3-1 Glycolysis Case SP2019 PDF

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BCMB3100 with Paula Lemons and Takahiro Ito...

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BCMB3100, 2019SP

Glycolysis and McArdle Disease Case Take Home Point: The breakdown of carbohydrates by an organism (e.g., human) involves series of chemical reactions called metabolic pathways. The directionality of each reaction in a pathway depends on the free energy and relative concentrations of reactants and products available. Observable “flux” of a pathway is the net result of forward and reverse processes. Enzymes control rates of forward and reverse reactions in a pathway, and their activity is highly regulated. Enzyme-mediated regulatory mechanisms allow pathways to be sensitive and responsive to the needs of the organism. In metabolic pathways, often a favorable process is coupled to drive a less-favorable process. Learning Objectives: 1) In terms of skeletal muscle cells, describe how glucose is imported, used and stored and how these processes are regulated. 2) Summarize the answers to the following questions for any given reaction in glycolysis: a) What is the G under typical physiological conditions? b) Is it readily reversible under typical physiological conditions? c) What enzyme catalyzes the reaction and what type of reaction is occurring? d) Is this enzyme regulated and if so how is it regulated? 3) Explain how coupling facilitates thermodynamically unfavorable reactions. [e.g. how do ATP hydrolysis and NAD+ reduction drive less favorable reactions?] 4) Explain the chemical reasons that ATP is the energy currency in biology, i.e., the negative ∆G of ATP hydrolysis and ATP’s high phosphoryl-transfer potential. 5) Explain and diagram oxidation-reduction reactions. 6) Describe substrate-level phosphorylation and explain why 1,3-bisphosphoglycerate and phosphoenolpyruvate are substrates for substrate-level phosphorylation. 7) Summarize how muscle cells store and release glucose from glycogen. 8) Describe McArdle disease and relate the symptoms observed with the enzyme deficiency present in an individual with McArdle disease. Part 1 Glycolysis is a critical metabolic pathway shared by virtually every cell. During exercise, glucose is an important fuel for muscle activities including primary active transport to maintain ion gradients (Na+/K+ ATPase and Ca+2 ATPase), basic cell processes (transcription and translation) and muscle contraction (myosin ATPase).

1. Glucose requires a transporter protein to go into the cell. In most cells, glucose moves across the plasma membrane via glucose transporters (GLUTs). GLUTs are proteins that consist of a single polypeptide chain ~500 amino acids long. a. Name one amino acid that you would expect to find in the membrane spanning region and one amino acid you would find in the region interacting with the cytoplasm. Valine- possible amino acid in membrane spanning region Glutamine- possible amino acid found in region interacting with the cytoplasm

BCMB3100, 2019SP b. Which GLUT protein is responsible for transport of glucose into muscle cells? What hormone regulates the transport of GLUT to the plasma membrane? GLUT4 (found in muscle and fat cells); insulin -

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Glycolysis: breaks down glucose into two molecules of pyruvate It occurs in the cytoplasm and has two major phases Energy investment o ATP needs to be used to activate glucose Energy payoff o More ATP generated, two NADH also produced to utilize later in the oxidative phosphorylation step It occurs whether O2 is present or not

2. Glucose is metabolized by glycolysis, a metabolic pathway with 10 chemical reactions, each catalyzed by a distinct enzyme. View the chart below, which details all 10 reactions. Step 1 2 3

4

5

6

7

8 9

Reactants & products? Glucose and ATP Glucose 6-phosphate Glucose 6-phosphate Fructose 6-phosphate Fructose 6-phosphate and ATP Fructose 1,6bisphosphate and ADP Fructose 1,6bisphosphate Dihydroxyacetone and Glyceraldehyde 3-phosphate Dihydroxyacetone Glyceraldehyde 3phosphate Glyceraldehyde 3phosphate, Pi and NAD+ 1,3bisphosphoglycerate, NADH and H+ 1,3bisphosphoglycerate and ADP 3-phosphoglycerate and ATP 3-phosphoglycerate 2-phosphoglycerate 2-phosphoglycerate

Free energy change? ∆G -33.5 kJ/mol

Enzyme

Type of reaction

Hexokinase

Phosphorylation

-22.2 kJ/mol

Phosphoglucose isomerase Phosphofructokinase

Isomerization (rearrangement) Phosphorylation

-1.3 kJ/mol

Aldolase

Cleavage

2.5 kJ/mol

Triose phosphate isomerase

Isomerization (rearrangement)

