Why is Patrick Paralyzed? A case study by: Maureen Knabb Department of Biology West Chester University

Directions Read through the following case study (you may work with ONE partner) Answer the Multiple choice questions AND question prompts on a separate sheet of paper Highlight key terms and circle any concepts/ideas that link metabolism to previously learned material Answer questions 1-4 in “Thinking Beyond” at the conclusion of the case study

Why did Patrick lose his ability to move? Patrick at 2: Patrick at 21: Movie in QuickTime (mov) ~ To view this at home, visit: http://www.sciencecases.org/patrick_paralyzed/patrick_paralyzed.mov Movie available at http://www.sciencecases.org/patrick_paralyzed/patrick_paralyzed.mov

Patrick’s History When Patrick was 16 years old, his hand started twitching as he picked up a glass at dinner. Five months later (in February 2001), he fell down the steps at his home and was unable to climb the steps to the bus. He went to the ER for his progressive weakness. At Children’s Hospital of Philadelphia he was initially diagnosed with a demyelinating disease (loss of the coating that surrounds neurons; myelin is involved in conduction of electrical impulses). He was treated with anti-inflammatory drugs and antibodies for 2 years with no improvement. What was wrong with Patrick?

Q1: What could be responsible for Patrick’s loss of mobility? A: His nervous system is not functioning properly. B: His muscles are not functioning properly. C: He cannot efficiently break down food for energy. D: All of the above are possible causes.

Q2: Which of the following processes requires energy? A: Creating ion gradients across membranes. B: Muscle shortening. C: Protein synthesis. D: All of the above.

Why do nerve and muscle cells need energy? Synthetic work = building macromolecules (e.g., Making protein) Mechanical work = moving molecules past each other (e.g., Muscle shortening) Concentration work = creating chemical gradients (e.g., Storing glucose) Electrical work = creating ion gradients (e.g., Unequal distribution of sodium and potassium ions)

What is energy? Potential Energy = stored energy Chemical bonds Concentration gradients Electrical potential Kinetic Energy = movement energy Heat = molecular motion Mechanical = moving molecules past each other Electrical = moving charged particles

Cycling between stored chemical versus movement energy Stored chemical energy must be released Processes that RELEASE energy Make ATP Catabolic/ Exergonic Movement requires energy Processes that REQUIRE energy Use ATP Anabolic/ Endergonic Energy released > Energy required ATP plays a central role

ATP plays a central role in energy cycling + Stored chemical energy is released in catabolic reactions to make ATP ATP is used in energy requiring reactions like muscle movement 10 10

Q3: The high energy phosphate bond in ATP is _____ and ____ energy to break the bond. A: Easy to break, releases B: Hard to break, requires C: Easy to break, requires D: Hard to break, releases

Adenosine triphosphate (ATP) Adenosine diphosphate (ADP) This bond is easy to break and requires energy! Adenosine triphosphate (ATP) H2O Hydrolysis of ATP Formation of these new bonds releases energy There is a common misconception that breaking the bond in ATP releases energy. Breaking bonds requires energy and making bonds releases energy. Because the bond on the terminal phosphate group is easy to break (requires less energy) and the bonds formed to make the phosphate ion release energy, there is a net energy release. H H Inorganic phosphate (Pi) Adenosine diphosphate (ADP) 12 12

ATP plays a central role in metabolism ATP is NOT the highest energy molecule intermediate energy ATP hydrolysis releases energy phosphate groups require low energy to break new bonds formed release more energy than the energy required to break the bond Phosphorylation by ATP increases the energy of other molecules

Q4: What would happen if Patrick lost his ability to make ATP? A: His muscles would not be able to contract. B: His neurons would not be able to conduct electrical signals. C: Both A and B.

How is ATP generated? ATP is formed through metabolic pathways. In metabolic pathways, the product of one reaction is a reactant for the next. Each reaction is catalyzed by an enzyme.

What are enzymes? Enzymes (usually proteins) are biological catalysts, highly specific for their substrates (reactants). Enzymes change reactants into products through transition state intermediates. Enzymes are not consumed in the reaction. http://commons.wikimedia.org/wiki/File:Simple_mechanism.svg.png This image has been (or is hereby) released into the public domain by its author, TimVickers at the wikipedia project. This applies worldwide. In case this is not legally possible: TimVickers grants anyone the right to use this work for any purpose, without any conditions, unless such conditions are required by law. 16 16

Enzymes as Catalysts Enzymes “speed up” reactions by lowering the “activation energy” of a reaction. Enzymes DO NOT change the overall energy released in a reaction. Click on the link to view an animation about enzyme action: http://www.wiley.com/legacy/college/boyer/0470003790/animations/catalysis_energy/catalysis_energy.swf 17 17

Q5: Which statement about enzymes is correct? A: Enzymes are always proteins. B: Enzymes are consumed in a reaction. C: Enzymes are always active. D: All are correct. E: None are correct. Note: It is a common misconception that enzymes are always active. This question leads to the next slides on enzyme regulation.

