Chapter Review in Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

Chapter Review

Key Terms

Terms in bold are defined in the glossary.

Problems

1. Is the Hexokinase Reaction at Equilibrium in Cells? For the reaction catalyzed by the enzyme hexokinase

$Glucose + ATP ⇌ glucose 6 -phosphate + ADP$ $Glucose plus ATP right harpoon over left harpoon glucose 6 hyphen phosphate plus ADP$

the equilibrium constant, $K_{eq}$ $upper K Subscript eq$ , is $7.8 times 10 squared$ $7.8 times 10 squared$ . In living E. coli cells, $left-bracket ATP right-bracket equals 5 m upper M$ $left-bracket ATP right-bracket equals 5 m upper M$ , $left-bracket ADP right-bracket equals 0.5 m upper M$ $left-bracket ADP right-bracket equals 0.5 m upper M$ , $left-bracket glucose right-bracket equals 2 m upper M$ $left-bracket glucose right-bracket equals 2 m upper M$ , and $left-bracket glucose 6 hyphen phosphate right-bracket equals 1 m upper M$ $left-bracket glucose 6 hyphen phosphate right-bracket equals 1 m upper M$ . Is the reaction at equilibrium in E. coli?
2. Equation for the Preparatory Phase of Glycolysis Write balanced biochemical equations for all the reactions in the catabolism of glucose to two molecules of glyceraldehyde 3-phosphate (the preparatory phase of glycolysis), including the standard free-energy change for each reaction. Then write the overall or net equation for the preparatory phase of glycolysis, with the net standard free-energy change.
3. Payoff Phase of Glycolysis: Fate of Pyruvate in Active Skeletal Muscle In working skeletal muscle under anaerobic conditions, glyceraldehyde 3-phosphate is converted to pyruvate (the payoff phase of glycolysis), and the pyruvate is reduced to lactate. Write balanced biochemical equations for all the reactions in this process, with the standard free-energy change for each reaction. Then write the overall or net equation for the payoff phase of glycolysis with fermentation to lactate, including the net standard free-energy change.
4. Energetics of the Aldolase Reaction Aldolase catalyzes the glycolytic reaction

$\begin{matrix} Fructose 1,6 -bisphosphate \to glyceraldehyde \\ 3-phosphate + dihydroxyacetone phosphate \end{matrix}$ $StartLayout 1st Row Fructose 1 comma 6 hyphen bisphosphate right-arrow glyceraldehyde 2nd Row 3 hyphen phosphate plus dihydroxyacetone phosphate EndLayout$

The standard free-energy change for this reaction in the direction written is $plus 23.8 kJ slash mol$ $plus 23.8 kJ slash mol$ . The concentrations of the three intermediates in the hepatocyte of a mammal are fructose 1,6-bisphosphate, $1.4 \times 10^{- 5} M$ $1.4 times 10 Superscript negative 5 Baseline upper M$ ; glyceraldehyde 3-phosphate, $3 \times 10^{- 6} M$ $3 times 10 Superscript negative 6 Baseline upper M$ ; and dihydroxyacetone phosphate, $1.6 \times 10^{- 5} M$ $1.6 times 10 Superscript negative 5 Baseline upper M$ . At body temperature $left-parenthesis 37 degree upper C right-parenthesis$ $left-parenthesis 37 degree upper C right-parenthesis$ , what is the actual free-energy change for the reaction?
5. Equivalence of Triose Phosphates A researcher adds $^{14} C$ $Superscript 14 Baseline upper C$ -labeled glyceraldehyde 3-phosphate to a yeast extract. After a short time, she isolates fructose 1,6-bisphosphate labeled with $^{14} C$ $Superscript 14 Baseline upper C$ at C-3 and C-4. What was the location of the $^{14} C$ $Superscript 14 Baseline upper C$ label in the starting glyceraldehyde 3-phosphate? Where did the second $^{14} C$ $Superscript 14 Baseline upper C$ label in fructose 1,6-bisphosphate come from? Explain.
6. Glycolysis Shortcut Suppose you discovered a mutant yeast whose glycolytic pathway was shorter because of the presence of a new enzyme catalyzing the reaction

