Teaching Unit Summary Title: Understanding Enzymes using a Forensic Hook Learning Objectives Learning Goals

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Teaching Unit Summary
Title: Understanding Enzymes using a Forensic Hook
Learning Objectives

Learning Goals

Students will learn binding specificity

Students will understand the role of inhibitors

Students will apply knowledge to novel situations

Learning Outcomes

Students will be able to predict reactions between given enzymes and of family of substrates.

Students will be able to predict the consequences of the addition of an inhibitor.

Students will be able to interpret (quantitative/qualitative) enzyme assay test results.

Students will be able to relate the theoretical understanding to real-life applications.
Target Audience

Introductory biology

Second semester general, organic & biochemistry

Second semester general chemistry

Sophomore level forensic science students
Brief Description of the teachable unit: We will start with a crime scene scenario and use it to introduce enzymes, inhibitors, and enzyme assay tests.

  1. Scenario

Students are presented with a forensic case study:
In 1968 a woman was found strangled to death in a NYC alley. During the autopsy, the medical examiner discovered extensive bruising around the lips and mouth, and several green fibers in her teeth and throat. Microscopical analysis revealed that the fibers were common in both composition (cotton) and color (green). A month later a suspect was apprehended. The investigators discovered a green handkerchief in the suspect’s van. The investigators are now interested in linking the handkerchief with the victim. What might you do to accomplish this?
Students are asked to identify the potential substance (saliva). They are further asked to identify how saliva is different from other body fluids,

  1. As a formative assessment, students are asked to select the component of saliva that is the most unique (salivary enzymes).

  2. The fact that the salivary enzymes == amylase, and that amylase breaks down starch, sugar, and other carbohydrate is introduced if the students did not do so during discussion.

  3. The affinity of amylase for starch (and not other compounds) is posited as a means for detecting the presence of saliva on the object from the case study.

Content: Use a colorimetric reaction to visualize the presence of an enzyme in a physiological fluid.

The Phadebas test is widely used in forensic science to identify physiological fluids that contain the enzyme alpha-amylase. To perform the test, a forensic analyst needs both a positive and negative control.
Student Activity & Assessment

Can anyone explain what a negative control is?

Make it.
Can anyone explain what a positive control is?
I need a volunteer.
The last test tube contains residues from the handkerchief and is referred to as the questioned sample.
The reaction works by combining sample with a few millimeters of water, and a single Phadebase tablet (tablets can be purchased from Phadebas.com). The tablet contains starch microspheres covalently bonded to a blue dye. The microsphere-blue dye complex is insoluble in water. In the presence of alpha-amylase (from saliva), enzymatic activity cleaves the starch microsphere from the blue dye, releasing the dye into solution, and turning the entire volume a dark blue color. In the absence of amylase, the Phadebas tablet remains as an insoluble precipitate that accumulates in the bottom of the test tube, leaving a clear water column above (centrifuging the vials will help to improve the clarity of the negative control).

If the positive and negative controls behave as expected, then the unknown or questioned sample can be interpreted by comparison to the knowns.

Can the students accurately predict what results would be expected for the positive and negative controls? Did the handkerchief presumptively contain residues of alpha-amylase (and therefore saliva)?

  1. Enzyme specificity

Context: Introduction to enzymes
Active Learning: On board and/or as handouts, students will have a model for enzyme activity. Students will be asked to describe what is going on in the diagrams, with call-outs from the class. As students present important aspects using chemical terms, the appropriate definitions in biochemistry will be given.

Once a simplistic lock-and-key understanding has been developed, the more accurate idea of induced fit will be introduced.

Additional Example: Alcohol dehydrogenase will be presented as an example. It catalyzes the oxidation of ethanol to acetaldehyde, the first step in clearing EtOH from the bloodstream. The acetaldehyde causes hangovers. It also oxidizes other small alcohols such as methanol, turning it into formaldehyde.

Formative assessment: Free response question

  1. Inhibition

Context: A student-centered activity to exhibit inhibition using starch blockers
Hook and Active Learning Exercise

You are a scientist at a pharmaceutical company and you are tasked with coming up with a pill that will mimic the Atkin’s diet (but, still allowing dieters to consume carbohydrates). Ideally, you would like to use something that is naturally occurring so that you can by-pass FDA regulations!

What do you need to know to do your job?

Instructions: Give the students 10 minutes to discuss in small groups. Elicit answers from each group and record them on the whiteboard.
Minimum expectations for discussion:

1. α - amylase is an enzyme that cleaves or hydrolyses α-linked polysaccharides such as starch and glycogen. In other words, α-amylase is involved with the catalysis of starch into simpler sugars (enzyme has a salivary and pancreatic origin).
2. What is something in nature that inhibits amylase and therefore allows carbohydrates to pass through the human body without being absorbed? Does it exist in nature. Yes!
Proteinaceous α - amylase inhibitors are widely distributed in seeds of cereal crops and some grain legumes. The inhibitor is assumed to be responsible for biochemical defense, especially against insects. Kidney beams (Phaseolus vulgaris) contain an inhibitor (phaseolamin) of insect and mammal α - amylase from (but not plant α - amylase).
pH level for salivary (optimal at ~5.5-5.6).
Temperature (enzyme-catalyzed reaction rates increase with temperature to a maximum and then decrease due to thermal degradation).

