mM, but in the presence of the substrate, the inhibitor K_d is decreased to 1 nM.

inv13

true

Results: Dr. Wight works on an inhibitor of XYZase that acts by binding to a lysine residue in the enzyme active site, forming a Schiff base. This lysine is critical to function, normally acting as an acid in the enzyme mechanism. Stability studies have confirmed that under physiological conditions, the inhibitor−Schiff base linkage formed in the enzyme active site is extremely stable, with a half-life of more than 24 hours.