Glycolysis,
Gluconeogenesis, and
the Pentose Phosphate
Pathway
S-160
1. 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 prepara-
tory 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.
2. The Payoff Phase of Glycolysis in 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 equa-
tion for the payoff phase of glycolysis (with lactate as the end product), including the net standard
free-energy change.
chapter
14
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Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway S-161
3. GLUT Transporters Compare the localization of GLUT4 with that of GLUT2 and GLUT3, and
explain why these localizations are important in the response of muscle, adipose tissue, brain, and
liver to insulin.
4. 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 C, given the standard
reduction potentials in Table 13–7.
5. Energetics of the Aldolase Reaction Aldolase catalyzes the glycolytic reaction
Fructose 1,6-bisphosphate 88n glyceraldehyde 3-phosphate dihydroxyacetone phosphate
The standard free-energy change for this reaction in the direction written is 23.8 kJ/mol. The con-
centrations of the three intermediates in the hepatocyte of a mammal are: fructose 1,6-bisphosphate,
1.4 10
5
M
; glyceraldehyde 3-phosphate, 3 10
6
M
; and dihydroxyacetone phosphate, 1.6 10
5
M
.
At body temperature (37 C), what is the actual free-energy change for the reaction?
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S-162 Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway
6. Pathway of Atoms in Fermentation A “pulse-chase“ experiment using
14
C-labeled carbon sources
is carried out on a yeast extract maintained under strictly anaerobic conditions to produce ethanol.
The experiment consists of incubating a small amount of
14
C-labeled substrate (the pulse) with the
yeast extract just long enough for each intermediate in the fermentation pathway to become labeled.
The label is then “chased” through the pathway by the addition of excess unlabeled glucose. The chase
effectively prevents any further entry of labeled glucose into the pathway.
(a) If [1-
14
C]glucose (glucose labeled at C-1 with
14
C) is used as a substrate, what is the location of
14
C in the product ethanol? Explain.
(b) Where would
14
C have to be located in the starting glucose to ensure that all the
14
C activity is
liberated as
14
CO
2
during fermentation to ethanol? Explain.
7. Heat from Fermentations Large-scale industrial fermenters generally require constant, vigorous cool-
ing. Why?
8. Fermentation to Produce Soy Sauce Soy sauce is prepared by fermenting a salted mixture of soy-
beans 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 vinegar taste (vinegar is dilute acetic acid),
oxygen must be kept out of the fermentation tank. Why?
9. Equivalence of Triose Phosphates
14
C-Labeled glyceraldehyde 3-phosphate was added to a yeast
extract. After a short time, fructose 1,6-bisphosphate labeled with
14
C at C-3 and C-4 was isolated.
What was the location of the
14
C label in the starting glyceraldehyde 3-phosphate? Where did the sec-
ond
14
C label in fructose 1,6-bisphosphate come from? Explain.
10. Glycolysis Shortcut Suppose you discovered a mutant yeast whose glycolytic pathway was shorter
because of the presence of a new enzyme catalyzing the reaction
Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway S-163
Would shortening the glycolytic pathway in this way benefit the cell? Explain.
11. Role of Lactate Dehydrogenase During strenuous activity, the demand for ATP in muscle tissue is
vastly increased. In rabbit leg muscle or turkey flight muscle, the ATP is produced almost exclusively
by lactic acid fermentation. ATP is formed in the payoff phase of glycolysis by two reactions, promoted
by phosphoglycerate kinase and pyruvate kinase. Suppose skeletal muscle were devoid of lactate dehy-
drogenase. Could it carry out strenuous physical activity; that is, could it generate ATP at a high rate
by glycolysis? Explain.
12. Efficiency of ATP Production in Muscle The transformation of glucose to lactate in myocytes re-
leases only about 7% of the free energy released when glucose is completely oxidized to CO
2
and H
2
O.
Does this mean that anaerobic glycolysis in muscle is a wasteful use of glucose? Explain.
Glyceraldehyde 3-phosphate H
2
3-phosphoglycerate
NAD
NADH H
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S-164 Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway
13. 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 (K
eq
0.08; G 6.3 kJ/mol), yet the flow
through this point in the glycolytic pathway proceeds smoothly. How does the cell overcome the unfa-
vorable equilibrium?
14. Arsenate Poisoning Arsenate is structurally and chemically similar to inorganic phosphate (P
i
), 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:
O
OO
O
AsOCR H
2
OO
OO
O
AsOCR HHO
On the other hand, acyl phosphates, such as 1,3-bisphosphoglycerate, are more stable and undergo fur-
ther enzyme-catalyzed transformation in cells.
(a) Predict the effect on the net reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase if
phosphate were replaced by arsenate.
(b) What would be the consequence to an organism if arsenate were substituted for phosphate?
Arsenate is very toxic to most organisms. Explain why.
15. 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
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 fermenta-
tion under these conditions, ethanol, CO
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
2
.
(a) Why did fermentation cease when the supply of phosphate was exhausted?
(b) Why did ethanol and CO
2
accumulate? Was the conversion of pyruvate to ethanol and CO
2
essen-
tial? Why? Identify the hexose bisphosphate that accumulated. Why did it accumulate?