Would shortening the glycolytic pathway in this way benefit the cell? Explain.
7. Role of Lactate Dehydrogenase During strenuous activity, the demand for ATP in muscle tissue vastly increases. In rabbit leg muscle or turkey flight muscle, ATP production is almost exclusively a product of lactic acid fermentation. Phosphoglycerate kinase and pyruvate kinase catalyze the two reactions that form ATP in the payoff phase of glycolysis. Suppose skeletal muscle were devoid of lactate dehydrogenase. Could it carry out strenuous physical activity; that is, could it generate ATP at a high rate by glycolysis? Explain.
8. Efficiency of ATP Production in Muscle The transformation of glucose to lactate in myocytes releases only about 7% of the free energy released when glucose is completely oxidized to ${CO}_{2}$ $CO Subscript 2$ and $upper H Subscript 2 Baseline upper O$ $upper H Subscript 2 Baseline upper O$ . Does this mean that glycolysis with lactate fermentation under anaerobic conditions in muscle is a wasteful use of glucose? Explain.
9. Free-Energy Change for Triose Phosphate Oxidation The oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, catalyzed by glyceraldehyde 3-phosphate dehydrogenase, proceeds with an unfavorable equilibrium constant $left-parenthesis upper K prime Subscript eq Baseline equals 0.08 semicolon$ $left-parenthesis upper K prime Subscript eq Baseline equals 0.08 semicolon$ $upper Delta upper G Superscript prime Baseline degree equals 6.3 kJ slash mol right-parenthesis$ $upper Delta upper G Superscript prime Baseline degree equals 6.3 kJ slash mol right-parenthesis$ , yet the flow through this point in the glycolytic pathway proceeds smoothly. How does the cell overcome the unfavorable equilibrium?
10. Arsenate Poisoning Arsenate is structurally and chemically similar to inorganic phosphate $left-parenthesis upper P Subscript i Baseline right-parenthesis$ $left-parenthesis upper P Subscript i Baseline right-parenthesis$ , and many enzymes that require phosphate will also use arsenate. Organic compounds of arsenate are less stable than analogous phosphate compounds, however. For example, acyl arsenates decompose rapidly by hydrolysis, as shown.

On the other hand, acyl phosphates, such as 1,3-bisphosphoglycerate, are more stable and undergo further enzyme-catalyzed transformation in cells.
1. Predict the effect on the net reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase if phosphate were replaced by arsenate.
2. What would be the consequence to an organism if arsenate were substituted for phosphate? Arsenate is very toxic to most organisms. Explain why.
11. Requirement for Phosphate in Ethanol Fermentation In 1906 Harden and Young, in a series of classic studies on the fermentation of glucose to ethanol and ${CO}_{2}$ $CO Subscript 2$ by extracts of brewer’s yeast, made the following observations: (1) Inorganic phosphate was essential to fermentation; when the supply of phosphate was exhausted, fermentation ceased before all the glucose was used. (2) During fermentation under these conditions (with no phosphate), ethanol, $CO Subscript 2$ $CO Subscript 2$ , and a hexose bisphosphate accumulated. (3) When arsenate was substituted for phosphate, no hexose bisphosphate accumulated, but the fermentation proceeded until all the glucose was converted to ethanol and $CO Subscript 2$ $CO Subscript 2$ .
1. Why did fermentation cease when the supply of phosphate was exhausted?
2. Why did ethanol and ${CO}_{2}$ $CO Subscript 2$ accumulate? Was the conversion of pyruvate to ethanol and ${CO}_{2}$ $CO Subscript 2$ essential? Why? Identify the hexose bisphosphate that accumulated. Why did it accumulate?
3. Why did the substitution of arsenate for phosphate prevent the accumulation of the hexose bisphosphate yet allow fermentation to ethanol and ${CO}_{2}$ $CO Subscript 2$ to go to completion? (See Problem 10.)
12. Role of the Vitamin Niacin Adults engaged in strenuous physical activity require an intake of about 160 g of carbohydrate daily but only about 20 mg of niacin for optimal nutrition. Given the role of niacin in glycolysis, how do you explain the observation?
13. Synthesis of Glycerol Phosphate The glycerol 3-phosphate required for the synthesis of glycerophospholipids can be synthesized from a glycolytic intermediate. Propose a reaction sequence for this conversion.
14. Severity of Clinical Symptoms Due to Enzyme Deficiency The clinical symptoms of two forms of galactosemia — galactokinase-deficiency galactosemia and transferase-deficiency galactosemia — show radically different severity. Although both types produce gastric discomfort after milk ingestion, deficiency of the transferase also leads to liver, kidney, spleen, and brain dysfunction and eventual death. What products accumulate in the blood and tissues with each type of enzyme deficiency? Estimate the relative toxicities of these products from the above information.
15. Ethanol Affects Blood Glucose Levels The consumption of alcohol (ethanol), especially after periods of strenuous activity or after not eating for several hours, results in a deficiency of glucose in the blood, a condition known as hypoglycemia. The first step in the metabolism of ethanol by the liver is oxidation to acetaldehyde, catalyzed by liver alcohol dehydrogenase:

