Chapter 1. “A likely story” - Enzyme Inhibition Case Study

1.1 Introduction Part 1

Stryer 7E

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A Likely Story

By Justin Hines, Lafayette College and Marcy Osgood, University of New Mexico

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Instructor Notes for “A Likely Story”, Enzyme Inhibition Case Study

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. The online assignment is made for students to work through individually or in groups and to answer the questions individually online, referring to their text. All assessments are automatically scored and a tutorial to guide students through using MS EXCEL is provided.

Topic Pre-requisites: Students should have exposure to the topics of sections 8.3-8.5 of Berg Biochemistry, 8^th ed. In particular, it is important to discuss mechanisms of reversible inhibition, irreversible inhibition, Lineweaver-Burk plots, and how to calculate k_cat.

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 identify 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 question about k_cat.

2) Students may mischaracterize the description of an irreversible inhibitor because they do not know what a highly 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) 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 in using MS EXCEL or a similar program. The case study can be started and stopped, so it is recommended to give students a window of 2-3 days in which to complete the assignment.

Hybrid (Online/In-class): (recommended approach; ~30 minutes of class-time expected)

1) Share the case study link with your students to work online outside of class and assign it 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) It’s recommended after using the case to select from the supplied assessment questions to use on exams or homework assignments to reinforce the difficult concepts covered. Please see the document “Exam Questions Case 1- Enzyme Inhibition”

Online only approach: (minimal in-class time required)

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.

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?
Students could be directed to brainstorm in groups or as a whole class.

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.
Could be easily used as a think-pair-share exercise.

How could an investigator distinguish between a noncompetitive inhibitor and an irreversible inhibitor?
Could be used when students have difficulty with questions 8 and 9 from the online activity.

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?
Could be used when students have difficulty with the k_cat question from the online activity.

Why does the measured K_M 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.

This activity has already been completed, however feel free to review the information contained within.

I am finished gathering information for this investigation and feel I am able to fully explain the reason for this man’s death in terms of a biochemical issue. I can fully justify and completely explain my reasoning based on the evidence I have gathered.

Figure 1.1

“I’m telling you I didn’t do it!” cried Mrs. Bleu. “He just fell on my knife!” The detective smirked, turning away to hold back a chuckle.

“I don’t know why,” she continued, “he ju—, he… he just sort of turned to me gasping and fell forward... I was just making dinner, chopping broccoli...” Mrs. Bleu trailed off and looked longingly at the floor of the interview room. They had been there for hours. “He was just standing at the end of the kitchen drinking his coffee and looking through the mail when it happened, but it was an accident. Why would I kill my husband?”

“We don’t know why,” the detective said calmly, “we’re hoping you’ll tell us. Your husband was found on the kitchen floor in a pool of blood...his blood. He had a knife-wound, a wound from a kitchen knife that was lying on the floor just feet away covered in his blood. His blood…and your fingerprints! Moreover, you told the 911 operator that you stabbed him—”

“Accidentally!” she gasped.

Figure 1.2

“Ok…ok, you admitted accidentally stabbing him…but do you really expect us to believe that he just fell onto your knife. Do you know how ridiculous that sounds?”

“I don’t care if it sounds ridiculous! It’s what happened!” she said, exasperated. “Look…I need to know what happened to my husband. One minute he was fine, and the next he was gasping! He had a heart attack I think, er--, I don’ t know, but he was MY husband. Why won’t you tell me?”

The detective looked annoyed. “We haven’t heard back yet from the coroner Mrs. Bleu…but this is an active homicide investigation. We will share that information with you when we get it. Believe me, when we get confirmation of the cause of death, we will be talking again….soon.”

He stepped out. He had heard some good ones, but “he fell on my knife”? Really?? At that moment, the coroner stepped off the elevator and into the empty hallway….she was out of breath and appeared to be sweating.

“Frank! Frank, listen! I just finished the autopsy. It missed!”