-1.7 kJ/mol

Glyceraldehyde 3phosphate dehydrogenase

Oxidation/Reductio n and phosphorylation

1.3 kJ/mol

Phosphoglycerate kinase

Substrate level phosphorylation

0.8 kJ/mol

Phosphoglycerate mutase Enolase

Isomerization (rearrangement) Dehydration

-2.5 kJ/mol

-3.3 kJ/mol

BCMB3100, 2019SP

10

Phosphoenolpyruvate and H2O Phosphoenolpyruvate and ADP Pyruvate and ATP

-16.7 kJ/mol

Pyruvate kinase

Substrate level phosphorylation

a. Highlight in yellow the step number of the three steps that you predict are the most energetically favorable under typical cellular conditions. For each of the steps highlighted, explain why the step is so energetically favorable. Very energetically favorable because has a very negative delta G; all phosphorylation reactions, which adds a phosphate to a molecule more bonds formed more energy released So spontaneous that essentially irreversible

b. Enzymes that catalyze essentially irreversible steps are often regulated in a metabolic pathway. Highlight in blue the names of the enzymes that you predict are regulated in glycolysis. For the enzymes you highlighted in blue, give an example for how each enzyme is regulated to control glycolysis. Hexokinase is hetero allosterically inhibited by its product, glucose 6-phosphate, and it is activated by ATP. Phosphofructokinase is allosterically inhibited by high levels of ATP because high levels of ATP by decreasing its affinity for fructose-6-phosphate, and activated by its Pyruvate kinase is inhibited by pyruvate PFK and pyruvate kinase are sensitive to ATP, inhibited by large ATP concentration because ATP isn’t being used PFK is activated by AMP (low energy change) and is inhibited by ATP (high energy change) Pyruvate kinase- fructose 1,6-bisphosphate is an activator, ATP is an inhibitor Regulation of glycolysis in muscle - Glycolysis is not activated at rest (glucose pyruvate) - It is stimulated during exercise (glucose pyruvateCO2 + H2O [long, slow run], lactate [sprint]) Allosteric enzymes - Enzymes that regulate the flux of biochemicals through metabolic pathways- allosteric enzymes - Regulation of catalytic activity by environmental signals - Kinetics more complex than Michaelis Menten - Quaternary structure with multiple active sites in each enzyme - Most allosterically regulated enzymes are made from polypeptide subunits - Each enzyme has an active and inactive form - The binding of an activator stabilizes the active form of the enzyme - The binding of an inhibitor stabilizes the inactive form of the enzyme c. Highlight in green the step that involves an oxidation-reduction reaction. In general, why is an oxidation-reduction reaction energetically favorable? Hint: The correct answer

BCMB3100, 2019SP is not that it’s favorable because the change in free energy is largely negative; that is the definition of favorable. The more a carbon is reduced, the more free energy released during oxidation (releasing more energy by forming stronger bonds) Two steps catalyzed by one enzyme: - Step 1: oxidation of carbon - Step 2: acyl-phosphate formation (phosphorylation) Redox reactions: oxidation and reduction - The transfer of electrons during chemical reactions releases energy stored in organic molecules - This released energy is ultimately used to synthesize ATP - More oxidized, less energetic; more reduced, most energetic Oxidation: Oxidation Is Losing, OIL substance loses electrons, is oxidized Reduction: Reduction Is Gaining, RIG substance gains electrons, is reduced (amount of positive charge is reduced) Oxygen- electrons held tightly, at low energy level, more stable, unlikely to react, high electronegativity, low energy Carbon- electrons held loosely at high energy level, likely to react, less stable, low electronegativity, high energy Lowest energy state-most stable d. Referring to figure 16.3, explain in your own words how glyceraldehyde 3-phosphate dehydrogenase overcomes the energetically unfavorable process of phosphorylation after oxidation.

The two processes must be coupled so that the favorable aldehyde oxidation can be used to drive the formation of the acyl phosphate. The key is an intermediate that is linked to the enzyme by a thioester after the aldehyde has been oxidized. Enzyme generates a thioester intermediate: avoids a thermodynamic pit because the thioester intermediate preserves much of the free energy released in the oxidation reaction. 3. ATP is an important energy currency inside most cells. Consider the structures of ATP, ADP, and AMP (Figure 15.4 in 3rd ed.).