Enzyme Regulation Enzymes turn “on” and “off” based on the need of the organism “ON” = Activators Positive allosteric regulation “OFF” = Inhibitors Irreversible = must make new enzyme! Reversible = inhibitor can “come off” Competitive = active site Noncompetitive = “other” site = allosteric site Feedback Inhibition Click on the link to view an animation of enzyme inhibition: http://www.chem.purdue.edu/courses/chm333/enzyme_inhibition.swf This information is needed to understand the potential treatments for Patrick’s paralysis. Instructors may wish to provide additional information about allosteric regulation based on their course.

Q6: In competitive inhibition… A: the inhibitor competes with the normal substrate for binding to the enzyme's active site. B: an inhibitor permanently inactivates the enzyme by combining with one of its functional groups. C: the inhibitor binds with the enzyme at a site other than the active site. D: the competing molecule's shape does not resemble the shape of the substrate molecule.

How are metabolic pathways regulated? Explain what feedback inhibition is. You may use your text to help. Be sure to identify and describe BOTH types. Click on the link to view an animation on feedback inhibition: http://programs.northlandcollege.edu/biology/Biology1111/animations/enzyme.html

DNA mutations can disrupt metabolic pathways Patrick suffered from a genetic disease that altered the structure of one protein. The protein was an enzyme. The enzyme could potentially: lose its ability to catalyze a reaction. lose its ability to be regulated.

Q7: Consider the following metabolic pathway: A C D B If the enzyme responsible for converting A to C was mutated and nonfunctional, what would happen? A: A levels would increase; B, C, and D levels would decrease. B: A and B levels would increase; C and D levels would decrease. C: A, B and C levels would increase; D levels would decrease. D: A, B, C, and D levels would all decrease.

Metabolic Pathways: Glycolysis Pathway present in almost every cell! Takes place in the cytoplasm of the cell. Occurs with or without oxygen. Oxidizes glucose (6 C) to 2 pyruvate (3 C). Overall yield = 2 ATP and 2 NADH + H+ Click on the link to view a simplified animation of the glycolytic pathway linked to fermentation. http://instruct1.cit.cornell.edu/Courses/biomi290/MOVIES/GLYCOLYSIS.HTML You may want to supplement this material with your own images of glycolysis and fermentation pathways.

Important Electron Acceptors Coenzymes NAD (Nicotinamide Adenine Dinucleotide) NAD+ + 2H+ + 2 e- --> NADH+ + H+ FAD (Flavin Adenine Dinucleotide) FAD + 2H+ + 2 e- --> FADH2 Both molecules serve as coenzymes in many reactions.

Fermentation: Recycles NADH Occurs in the cytoplasm without O2 NADH + H+ is reoxidized to NAD+ Alcoholic Fermentation = yeast cells Converts pyruvate to ethanol and CO2 Overall yield = 2 ATP Lactate Fermentation = animal cells Converts pyruvate to lactate

Q8: Consider the following metabolic pathway: Pyruvate Acetyl CoA TCA cycle Lactate If Patrick’s enzyme responsible for converting pyruvate to acetyl CoA was inhibited, what would happen? A: Pyruvate levels would increase; acetyl CoA and lactate levels would decrease. B: Pyruvate and lactate levels would increase; acetyl CoA levels would decrease. C: Pyruvate, acetyl CoA, and lactate levels would increase. D: Pyruvate, acetyl CoA, and lactate levels would all decrease. This question leads specifically to some of the clinical symptoms that Patrick suffered due to his enzyme deficiency.

Patrick suffered from lactate acidosis Lactate (lactic acid) and pyruvate accumulated in his blood. Acidosis led to: Hyperventilation Muscle pain and weakness Abdominal pain and nausea The enzyme deficiency must be between the conversion of pyruvate to acetyl CoA.