“What? What missed? Was it homicide?”

“No! I mean, maybe. Er—I don’t know for sure yet, we need the Tox. results.”

“Toxicology results? …for a stabbing? And what do you mean it missed?”

“That’s just it, the knife! It missed the brachial artery! It was only muscle damage, mostly venous bleeding…seeping blood…comes out slow. It was halfway coagulated before he died and wasn’t even that deep.”

“What? Will you just calm down and start talking in plain English! What are you trying to say?”

“It was a flesh wound…plenty of blood, but not enough to kill a person. Without arterial damage it would take a long time to bleed out from this wound. But there is more to it…this guy died shortly after he was stabbed, but it wasn’t from the knife, that much is for sure!”

The detective cursed under his breath… it was going to be a long day. “Okay, so the guy didn’t die from the knife wound, but he is dead, so from what?!!”

1.2 Introduction Part 2

Figure 1.3

Two weeks have passed and you have been brought in to help investigate this case. You have heard the interrogation tapes and been briefed by both the lead detective and the coroner. The coroner has ruled the cause of death of Dr. John Bleu a homicide, but by poisoning, rather than because of the knife-wound inflicted by Mrs. Bleu, presumably now by accident. Small quantities of an unknown substance, hereafter referred to as Compound X, were discovered in the decaf-coffee Dr. Bleu was drinking when he died. Dr. Bleu had brought the coffee home with him from work as he did everyday according to co-workers and his wife. His wife is still under suspicion, but it has not escaped the notice of the investigators that Dr. Bleu leads a large research group studying potentially lethal enzyme inhibitors for the drug company Hinesbiopharma.

Figure 1.4

The company is working with the U.S. government in a counter-terrorism effort to study the effects of chemical weapons so that potential treatments can be created in advance. Three scientists who Dr. Bleu supervised are running independent projects studying different inhibitors toward the same enzyme, referred to in company documents only as XYZase. XYZase is critical to human metabolism, and so development of chemical inhibitors of this enzyme by terrorist groups is viewed as a real and present threat by counter-terrorism experts. The three scientists are Dr. Greene, Dr. Gray, and Dr. Wight. According the company policies, only Dr. Bleu and the single scientist assigned to each project have access to the highly valuable but extremely lethal compounds each one is studying.

To complete the case, you may choose which action you would like to complete next. You will be given enzyme kinetics data, and you will need to use Microsoft EXCEL (or another graphing program) to graph the data in order to determine the kinetic parameters and the type of inhibition. Tutorials to guide students through using MS EXCEL are provided in the Instructors Resources folder in LaunchPad.

1.3 Investigations

Recommended Initial Investigation

Run kinetics experiments on XYZase without inhibitors and with Compound X.

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.

[S]	V₀ (No inhibitor)	V₀ (Murder Weapon - Cmpd. X)
50	0.14	0.040
75	0.19	0.050
150	0.32	0.080
400	0.47	0.14

Table

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Question 1.1

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Correct.

Incorrect.

true

Question 1.2

9OJ4ThJGCuoyMmqxemH5mYy6wnhaOU+CGlOFBAmhCwBBWcp4Col2A99ygtQiiUvqngfBN540oUhaS2RJdo/lz87RWvNk0Syv4YcvFe9iK5fU8QYoyYFuhB5sVUuULibhx/I4vMMRiwiGqaWo

Correct.

Hint: Refer to pg. 228-229 and Figure 8.12 of Berg Biochemistry, 8^th ed.; K_M can be determined quickly and accurately using a Lineweaver-Burk plot.

