Chapter 9

Consider the two metabolic reactions below:Reaction 1: A + B → C ΔG˚ = 8.8 kJ/molReaction 2: C → D ΔG˚ = -15.5 kJ/mol If reaction 1 and 2 are coupled, what would the net reaction be?

-6.7 kJ/mol The net reaction has ΔG˚ = (8.8 kJ/mol + -15.5 kJ/mol) = -6.7 kJ/mol.

6. Give three reasons why glycolysis is considered a core metabolic pathway.

1. Glycolytic enzymes are evolutionarily conserved in essentially all living organisms. 2. Glycolysis is the only anaerobic pathway for ATP generation. 3. Glycolytic intermediates are shared metabolites in numerous pathways.

2. What is meant by the term metabolic flux, and how is it regulated?

Metabolic flux describes the overall reaction rates in metabolic pathways and is primarily controlled by available enzyme activity, a function of enzyme abundance and catalytic activity. Flux is also affected by availability of substrates and utilization of products.

Again consider the pathway above. Under what conditions would adding more A to the system not lead to an increase in the flux of the pathway from A to E?

-An allosteric inhibitor of the enzyme that catalyzes C → D is also present in the system. -The enzyme that catalyzes C → D has already reached 100% of Vmax. The addition of more A to the system will lead to a corresponding increase in the concentration of C. However, if the enzyme that catalyzes C → D is operating near 100% of the maximum velocity, the addition of more substrate will not increase the rate of the reaction. The increase in C will also not alleviate the inhibition of the C → D reaction if an allosteric inhibitor is present.

Glycolytic reactions can either be described as reversible or irreversible. Looking at all 10 reactions in glycolysis, what conclusions can you make about irreversible reactions in general?

-They operate far from equilibrium. -The activity of the enzymes that catalyze irreversible reactions can be increasedor decreased. -The enzymes that catalyze these reactions are not shared in opposingpathways (i.e., glycolysis and gluconeogenesis). Irreversible reactions have a large free energy change and therefore operate far from equilibrium. The enzymes that catalyze these reactions also do not operate near their maximum activity level and are not shared in opposing pathways.

The flux of which of the following reactions would depend only on substrate availability?

-dihydroxyacetonephosphate → glyceraldehyde-3-phosphate -glucose-6-phosphate → fructose-6-phosphate The free energy change of both reactions is near zero, and the enzymes that catalyze these reactions operate near their highest activity levels. Therefore the flux of these reactions is controlled by the availability of substrate.

Which of the following enzymes catalyze rate-determining steps in glycolysis?

-phosphofructokinase -pyruvate kinase -hexokinase These three reactions are all irreversible in the cell and are the rate-determining steps for the pathway.

Two reactions, A → B and B → C, are coupled. If A → B has ΔG˚= -22.6 kJ/mol and A → C has ΔG˚= 5.7 kJ/mol, then B → C will have ΔG˚= _____ kJ/mol.

28.3 kJ/mol ΔG˚ = 5.7 kJ/mol for the coupled reaction. This value is obtained by adding ΔG˚ for both individual reactions. If A → B has ΔG˚ = -22.6 kJ/mol, B → C has ΔG˚ = 28.3 kJ/mol.

A net yield of ____ ATP would be produced from the conversion of three molecules of glucose into pyruvate.

6 Completion of the 10 glycolytic reactions starting with glucose produces a net yield of 2 ATP. Three glucose molecules would therefore produce three times as much ATP.

Consider the two metabolic reactions below:Reaction 1: A + B → C ΔG˚ = 8.8 kJ/molReaction 2: C → D ΔG˚ = -15.5 kJ/mol If reaction 1 and 2 are coupled, what would the net reaction be?

A + B → D C is a product of reaction 1 and a substrate in reaction 2. In the net reaction, C would cancel out.

Consider the pathway A → B → C → D → E. Reactions A → B, B → C, and D → E have a ΔG near equilibrium, while C → D has a ΔG << 0. Which of the following is true about this pathway if it were operating within a cell?

Adding more A to the system will increase the amount of B and C in the system. Reactions A → B → C all operate near equilibrium. Adding more A to the system could push the reaction toward production of B, which could in turn push the B → C reaction forward.

5. How can a chemical reaction that is energetically unfavorable under standard conditions (delta G standard > 0) occur in a cell?

An unfavorable reaction (one with a positive delta G) may occur if the sum of the standard free energy change and RTlnQ is sufficiently less than zero (Q is the mass action ratio), resulting in an overall negative delta G.

3. Describe how glucagon and insulin signaling alter metabolic flux through glucose metabolizing pathways before and after breakfast.

Before: glucagon signaling in the liver elevates glucose efflux by increasing flux through liver glycogen degradation and gluconeogenesis. After: insulin signaling stimulates glucose influx into the liver by increasing flux through the glycogen synthesis and glycolysis,

10. Compare and contrast the enzymatic properties of hexokinase and glucokinase?

Both phosphorylate glucose on the C-6 position in the first reaction of the glycolytic pathway. Hexokinase, however, has a very high affinity for glucose (199 times higher than that of glucokinase); can phosphorylate fructose; is allosterically inhibited by glucose-6-phosphate; and is expressed in all tissues. Glucokinase is highly specific for glucose, but binds with low affinity; is not allosterically inhibited by glucose-6-phosphate; is expressed primarily in liver and pancreas; and functions as a glucose sensor in pancreatic Beta cells.