Anaerobic versus aerobic metabolism Pyruvate dehydrogenase enzyme Na+ Cell membrane Glucose No O2 Glycolysis 2 ATP 2 Lactate (fermentation) O2 Glucose 2 Pyruvate Oxygen diffuses into the cell 2 NADH + H+ Mitochondria Pyruvate dehydrogenase enzyme With O2 H+ H+ cytoplasm e- H+ e- e- O2 e- e- H+ H2O Electron transport carriers NAD+ H+ Pyruvate ATP NADH + H+ H+ Outer membrane 3 NADH + H+ FAD CO2 FADH2 ADP + Pi CO2 diffuses out of the cell Acetyl CoA 3 NAD+ F0F1 ATPase GDP + Pi This particular slide is rich in detail and contains a custom animation that requires many clicks in “Slide Show” view. Instructors can easily modify or completely remove the animation by using Powerpoint‘s Custom Animation task pane, which can be accessed in “Normal” view by going to the menu bar and selecting Slide Show→Custom Animation. citrate Intermembrane space GTP Krebs cycle Oxaloacetate ATP 2 CO2 matrix Inner membrane

What happened to Patrick? He inherited a mutation leading to a disease called pyruvate dehydrogenase complex disease (PDCD) – an enzyme deficiency in the mitochondria. Pyruvate dehydrogenase is an enzyme that converts pyruvate to acetyl CoA inside the mitochondria. The brain depends on glucose as a fuel. PDCD degenerates gray matter in the brain. Pyruvate accumulates, leading to alanine and lactate accumulation in the blood (lactate acidosis). The pyruvate dehydrogenase complex disease link provides more extensive information about PDCD. It is important to emphasize that the enzyme deficiency is within the mitochondria. However, alanine and lactate accumulate in the cytoplasm. The “mitochondrial membrane” box delineates the different compartments (above the box = cytoplasm, below the box = mitochondria). This image can be found at: http://www.ehponline.org/realfiles/members/1998/Suppl-4/989-994stacpoole/stacfig1.GIF

Q9: Why did Patrick become paralyzed? A: He inherited a genetic disease that resulted in the partial loss of an enzyme necessary for aerobic breakdown of glucose. B: The enzyme that is necessary for metabolizing fats was defective. C: He was unable to synthesize muscle proteins due to defective ribosomes. D: He suffered from a severe ion imbalance due to a high salt diet.

Q10: Which food(s) can be metabolized to generate acetyl CoA? A: Carbohydrates B: Fats C: Proteins D: Both carbohydrates and fats E: Carbohydrates, fats and proteins This question leads the students to understand why a ketogenic diet may be used to treat this enzyme deficiency. It also emphasizes that other types of fuel can be used for aerobic metabolism. Image from http://commons.wikimedia.org/wiki/File:ATP_Production_Pathways.jpg

Are there any treatment options for PDH deficiency? High fat, low carbohydrate diet (ketogenic diet) Fatty acids can form acetyl CoA which can enter the Krebs cycle Fatty acids 

Are there any treatment options for PDH deficiency? Dichloroacetate (DCA) blocks the enzyme that converts PDH from active to inactive forms PDH remains in the active form DCA blocks here The use of DCA, an enzyme inhibitor, to treat this disorder links the previous information about enzyme inhibition with the treatment. By preventing the conversion of active PDH to inactive PDH, the levels of active PDH increase. This leads to more active form of the enzyme.

Q11: Dichloroacetate (DCA) administration would lead to… A: Increased production of acetyl CoA. B: Decreased lactate accumulation. C: Increased ATP production. D: All of the above. Images from previous slides are included to show the relationships between the overall metabolic pathway, the specific enzyme deficiency, and the treatment.

Q12: The loss of which of the following molecules was the most critical for Patrick’s paralysis? A: Pyruvate dehydrogenase B: Acetyl CoA C: Lactate D: ATP Even though Patrick lost PDH activity which resulted in decreased levels of acetyl CoA, ultimately Patrick’s disease was due to his inability to make ATP. This clicker question links the case back to the importance of ATP formation for cellular work.

What happened to Patrick? Although his family tried to care for him at home, Patrick remained in hospitals and nursing homes until he died in 2006. Patrick died due to pneumonia, sepsis, and renal failure when he was only 21 years old. His family mourns his loss but feels grateful that he was able to survive for 5 years on a respirator, 4 years beyond his doctor’s predictions.

Thinking Beyond How can Patrick’s case help explain the importance of cellular respiration in organisms? Explain how a single mutation in the production of one enzyme led to Patrick’s death. What if there was a defect in the gene coding for ATP synthase or phosphofructokinase in plants. Predict the implications of such mutations. Brainstorm and describe possible treatment options.