Incorrect.

true

Question 1.3

Sample k_cat calculation:

In an enzyme assay, you determine the K_M for your enzyme to be 0.50 mM. You measure the maximum rate of the reaction to be 0.050 mmol/second. Calculate k_cat for this reaction if the above experiment was conducted with a 1 mL reaction volume at an enzyme concentration of 100 nM.

k_cat = V_max/E_total

[E] = 100 nM

E_total = [E] x volume

E_total = (100 nM)(1mL)

E_total = (1 x 10^-7 M/L)(1 x 10^-3 L)

E_total = 1 x 10^-10 mol

V_max = 0.05 mmol/second

k_cat = V_max/E_total

k_cat = (0.05 mmol/second) / (1 x 10^-10 mol)

k_cat = (5 x 10-5 mol/second) / (1 x 10^-10 mol)

k_cat = 500,000 s^-1

Note: In this problem knowing the value of K_M was irrelevant…this is true because the value of V_max was given. If in another problem we were instead given the initial velocity (V₀) at some concentration of substrate, then we could use the Michaelis-Menten equation, and the value of K_M, to first calculate V_max!

Also Note: When V₀ is given in units of molarity per unit time, then k_cat = V_max/[E_t] where [E_t] is the concentration of the enzyme in the assay; however, when V_max is given in mol per unit time, then k_cat = V_max/E_t, where E_t is the number of moles of E_t in the assay.

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

Correct.

Hint: Refer to pg. 230 and Equation 31 of Berg Biochemistry, 8^th ed. (Note: k_cat is referred to as k₂ in Equation 31). Be certain that you use correct units in your calculations: k_cat should be in units of s^-1 and all other units must cancel out in the calculation. Note: when V_o is given in units of M/min., then k_cat = V_max/[E_t] where [E_t] is the concentration of the enzyme in the assay; however, when V_max is given in mol/min, then k_cat = V_max/E_t, where E_t is the number of moles of E_t in the assay. Try your calculation again.

Incorrect.

Given that Dr. Bleu appears to have been killed by an inhibitor to XYZase, you now have sufficient cause to search the labs of Drs. Greene, Gray, and Wight so that you can recover and test samples of the compounds they are each working on developing. This discovery has opened up a new investigation option! The new option is:

true

Search the labs and run kinetics experiments on suspicious samples.

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.

[S]	V₀ (Sample 1)	V₀ (Sample 2)	V₀ (Sample 3)
50	0.080	0.067	0.055
75	0.11	0.080	0.063
150	0.19	0.14	0.086
400	0.36	0.24	0.10

Table

true

Question 1.4

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

Correct.

Try again.

Incorrect.

true

Question 1.5

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Correct.

Incorrect.

You have incorrectly matched one or more samples. Please reexamine your data and review pgs. 234-236 of Berg Biochemistry, 8^th ed. and try again

You notice, however, that the samples you collected from the labs are now labeled 1, 2 and 3. You ask your lab assistants which samples correspond to which labs, but they mixed up the samples (somebody didn’t label the tubes when running the assays!). This is a disaster! Now, in addition to determining which sample (if any) matches the murder weapon, you are going to have to testify in court as a biochemist to convince a jury that the guilty sample (1, 2, or 3) belongs to the guilty person (Drs. Greene, Wight, or Gray) based on what you know about enzyme kinetics! Are you up to the challenge? Who killed Dr. Bleu?

The good news is that you may now go back to Hinesbiopharma and interview the three scientists about the molecular mechanism of the inhibitor each is working with. This has opened three new investigation options which are:

Interview Dr. Greene.

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.