(c) Why did the substitution of arsenate for phosphate prevent the accumulation of the hexose bis-
phosphate yet allow fermentation to ethanol and CO
2
to go to completion? (See Problem 14.)
16. 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?
17. Synthesis of Glycerol Phosphate The glycerol 3-phosphate required for the synthesis of glyc-
erophospholipids can be synthesized from a glycolytic intermediate. Propose a reaction sequence for
this conversion.
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18. Severity of Clinical Symptoms Due to Enzyme Deficiency The clinical symptoms of two forms
of galactosemia—deficiency of galactokinase or of UDP-glucose:galactose 1-phosphate uridylyltransferase—
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.
19. Muscle-Wasting in Starvation One consequence of starvation is a reduction in muscle mass. What
happens to the muscle proteins?
20. Pathway of Atoms in Gluconeogenesis A liver extract capable of carrying out all the normal meta-
bolic reactions of the liver is briefly incubated in separate experiments with the following
14
C-labeled
precursors:
(a) [
14
C]Bicarbonate,
(b) [1-
14
C]Pyruvate,
HO
14
C
O
C
O
14
COCH
3
O
O
Trace the pathway of each precursor through gluconeogenesis. Indicate the location of
14
C in all inter-
mediates and in the product, glucose.
Answer
Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway S-167
21. Energy Cost of a Cycle of Glycolysis and Gluconeogenesis What is the cost (in ATP equiva-
lents) of transforming glucose to pyruvate via glycolysis and back again to glucose via gluconeogenesis?
22. Relationship between Gluconeogenesis and Glycolysis Why is it important that gluconeogenesis
is not the exact reversal of glycolysis?
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S-168 Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway
CH
2
OH
C
OH
H
CH
2
OH
CH
3
C S-CoA
CH
3
C
O
O
COO
᎐
CH
3
CH
2
CH
2
COO
᎐
᎐
OOC CH
2
CH
2
COO
᎐
(a) Succinate,
(b) Glycerol,
(c) Acetyl-CoA,
(d) Pyruvate,
(e) Butyrate,
23. 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.
24. 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 glyco-
gen is termed glucogenic because it must first be converted to glucose 6-phosphate. Show by means
of known enzymatic reactions which of the following substances are glucogenic:
Answer
25. Ethanol Affects Blood Glucose Levels The consumption of alcohol (ethanol), especially after peri-
ods 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:
CH
3
CH
2
OH NAD
88n CH
3
CHO NADH H
Explain how this reaction inhibits the transformation of lactate to pyruvate. Why does this lead to
hypoglycemia?
26. Blood Lactate Levels during Vigorous Exercise The concentrations of lactate in blood plasma
before, during, and after a 400 m sprint are shown in the graph.
Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway S-169
(a) What causes the rapid rise in lactate concentration?
(b) What causes the decline in lactate concentration after completion of the sprint? Why does the
decline occur more slowly than the increase?
(c) Why is the concentration of lactate not zero during the resting state?
Answer
Blood [lactate] (M)
0
150
Time (min)
100
50
60
40
Before
0
200
Run
After
20
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S-170 Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway
27. 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.
28. 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 is the succinate transformed to glucose? Explain the stoichiometry.
OC
O
OH
OH
OH
HO HOH
HH
OHH
HOCH
2
CH
2
H
O
Phloridzin
CH
2
29. Excess O
2
Uptake during Gluconeogenesis Lactate absorbed by the liver is converted to glucose,
with the input of 6 mol of ATP for every mole of glucose produced. The extent of this process in a rat
liver preparation can be monitored by administering [
14
C]lactate and measuring the amount of
[
14
C]glucose produced. Because the stoichiometry of O
2
consumption and ATP production is known
(about 5 ATP per O
2
), we can predict the extra O
2
consumption above the normal rate when a given
amount of lactate is administered. However, when the extra O
2
used in the synthesis of glucose from
lactate is actually measured, it is always higher than predicted by known stoichiometric relationships.
Suggest a possible explanation for this observation.
30. 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
31. 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
emissions that lead to global
warming. Many microorganisms can break down cellulose then ferment the glucose to ethanol. How-
ever, many potential cellulose sources, including agricultural residues and switchgrass, also contain
substantial amounts of arabinose, which is not as easily fermented.
D–Arabinose
HO
C
C
C
C
CH
2
OH
H
H
HO H
OH
OH
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.
(a) 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.
(b) 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. 14–23), where it was converted to glucose 6-phosphate
and fermented to ethanol.
(c) The three ara enzymes eventually converted arabinose into which sugar?
(d) The product from part (c) feeds into the pathway shown in Figure 14–23. Combining the five
E. coli enzymes listed above with the enzymes of this pathway, describe the overall pathway for
the fermentation of 6 molecules of arabinose to ethanol.
(e) What is the stoichiometry of the fermentation of 6 molecules of arabinose to ethanol and CO
2
?
How many ATP molecules would you expect this reaction to generate?
(f) Z. mobilis uses a slightly different pathway for ethanol fermentation 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?
S-172 Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway
D–Xylose
HO
C
C
C
C
CH
2
OH
H
HO
HOH
OH
H
(g) What additional enzymes would you need to introduce into the modified Z. mobilis strain de-
scribed 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.
Answer
Another sugar commonly found in plant matter is xylose.