1. Describe the functional role of catabolic and anabolic pathways in metabolism.

Catabolic: capture energy as ATP and reducing potential (NADH and FADH2) by degrading macromolecules obtained in food. Anabolic: build macromolecules from small metabolites using ATP and reducing ability (NADPH) as source of energy.

8. Write the net reaction for the glycolytic pathway and describe the primary functions of stage 1 and stage 2.

Glucose + 2 NAD+ + 2 ADP + 2 Pi --> 2 pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O Stage 1: ATP investment phase includes 5 reactions, requiring the input of 2 ATP to generate fructose-1,6-biphosphate, which is cleaved, followed by an isomerization reaction to yield two molecules of glyceraldehyde-3-phosphate for each glucose. Stage 2: ATP earnings phase consists of five reactions and generates a total of four molecules of ATP fore each molecule of glucose. Because 2 ATP were invested in stage 1, the net ATP yield of glycolysis is 2 ATP per glucose.

Why is HFCS preferred over corn syrup for sweetening beverages and processed foods?

HFCS is sweeter than corn syrup. Fructose is about three times sweeter than glucose.

Which of the following statements best describes the effect of ADP on the activity of PFK-1?

High concentrations of ADP block the inhibitory effect of ATP by competing for the same allosteric site on PFK-1. Both ADP and ATP bind to the same allosteric site on PFK-1 and compete for binding. High concentrations of ADP will prevent ATP from binding to PFK-1.

If 2 moles of glucose were converted into 4 moles of pyruvate through the glycolytic pathway, there would be a net yield of ____ ATP.

In stage I, 2 moles of glucose would require an investment of 4 ATP to produce 4 glyceraldehyde-3-phosphate. In stage II, these 4 glyceraldehyde-3-phosphate would be converted to 4 pyruvate with an ATP yield of 8 ATP.

4. List the key macromolecules, primary metabolites, and small molecules in metabolism.

Key macromolecules: proteins, nucleic acids, carbohydrate, and lipids. Primary metabolites: amino acids, nucleotides, fatty acids, glucose, pyruvate, and acetyl-CoA. Key small molecules: NH4+, CO2, NADH, FADH2, O2, ATP, and H2O.

12. Briefly explain how the abundant dietary disaccharides maltose, lactose, and sucrose are metabolized by the glycolytic pathway.

Maltose is a disaccharide of glucose, cleaved by maltase to yield two molecules of glucose used by glycolysis. Lactose is cleaved by lactase to yield glucose and galactose; galactose is phosphorylated by galactokinase, converted to glucose-1-phosphate, and then isomerize to glucose-6-phosphate used by glycolysis. Sucrose is cleaved by sucrase into glucose and fructose. In muscle, fructose is directly phosphorylated by hexokinase to fructose-6-phosphate for glycolysis, whereas in liver cells, fructose is phosphorylated by fructokinase to yield fructose-1-phosphate, which is converted to eventually yield two molecules of glyceraldehyde-3-phosphate.

Phosphorylation of glucose requires 1 ATP. Can the reverse reaction be used for substrate-level phosphorylation?

No, phosphate hydrolysis of glucose-6-phosphate is not spontaneous enough to couple to ATP synthesis. The standard free energy of phosphate hydrolysis from glucose-6-phosphate is less favorable than phosphate hydrolysis of ATP.

A student on the cross country team wishes to improve her performance in an upcoming race. She has taken an introductory science course and knows that oxidation of glucose produces ATP for muscle contraction. The student normally eats small amounts of glucose gel for energy during a long run, and she decides that the best course of action is to triple the amount of glucose gel she eats each time so that her muscles can have access to more glucose. Will this strategy work?

No; ingesting additional glucose gel will raise the blood glucose level but will not increase the reaction rate of hexokinase. Instead, the increase in glucose will increase the reaction rate of glucokinase, which could have negative effects on the athlete's performance. The excess glucose will increase the reaction rate of glucokinase but not hexokinase. The increase in glucokinase activity in the pancreas could cause insulin release. Insulin activates many fuel storage pathways, which may slow down the ability of muscle cells to produce ATP.

Sucrose, maltose, and lactose are common dietary disaccharides. Their monosaccharide components are substrates for the glycolytic pathway, but some must participate in additional reactions before they can act as substrates for a glycolytic enzyme. As a result, all do not enter glycolysis at the same step. In muscle cells, which enzyme would act as the rate-limiting step to regulate entry of products from all three dietary disaccharides into glycolysis?

PFK-1 Monosaccharides from sucrose and maltose are substrates for hexokinase, but the galactose component of lactose is not. Galactose is converted to glucose-6-phosphate, bypassing the hexokinase reaction. Phosphofructokinase-1 would be the only rate-limiting step affecting entry of all sugars into the glycolytic pathway.