BCMB3100, 2019SP

a. Which of these molecules is least stable because of electrostatic repulsion? How does this help to explain why ATP has a high phosphoryl transfer potential, i.e., ATP hydrolysis is energetically favorable? -

ATP is the cell’s energy shuttle ATP is least stable because it has three negative charges around the oxygens (three phosphate groups); this explains why ATP has a high phosphoryl transfer potential. The most stable molecule is AMP because it has the least amount of phosphate groups and it also has the least number of negative charges. ATP releases one phosphate and releases 30 kJ if it releases two phosphate groups it releases 45.6 kJ Why does ATP have a high phosphoryl transfer potential? Structure! Delta G naught prime depends on the difference in free energy of the products and the reactants of a reaction To understand ATP’s high phosphoryl transfer potential must understand free energy differences between ATP and ADP + Pi o Electrostatic repulsion o Stabilization due to hydration o Increase in entropy (one molecule two molecules)

b. But wait, I thought you told me bond breaking required energy. Why does the hydrolysis of ATP have a negative ∆G’? Why doesn’t ATP spontaneously hydrolyze in the cell?

The hydrolysis of ATP is used to couple; the negative delta G value is used to further carry out a reaction after the hydrolysis that is unfavorable, such as phosphorylation. Energy required to break the bond “activation energy” - High in solution (uncatalyzed) - Lower in an enzyme (catalyzed) - In solution, the change of the transition state is large enough to inhibit the reaction

BCMB3100, 2019SP 4. Name the substrates for the enzymes phosphoglycerate kinase and pyruvate kinase? Using the figure below (Figure 15.7, 3rd ed), explain why these substrates can be used for substrate-level phosphorylation.

Substrates for enzymes phosphoglycerate kinase and pyruvate kinase: 1,3-biphosphateglycerate and ADP and phosphoenolpyruvate and ADP. All of these substrates contain phosphorus, therefore being able to donate a P during phosphorylation. They all have high phosphoryl transfer

Why is step 10 so energetically favorable? - Because a high phosphoryl transfer molecule has its phosphate transferred to ATP, which has a lower phosphoryl transfer Step 1? - ATP is hydrolyzed, the phosphate group is moved from ATP to a molecule with a much lower free energy, which is energetically favorable Step 3? - ATP is hydrolyzed, the phosphate group is moved from ATP to a molecule with a much lower free energy, which is energetically favorable

Part 2 Skeletal muscle cells use a mixture of carbohydrates and lipids to make ATP for normal function. At rest and during low intensity exercise, skeletal muscle predominately uses fatty acids. As exercise intensity increases, skeletal muscle predominately utilizes glucose, as shown in the diagram below. Not shown below but of note is that at moderate intensity exercise for long duration (~1 hour), skeletal muscle switches from predominately using glucose to using fatty acids.

BCMB3100, 2019SP

Image from Dr. Ross Tucker, UCT, Cape Town

5. When skeletal muscles metabolize glucose, the pyruvate that is produced via glycolysis has several potential fates. a. What is the main fate of pyruvate in skeletal muscle when oxygen is limited? What is the purpose of this reaction in muscle during exercise? When oxygen is limited, exercise intensity is most likely increasing. This means that the skeletal muscle predominantly uses glucose, which means more pyruvate will be formed as a product. This means that under lack of oxygen conditions, the pyruvate will be converted to lactate, which makes lactic acid, the acid in the body that causes muscle fatigue and post-exercise soreness. Pyruvate (no oxygen present: fermentation) lactate (mammals), ethanol (microorganisms due to fermentation) or other products Pyruvate (oxygen present; aerobic cellular respiration) acetyl coA citric acid cycle By making lactate from pyruvate, we reduce the amount of pyruvate and made more NAD+ available for glycolysis to proceed. b. What is the main fate of pyruvate in skeletal muscle when oxygen is readily available, such as during moderate intensity exercise? What pathway does this then enter and how does the further metabolism of this compound result in ATP synthesis? During moderate intensity exercise, the skeletal muscle transitions from primarily using glucose to using fatty acids. This will decrease the amount of pyruvate formed, since glucose is not being used as much. This is under aerobic conditions; pyruvate will be converted to Acetyl CoA which will then be expended from the body as CO2. p.175, ch24 6. Glucose can be stored in several tissue/organs in the human body.