$\begin{matrix} {CH}_{3} {CH}_{2} OH + {NAD}^{+} \to \\ {CH}_{3} CHO + NADH + H^{+} \end{matrix}$ $StartLayout 1st Row CH Subscript 3 Baseline CH Subscript 2 Baseline OH plus NAD Superscript plus Baseline right-arrow 2nd Row CH Subscript 3 Baseline CHO plus NADH plus upper H Superscript plus EndLayout$

Explain how this reaction inhibits the transformation of lactate to pyruvate. Why does this lead to hypoglycemia?
16. Blood Lactate Levels during Vigorous Exercise The graph shows the concentrations of lactate in blood plasma before, during, and after a 400 m sprint.

The horizontal axis is labeled time (min) and ranges from below 0 to 60, labeled in increments of 20, and the vertical axis is labeled blood [lactate] (mu M) and ranges from 0 to 200, labeled in increments of 200. The word run has an arrow pointing down to the horizontal axis at 0. The region to the left of this arrow is labeled before and the region to the right of this arrow is labeled after. The curve begins at 30 on the vertical axis before 0 on the horizontal axis, then rises vertically at (0, 30) at the point labeled run. The curve bends slightly beginning at (0, 170), peaks at (2, 200), then curves downward to form a concave shape that opens upward and ends at (60, 50). All data are approximate.
1. What causes the rapid rise in lactate concentration?
2. What causes the decline in lactate concentration after completion of the sprint? Why does the decline occur more slowly than the increase?
3. Why is the concentration of lactate not zero during the resting state?
17. Relationship between Fructose 1,6-Bisphosphatase and Blood Lactate Levels A congenital defect in the liver enzyme fructose 1,6-bisphosphatase results in abnormally high levels of lactate in the blood plasma. Explain.
18. Effect of $O_{2}$ $bold upper O Subscript 2$ Supply on Glycolytic Rates The regulated steps of glycolysis in intact cells can be identified by studying the catabolism of glucose in whole tissues or organs. For example, the glucose consumption by heart muscle can be measured by artificially circulating blood through an isolated intact heart and measuring the concentration of glucose before and after the blood passes through the heart. If the circulating blood is deoxygenated, heart muscle consumes glucose at a steady rate. When oxygen is added to the blood, the rate of glucose consumption drops dramatically, then is maintained at the new, lower rate. Explain.
19. Regulation of PFK-1 The graph shows the effect of ATP on the allosteric enzyme PFK-1. For a given concentration of fructose 6-phosphate, the PFK-1 activity increases with increasing concentrations of ATP, but there is a point beyond which increasing the concentration of ATP inhibits the enzyme.