11. Describe allosteric regulation of phosphofructokinase-1.

PFK-1 is a homotetramer with two conformational structures: the T state (inactive) and the R state (active). Allosteric regulation shifts the equilibrium between states. PFK-1 activity is stimulated by a low energy charge (low ATP relative to AMP and ADP), and inhibited by high energy charge. PFK-1 is allosterically activated by AMP, ADP, and fructose-2,6-biphosphate, and allosterically inhibited by ATP and citrate.

Consider two reactions. Reaction 1 (P → Q) has ΔG = 2.3 kJ/mol. Reaction 2 (Q → R) has ΔG = 12.3 kJ/mol. Which reaction is more likely to require coupling to ATP or the equivalent in order to be spontaneous under cellular conditions?

Reaction 2 (Q → R). This reaction is nonspontaneous and operates far from equilibrium in the cell. It will not be able to proceed as written in the cell without coupling to a process with a large negative free energy change (such as ATP hydrolysis). Using only the value of ΔG, both reactions would be considered nonspontaneous as written. The ΔG of P → Q is smaller than that of R → S, indicating that P → Q is closer to equilibrium. A nonspontaneous reaction close to equilibrium in the cell is more likely to become spontaneous with the small fluctuations in substrate and product concentrations than one far from equilibrium. Consider the isomerization of glucose-6-phosphate to fructose-6-phosphate versus the phosphorylation of glucose to glucose-6-phosphate.

Reactions that occur in glycolysis can all be described in terms of the chemical changes that occur within each. Place the descriptions of these chemical reactions in the order that they occur in stage I of glycolysis (from beginning to end).

Stage I of glycolysis begins with glucose and ends with two molecules of glyceraldehyde-3-phosphate. There are two reactions in which energy investment from ATP is used to drive phosphoryl transfer. The end result is two phosphorylated products that can enter stage II.

What is the net reaction for stage I of glycolysis if the pathway begins with 2 moles of glucose?

Stage I of glycolysis produces 2 glyceraldehyde-3-phosphate and requires an investment of 2 ATP for every glucose molecule that enters the pathway. These values are doubled for 2 moles of glucose.

Reactions that occur in glycolysis can all be described in terms of the chemical changes that occur within each. Place the descriptions of these chemical reactions in the order that they occur in stage II of glycolysis (from beginning to end).

Stage II of glycolysis begins with a three-carbon phosphorylated aldose. In the first reaction, this aldose is phosphorylated to produce a molecule with high phosphoryl transfer potential, which leads to the production of ATP in the next reaction. An additional ATP is produced in the last reaction.

9. Which reactions in the glycolytic pathway are substrate-level phosphorylations?

Substrate-Level phosphorylation is a reaction in which ATP is generated from ADP by transfer of a phosphorylation group from a phosphorylated metabolite (Does not require ATP synthase complex). The substrate-level phosphorylation reactions in glycolysis are reaction 7, catalyzed by phosphoglycerate kinase, and reaction 10, catalyzed by pyruvate kinase.

Erythrocytes do not contain mitochondria. How is ATP produced in these cells?

Substrate-level phosphorylation Substrate-level phosporylation reactions in glycolysis occur in the cytosol. The absence of mitochondria would not affect these reactions or their production of ATP.

Label the graph below to show the effect of high ATP, ADP, or fructose-2,6-bisphosphate on phosphofructokinase-1 (PFK-1) activity.

The activity of PFK-1 is inhibited by high ATP concentrations, while fructose-2,6 bisphosphate has the opposite effect. High ADP concentrations increase the activity of PFK-1 as compared to when ATP is present.

The structure of D-arabinose is shown below as a Fischer projection. Identify the correct structure of L-arabinose.

This structure is the mirror image of D-arabinose.

13. What are the three metabolic fates of pyruvate? Include in your answer the conditions dictating which of these three metabolic fates is most likely to occur.

Under aerobic conditions, pyruvate is decarboxylated and converted to acetyl-CoA in organisms that use the citrate cycle. This yields the maximum number of ATP from glucose oxidation. Under anaerobic conditions, pyruvate is converted to lactate by lactate dehydrogenase in order to regernate NAD+ for the glyceraldehyde-3-phosphate dehydrogenase reaction in glycolysis. Alternatively, fermenting organisms, such as yeast, convert pyruvate to CO2 and ethanol under anaerobic conditions.

The two most common nutrative (caloric) sweeteners used in the manufacturing of food and beverages are sucrose and high-fructose corn syrup (HFCS). The latter is produced from corn syrup in a process that enzymatically converts a percentage of the glucose, which is the predominant sugar component, into fructose. HFCS used in most commercial food and beverage production is either 55% or 42% fructose. Which enzyme can be used in the production of HFCS to convert glucose to fructose in a single step?

glucose isomerase Fructose is an isomer of glucose; therefore, an enzyme from the isomerase class could catalyze the conversion of one to another.