Marini, I. Biochemistry and Molecular Biology Education, Vol. 33, No. 2, pp. 112-116, 2005.

3. Dietary supplements termed ‘starch-blockers’ are “used” to control weight. They are based on the proteins that are concentrated from “seeds” or “beans” such as the kidney bean, and known to contain high levels of the α - amylase. One example is the inhibitor phaseolamin, which has the potential to hinder digestion of complex carbohydrates, thereby promoting weight loss. Mosca, M. Analytica Chimica Acta, 617 (2008) 192–195.
Caveat: α - amylase inhibitors are marketed for weight control under the generic name “starch blockers”, but results are ambiguous (studies show a drop in blood glucose, but the following also suggests lower efficacy). Synopsis: using a one-day calorie-balanced technique and a high starch meal, research attempted to measure the excretion of fecal calories after consuming a placebo or starch blocker tablet (where expected fecal calorie excretion should decrease by 400kcal if the tablet had an impact over a placebo). Results: No change. Why? The authors believe that the pancreas likely creates much more α - amylase than necessary, consuming 3 tablets is still insufficient to inhibit the enzymatic activity for digestion! Bo-Linn, G., et al. The New England Journal of Medicine, Vol. 307, No. 23, 1982, 1414-1416.

Over 1 million starch blocker tablets were consumed daily in the US in the first part of 1982. Random fact (ref. not provided).
Optimal activity of the inhibitor is at a pH of 5.5 and temperature of 37 deg. Stoichiometry of inhibition shows 1:1 complex of α - amylase and inhibitor. The phaseolamin-amylase complex is dissociated at low pH values, but not dissociated at low temperatures of high pH. Inhibits hog pancreatic alpha-amylase in a noncompetitive manner. (Taken nearly verbatim from Marshall, J. and Lauda, C. The Journal of Biological Chemistry, Vol. 250, No. 20, pp. 8030-8037).
Since the catalytic site of the a-amylase remains accessible to substrate after complexation (the inhibitory effect is thought to be mediated by a long range conformational change). However, the molecular determinants for the selective recognition of amylases have not been elucidated yet. Indeed, to discriminate between plant and animal enzymes is no minor achievement because a-amylases of both origins are homologous and their catalytic sites share similar structural and mechanistic features. (Copied verbatim from: Moreno, J. et al. Journal of Chemical Education, pp350-351, vol. 71, no. 4, 1994).

  1. Teach inhibitors with model – The action of an enzyme

Active Learning:

Ask students: Which of the following molecules can interfere with the action of an enzyme?

  1. A molecule that binds to the active site of the enzyme and blocks the entrance for a substrate.

  2. A molecule that binds to another site of the enzyme but changes the structure of the active site.

  3. A molecule that binds to another site of the enzyme and does not affect the active site.

  4. A molecule that binds to enzyme-substrate complex and slows down the conversion of substrate to product.

  5. A molecule that binds to the product after it is released from the enzyme, and prevents the product from binding to any other proteins, including the enzyme.

Active Learning

Ask students to halt the rate of reaction

What is an inhibitor for an enzyme?

  • An inhibitor is a molecule that binds to an enzyme and causes decreased enzyme activity.

  • Inhibition can be either reversible or irreversible.

  • Inhibitors bind to enzymes with some specificity. They are not to be confused with denaturants such as urea and guanidine.

  • Enzyme inhibitors can be harmful or beneficial. Examples: Diisopropyl fluorophosphate, or DFP, is a neurotoxin that inhibits acetylcholinesterase. Synthetic enzyme inhibitors are also widely used in clinical medicine.

  1. Real-life example- Dopamine -b-hydroxylase (DBH) inhibitors

Context: An application on enzyme inhibition in therapeutic and disease treatment

The students will be exposed to the structures of both dopamine (DA) and epinephrine to identify them

Hook: Dopamine function is converting Dopamine (DA) to norepinephrine( NE). DBH has been shown to be associated with decision making and addictive behaviors such as alcohol and smoking, attention deficit hyperactivity disorder, and also with neurological diseases such as Schizophrenia and Alzheimer’s

Active Learning:

Dopamine is catecholamine, a member of phenethylamine class of compounds. Competitive DBH is reversibly inhibited by l-2H-Phthalazine hydrazone (hydralazine; HYD), 2-1H-pyridinone hydrazone (2-hydrazinopyridine; HP), 2-quinoline-carboxylic acid (QCA), l-isoquinolinecarboxylic acid (IQCA), 2,2'-bi-lH-imidazole (2,2'-biimidazole; BI).

Student activity:

What type of reaction is conversion of oxygen to water?

Based on your answer

  1. What kind of reaction is the transformation of DA to NE?

  2. What is the role of ascorbic acid in this mechanism?

  3. Given the structure of DA can you suggest a competitive inhibitor for DBH.

Assessment questions:

What compounds would you expect to be DBH inhibitors?

The role of DA in in the body and the imbalance in it levels in the body of human influences all populations so it a diverse in all races, sexes, and ages.

Students will be formatively assessed through

  1. Clicker questions of varying structures of inhibitors. To find out which ones are expected to be inhibitors

  2. Take home problem for literature search to find other diseases that may be caused by DA depletion or imbalance. DA being a neurotransmitter which tissues in the body are expected to contain significant amounts of the neurotransmitter DA.

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