BCMB3100, 2019SP a. What organs/tissues are the two major sites of glucose storage in the body? Explain if these organs/tissues contribute to blood glucose levels. The two major sites of glucose storage in the body are skeletal muscle and the liver. The concentration of glycogen is higher in the liver than in muscle, but more glycogen is stored in skeletal muscle overall. In the liver, glycogen synthesis and degradation are regulated to maintain the concentration of glucose in the blood required to meet the needs of the organism as a whole. In contrast, in muscle, the processes are regulated to meet the energy needs of the muscle itself. LIVER utilizes glucose for BLOOD GLUCOSE LEVELS MUSCLE utilizes glucose for ATP SYNTHESIS FOR MOVEMENT Glycogen and fat are both forms of glucose storage Glycogen - Large up to 50,000 Glc residues - Granules found in cytosol - Allow rapid release of glucose b. In what form is glucose stored in the body? Summarize the structure and size of this molecule. Glucose is stored in the body in the form of glycogen, a homopolymer. Glycogen controls the release of glucose from it to maintain the blood-glucose concentration between meals. ____ c. Briefly describe the process by which glucose is released from its stored form to be utilized by glycolysis? Does this process require ATP? Glycogen phosphorylase (key regulatory enzyme in glycogen breakdown) cleaves its substrate by the addition of orthophosphate to yield glucose-1-phosphate (cleavage of a bond by the addition of orthophosphate is referred to as phosphorylsis) phosphorylase catalyzes sequential removal of glucosyl residues from the nonreducing ends of the glycogen molecule orthophosphate splits the glycosidic linkage between the C-1 carbon atom and the new glycosidic oxygen atom alpha configuration at C-1 of the newly released glucose 1-phosphate is retained glucose 1-phosphate released from glycogen can be readily converted into glucose 6-phosphate. Glycogenolysis - Glycogen phosphorylase breaks glycosidic bond with Pi - Glucose 1-phosphate isomerization to glucose 6-phosphate) by phosphoglucomutase) - Branching removed by glucosidase which releases free glucose Phosphorylase regulates glycogen usage 7. Muscle phosphorylase is the key regulator step for glycogenolysis. Several important regulators of muscle phosphorylase are listed below. For each of these describe how they regulate glycogenolysis. Phosphorylase a is the active form a. ATP Negative allosteric effector- competes with AMP Inhibitorb. AMP Activates muscle phosphorylase b by binding to nucleotide binding site and stabilizing the conformation of phosphorylase b in the active R state

BCMB3100, 2019SP Activates; more AMP, cell needs more ATP so it activates glycogenolysis to have more ???????????????????????????? available for glycogenolysis c. Glucose-6-phosphate Feedback inhibitor; binds at same site as ATP and stabilizes the less active state of phosphorylase b Inhibits- no need to make more glucose-6-phosphate d. Ca+2 Activator; initiates phosphorylase kinase to work when it binds to the sigma subunit Activating glycogenolysis because it’s making kinase active- presence of calcium activates glycogenolysis e. Epinephrine Activator; rise in epinephrine concentration results in phosphorylation of the enzyme to the phosphorylase a form in liver and muscle Stimulating phosphorylase kinase; activating glycogenolysis- glycogen increases 8. A 10-year-old boy presents with muscle fatigue and cramping during the first 5 minutes of vigorous exercise. His parents report that he has recently joined a soccer team, but has been unable to keep up with the other kids. This child has dark brown urine following practice due to rhabdomyolysis. The child undergoes an ischemic forearm exercise test which shows no elevation in blood lactate levels. A muscle biopsy shows normal glycogen stores. He is diagnosed with glycogen storage disease type V or McArdle disease. Cramps, fatigue (lack of ATP) Dark brown urine (myoglobinuria, due to myolysis) Ischemia means no oxygen supply a. What is McArdle Disease? b. Which enzyme is deficient in McArdle Disease? Glycogen phosphorylase c. Outline the metabolic impact that the above enzyme deficiency has on metabolism during exercise and relate these to the symptoms observed (cramps, fatigue and dark brown urine). In the absence of oxygen, a normal body would create lactate from glycose. With McArdle, the body cannot convert glucose to lactate 9. Several diagnostic tests were used to diagnosis this child. Explain the ischemic forearm exercise test and the results of the test for this child.

10. What are the treatment options for an individual with McArdle disease? No cure- suggestions are to avoid high intensity exercise...