The low [A D P] curve begins at the origin and rises slowly, then more rapidly, to peak at about one-third of the way across the horizontal axis and 100 on the vertical axis. It briefly runs horizontally along the top horizontal axis before symmetrically decreasing to end at 3 on the vertical axis at two-thirds of the length of the horizontal axis. The high [A D P] curve appears at almost halfway across the horizontal axis right where the low [A D P] curve begins to drop after peaking. The high [A D P] curve decreases slowly until it reaches 78 on the vertical axis at about three-quarters of the length of the horizontal axis, then drops rapidly to 20 on the vertical axis near the end of the horizontal axis, before slowly descending to end at 5 on the vertical axis at the right end of the horizontal axis. All data are approximate.
1. Explain how ATP can be both a substrate and an inhibitor of PFK-1. How is the enzyme regulated by ATP?
2. How do ATP levels regulate glycolysis?
3. The inhibition of PFK-1 by ATP diminishes when the ADP concentration is high, as shown in the graph. What explains this observation?
20. Cellular Glucose Concentration Homeostatic mechanisms maintain the concentration of glucose in human blood at about 5 mm. The concentration of free glucose inside a myocyte is much lower. Why is the concentration so low in the cell? What happens to glucose after entry into the cell? Physicians administer glucose intravenously as a food source in certain clinical situations. Given that the transformation of glucose to glucose 6-phosphate consumes ATP, why not administer intravenous glucose 6-phosphate instead?
21. Ethanol Production in Yeast When grown anaerobically on glucose, yeast (S. cerevisiae) converts pyruvate to acetaldehyde, then reduces acetaldehyde to ethanol using electrons from NADH. Write the equation for the second reaction, and calculate its equilibrium constant at $25 degree upper C$ $25 degree upper C$ , given the standard reduction potentials in Table 13-7.
22. Pathway of Atoms in Fermentation An investigator carries out a “pulse-chase” experiment using $^{14} C$ $Superscript 14 Baseline zero width space upper C$ -labeled carbon sources on a yeast extract maintained under strictly anaerobic conditions to produce ethanol. The experiment consists of incubating a small amount of $^{14} C$ $Superscript 14 Baseline zero width space upper C$ -labeled substrate (the pulse) with the yeast extract just long enough for each intermediate in the fermentation pathway to become labeled. The addition of excess unlabeled glucose then “chases” the label through the pathway. The chase effectively prevents any further entry of labeled glucose into the pathway.
1. If the investigator uses $[1 -^{14} C]$ $left-bracket 1 hyphen Superscript 14 Baseline upper C right-bracket$ glucose (glucose labeled at $C- 1$ $upper C hyphen 1$ with $^{14} C$ $Superscript 14 Baseline zero width space upper C$ ) as a substrate, what is the location of $^{14} C$ $Superscript 14 Baseline zero width space upper C$ in the product ethanol? Explain.
2. Where would $^{14} C$ $Superscript 14 Baseline zero width space upper C$ have to be located in the starting glucose to ensure that all the $^{14} C$ $Superscript 14 Baseline zero width space upper C$ activity is liberated as $^{14} {CO}_{2}$ $Superscript 14 Baseline zero width space CO Subscript 2 Baseline$ during fermentation to ethanol? Explain.
23. Heat from Fermentations Large-scale industrial fermenters generally require constant, vigorous cooling. Why?
24. Fermentation to Produce Soy Sauce Soy sauce preparation involves fermenting a salted mixture of soybeans and wheat with several microorganisms, including yeast, over a period of 8 to 12 months. The resulting sauce (after solids are removed) is rich in lactate and ethanol. How are these two compounds produced? To prevent the soy sauce from having a strong vinegary taste (vinegar is dilute acetic acid), oxygen must be kept out of the fermentation tank. Why?
25. Glucogenic Substrates A common procedure for determining the effectiveness of compounds as precursors of glucose in mammals is to starve the animal until the liver glycogen stores are depleted and then administer the compound in question. A substrate that leads to a net increase in liver glycogen is termed glucogenic, because it must first be converted to glucose 6-phosphate. Show by means of known enzymatic reactions which of these substances are glucogenic:

Molecule a is Succinate. C H 2 is bonded to C O O minus to the left and to C H 2 to the right further bonded to C O O minus. Molecule b is Glycerol. C H 2 is bonded to O H above and to C H to the right bonded to O H above and to C H 2 to the right bonded to O H above. Molecule c is Acetyl Co A. C H 3 is bonded to C to the right double bonded to O above and bonded to S to the right further bonded to C o A. Molecule d is Pyruvate. C H 3 is bonded to C double bonded to O above and bonded to C O O minus to the right. Molecule e is Butyrate. C H 3 is bonded to C H 2 bonded to C H 2 bonded to C O O minus.
26. Pathway of Atoms in Gluconeogenesis An investigator briefly incubates a liver extract capable of carrying out all the normal metabolic reactions of the liver in separate experiments with two different $^{14} C$ $Superscript 14 Baseline zero width space upper C$ -labeled precursors: $[^{14} C]$ $left-bracket Superscript 14 Baseline zero width space upper C right-bracket$ bicarbonate and $[^{14} C]$ $left-bracket Superscript 14 Baseline zero width space upper C right-bracket$ pyruvate.

