Case Study Section 2 – Enzymes Adapted from “Case Study on Enzyme Inhibition: A
ID: 202988 • Letter: C
Question
Case Study Section 2 – Enzymes
Adapted from “Case Study on Enzyme Inhibition: A Likely Story” (Lehninger Principles of Biochemistry)
The case:
“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.
“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?!!”
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.
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 to 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 will need to investigate. 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.
Run kinetics experiments on XYZase without inhibitors and with Compound X.
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] (nM)
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
Question 1:
From the data you have gathered, does it appear that XYZase follows Michaelis-Menten kinetics?
A. Yes
B. No
Question 2:
Based on the data gathered, what type of enzyme inhibition best describes the action of Compound X?
A. Competitive
B. Noncompetitive
C. Mixed
D. Uncompetative
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] (nM)
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
Question 3:
Based on the information you have gathered, match the samples collected to the observed type of enzyme inhibition.
Sample s Inhibitor types
Sample 1 Competative
Sample 2 Noncompetative
Sample 3 Mixed
Uncompetative
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 the following three new investigation options:
Interview Dr. Gray
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.
Question 4:
What type of inhibitor is being studied by Dr. Gray?
A. Competative
B. Noncompetative
C. Mixed
D. Uncompetative
E. Irreversible
Interview Dr. Greene
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 Kd of 10 mM, but in the presence of the substrate, the inhibitor Kd is decreased to 1 nM.
Question 5:
What type of inhibitor is being studied by Dr. Greene?
A. Competative
B. Noncompetative
C. Mixed
D. Uncompetative
E. Irreversible
Interview Dr. Wight
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 inhibitorSchiff base linkage formed in the enzyme active site is extremely stable, with a half-life of more than 24 hours.
Question 6:
What type of inhibitor is being studied by Dr. Wight?
A. Competative
B. Noncompetative
C. Mixed
D. Uncompetative
E. Irreversible
If you were to analyze Dr. Wright’s inhibitor using enzyme kinetics, you would find that the numbers look very much like those that you would get if you had a certain type of reversible inhibitor. Irreversible inhibitors render the enzyme molecules they bind completely unable to perform catalysis. The effect is similar to decreasing the concentration of the enzyme in the enzyme assay.
Question 7:
How does an irreversible inhibitor affect Km and Vmax? What type of reversible inhibitor does an irreversible inhibitor resemble in this manner?
A. Vmax unchanged, Km increased; looks like a competitive inhibitor
B. Vmax decreased, Km decreased; looks like an uncompetitive inhibitor
C. Vmax decreased, Km increased; looks like a mixed inhibitor
D. Vmax decreased, Km unchanged; looks like a noncompetitive inhibitor
Based on the information you have gathered answer the following questions.
Question 8:
Match the samples to their respective scientists.
Sample s Scientists
Sample 1 Gray
Sample 2 Greene
Sample 3 Wight
Question 9:
Based on their kinetic mechanisms of inhibition, which sample matches compound X?
A. Sample 1
B. Sample 2
C. Sample 3
Question 10:
Based on your answers to the previous questions, who is the murderer?
A. Dr. Gray
B. Dr. Greene
C. Dr. Wight
[S] (nM)
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
Explanation / Answer
1. Yes , the sample follows MM kinetics. Because , the reaction rate increases as substrate concentration increases.
2. Compound X must be a competitive inhibitor. Because the Vmax doesnot change in the competitive inhibition. So, the rate of raection will be normal but only the products will be changed.
3.
4.
5.
6.
7.
8. Sample 1 - wight
sample 2- Greene
sample 3 - Gray
9. Sample 1 is compound X.
10. Dr. GRay is murderer.
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