true

Question 1.6

lb6Ipt19jnX/rQ380VWagXBrL8mVjJ8CfPyIxW+HaOS5XUj4+QySCTlDkhVimg2lh8croHAz8rgy9K7F/CDgvrrLWzI4ZDtFaRtBo29zqEvwHYap2N/1Bip97d7I4XNXns8ktI8PrH79FloE892JoIzH6JzgJidCz8cMvMZDMHVrfLZxNZCfT9hWbGwZYRXB5uonDQRPDDosYp5z5Y+NQjs3Z5tvRVnpN0FKkzSw+v9L1SgpRJ/6IyGVOsYtNG+zSicEKF0rdIpNwxtMTd4gQMQJHj5JOmOS1lxLceum7LVGS6DV+YJ23N1REJY7QGIs2tbMuui+PA0GTrPsw0o9ooBWGDHUR9V42IKOFPSnBOmhNahbGb1GnLBS778EeApG+2arVxcqHU6rte8NcHliI5dmVx2HRv9XylpidqVfDmbN83QlpjnJcphCldqhwhvi4OhQEZXYfGlEpf4XWeMTK0RKr2lJ4oo5bAlO3D6E3XSbP9xyJY5GW6pb8hx/aLtTG21rKoW+V06M2jtI01uZzWGWRqyrhuYR9d9vRfnRX9uOZG7aWzMF7G9ObwlI8gHj3uiLr0Vx6AI8pt+lbJOhh0jkzPKM4dILz5tbZphbTLcgPdubJiqNDtt+IKAMuvm8DzIZvqcXurx/OOds5Hf3+E8wmtMxxMZLW2OZ+w7M86Vb2PUobJuO4D8aZyfJYY7AnQmQJ9N8sw49r4BQ7L7VUoeJ1dUnamJSned4FrlwX+toGmk8Pf/7qVzwyLBHJAnINZAHd+lCROWczm+Tc0IBtg==

Correct.

Try again.

Incorrect.

Interview Dr. Gray.

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. This result triggers the following assessment question:

true

Question 1.7

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

Correct.

Try again.

Incorrect.

Interview Dr. Wight.

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.

true

Question 1.8

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

Correct.

Try again.

Incorrect.

true

Question 1.9

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

Correct.

Try again.

Incorrect.

1.4 Assessment

Final Assessment

You have now completed the investigations and can go on to answer the final assessment questions:

inv121

true

Question 1.10

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

Try Again.

Correct.

Incorrect.

true

inv109

true

Question 1.11

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

Correct.

Incorrect.

true

inv110

true

Question 1.12

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

Correct.

Incorrect.

true

inv111

true

Question 1.13

GBX6nP9gurYkTsjRiaWw3PVp4OuXenCpntdr2KK3HmU++KIZYNSFRB3eJgLQtxJF4je/4RKE0dnLX0/qRghkE/SsHnA+c3eZ6+7fCXlONIUQdHwk3Z7HTV7ruC2dBz+mM7FZb800lOK54OurhYu3XfIXR5cTpYBHXivY0CWdMp4WVdBm+I3hgrv9L76ofp1PAsUzIXuAbfKHfDOcvXePX3PCH2fA5PKDyhZz8y2aJI3T6hjlXTDwhXoh9LRYJDIkK9bEch8Pw8LIzt4N/9w1DHXFf76H6vthQYeToi/07ygI0vh5P/6epA==

Correct.

Incorrect.

true

Activity results are being submitted...

1.5 Case Summary

Congratulations on completing this Case Study! The following Case Summary gives a full explanation of the murder of Dr. John Bleu.

Case Summary

Dr. Bleu was indeed murdered by one of his colleagues at Hinesbiopharma, and not by poor Mrs. Bleu, who was wrongfully accused. Dr. Wight was working on an irreversible inhibitor that forms a stable Schiff base linkage in the active site of XYZase. This irreversible inhibitor makes a covalent bond with the enzyme in the active site, thereby converting an enzyme permanently into an inactive form. Adding this inhibitor to the enzyme assay would be equivalent to removing some of the enzyme from the assay. As such, the apparent K_M value measured with the inhibitor present does not differ significantly from the uninhibited enzyme, but the V_max value observed does decrease significantly (as it would if less enzyme were used). The result is that an irreversible inhibitor has the same apparent effects on K_M and V_max as a noncompetitive inhibitor, but no scientist in the case was working on a noncompetitive inhibitor based on the interviews, leaving only Dr. Wight’s irreversible inhibitor as the possible explanation for the kinetics observed with the murder weapon. It was Dr. Wight, in the kitchen, with the irreversible inhibitor.