Trace the pathway of each precursor through gluconeogenesis. Indicate the location of $^{14} C$ $Superscript 14 Baseline zero width space upper C$ in all intermediates and in the product, glucose.
27. Energy Cost of a Cycle of Glycolysis and Gluconeogenesis What is the cost (in ATP equivalents) of transforming glucose to pyruvate via glycolysis and back again to glucose via gluconeogenesis?
28. Relationship between Gluconeogenesis and Glycolysis Why is it important that gluconeogenesis is not the exact reversal of glycolysis?
29. Energetics of the Pyruvate Kinase Reaction Explain in bioenergetic terms how the conversion of pyruvate to phosphoenolpyruvate in gluconeogenesis overcomes the large, negative, standard free-energy change of the pyruvate kinase reaction in glycolysis.
30. Muscle Wasting in Starvation One consequence of starvation is a reduction in muscle mass. What happens to the muscle proteins?
31. Effect of Phloridzin on Carbohydrate Metabolism Phloridzin, a toxic glycoside from the bark of the pear tree, blocks the normal reabsorption of glucose from the kidney tubule, thus causing blood glucose to be almost completely excreted in the urine. In an experiment, rats fed phloridzin and sodium succinate excreted about 0.5 mol of glucose (made by gluconeogenesis) for every 1 mol of sodium succinate ingested. How do rats transform the succinate to glucose? Explain the stoichiometry.
32. Excess $O_{2}$ $bold upper O Subscript bold 2$ Uptake during Gluconeogenesis The conversion of lactate to glucose in the liver requires the input of 6 mol of ATP for every mol of glucose produced. Investigators can monitor the extent of this process in a rat liver preparation by administering $[^{14} C]$ $left-bracket Superscript 14 Baseline zero width space upper C right-bracket$ lactate and measuring the amount of $[^{14} C]$ $left-bracket Superscript 14 Baseline zero width space upper C right-bracket$ glucose produced. Because the stoichiometry of $O_{2}$ $upper O Subscript 2$ consumption and ATP production is known (about 5 ATP per $O_{2}$ $upper O Subscript 2$ ), investigators can predict the extra $O_{2}$ $upper O Subscript 2$ consumption above the normal rate after administering a given amount of lactate. However, when they actually measure extra $O_{2}$ $upper O Subscript 2$ used in the synthesis of glucose from lactate, it is always higher than what the stoichiometric relationships predict. Suggest a possible explanation for this observation.
33. Role of the Pentose Phosphate Pathway If the oxidation of glucose 6-phosphate via the pentose phosphate pathway were being used primarily to generate NADPH for biosynthesis, the other product, ribose 5-phosphate, would accumulate. What problems might this cause?

Data Analysis Problem

34. Engineering a Fermentation System Fermentation of plant matter to produce ethanol for fuel is one potential method for reducing the use of fossil fuels and thus the ${CO}_{2}$ $CO Subscript 2$ emissions that lead to global warming. Many microorganisms can break down cellulose, then ferment the glucose to ethanol. However, many potential cellulose sources, including agricultural residues and switchgrass, also contain substantial amounts of arabinose, which is not as easily fermented.

Escherichia coli is capable of fermenting arabinose to ethanol, but it is not naturally tolerant of high ethanol levels, thus limiting its utility for commercial ethanol production. Another bacterium, Zymomonas mobilis, is naturally tolerant of high levels of ethanol but cannot ferment arabinose. Deanda, Zhang, Eddy, and Picataggio (1996) described their efforts to combine the most useful features of these two organisms by introducing the E. coli genes for the arabinose-metabolizing enzymes into Z. mobilis.
1. Why is this a simpler strategy than the reverse: engineering E. coli to be more ethanol-tolerant?
  Deanda and colleagues inserted five E. coli genes into the Z. mobilis genome: araA, coding for l-arabinose isomerase, which interconverts l-arabinose and l-ribulose; araB, l-ribulokinase, which uses ATP to phosphorylate l-ribulose at C-5; araD, l-ribulose 5-phosphate epimerase, which interconverts l-ribulose 5-phosphate and l-xylulose 5-phosphate; talB, transaldolase; and tktA, transketolase.
2. For each of the three ara enzymes, briefly describe the chemical transformation it catalyzes and, where possible, name an enzyme discussed in this chapter that carries out an analogous reaction.
  The five E. coli genes inserted in Z. mobilis allowed the entry of arabinose into the nonoxidative phase of the pentose phosphate pathway (Fig. 14-31), where it was converted to glucose 6-phosphate and fermented to ethanol.
3. The three ara enzymes eventually converted arabinose into which sugar?
4. The product from part (c) feeds into the pathway shown in Figure 14-31a. Combining the five E. coli enzymes listed above with the enzymes of this pathway, describe the overall pathway for the fermentation of six molecules of arabinose to ethanol.
5. What is the stoichiometry of the fermentation of six molecules of arabinose to ethanol and ${CO}_{2}$ $CO Subscript 2$ ? How many ATP molecules would you expect this reaction to generate?
6. Z. mobilis uses a pathway for ethanol fermentation that is slightly different from the one described in this chapter. As a result, the expected ATP yield is only 1 ATP per molecule of arabinose. Although this is less beneficial for the bacterium, it is better for ethanol production. Why?
  Another sugar commonly found in plant matter is xylose.
7. What additional enzymes would you need to introduce into the modified Z. mobilis strain described above to enable it to use xylose as well as arabinose to produce ethanol? You don’t need to name the enzymes (they may not even exist in the real world); just give the reactions they would need to catalyze.

Reference

Deanda, K., M. Zhang, C. Eddy, and S. Picataggio. 1996. Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering. Appl. Environ. Microbiol. 62:4465–4470.