Download Energy Conversion Pathways 1. Substrate level phosphorylation

Dr. Roger Miesfeld Bioc 460 Spring 2005
Energy Conversion Pathways
1. Substrate level phosphorylation can be used in the cell to generate a high energy nucleotide
triphosphate such ATP or GTP. Give one example of this from the glycolytic pathway, and a
second example from the citrate cycle.
2. Is substrate level phosphorylation restricted to aerobic metabolism? Explain.
3. Write the net reaction derived from the 9 steps of the citrate cycle.
4. What key differences exist between hexokinase and glucokinase to ensure that glucose is
properly apportioned between muscle and liver?
5. Why can't an individual with a deficiency in lactate dehydrogenase activity easily run up three
flights of stairs?
6. Write the net reaction describing the conversion of pyruvate ---> CO2 that takes place in a test
tube lacking mitochondrial membranes, but containing all the enzymes found inside the
mitochondrial matrix.
7. A biochemist set-up an in vitro system that measured oxygen consumption under conditions
in which glucose was completely metabolized. She found that the addition of pyruvate to this
glucose-metabolizing system had little effect on the amount of oxygen consumed, whereas,
addition of fumarate resulted in a very large increase in oxygen consumption. Explain this
observation.
8. Describe the mechanism by which proton-motive force in the mitochondria drives the
synthesis of ATP.
9. How many ATP are synthesized by oxidative phosphorylation from each NADH produced
during aerobic glycolysis in muscle cells. Explain.
10. Write the basic chemical reaction of photosynthesis. Where does this reaction take place on
the planet Earth?
11. Write the net reaction of the Calvin cycle. What is the key enzyme in this cycle and how is it
activated?
12. Name the three primary mechanisms that have been found to regulate the flux of metabolic
pathways. Give one example for each mechanism that illustrates how the rate of metabolic flux
would be regulated.
13. Write the net reaction for the metabolism of glucose by the glycolytic pathway under aerobic
conditions. Is this a reversible pathway?
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Dr. Roger Miesfeld Bioc 460 Spring 2005
14. A muscle biopsy from an individual who was incapable of carrying out prolonged intense
exercise contained a severe deficiency in the glycolytic enzyme phosphoglycerate mutase which
converts 3-phosphoglycerate to 2-phosphoglycerate.
a. What is the explanation for the inability of this individual to exercise intensely?
b. Does this individual suffer from lactic acid build-up in the muscle? Explain.
15. All cells contain hexokinase. Liver cells contain both hexokinase and glucokinase. The
curve below shows the activity of each of these enzymes in a liver cell extract in response to
increasing amounts of glucose. Label the two curves with respect to either hexokinase or
glucokinase.
A
100
B
% max.
rate
0
0
10
[glucose]
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16. The liver stores excess glucose as glycogen (a glucose polymer). Why is it advantageous
for liver cells to have both hexokinase and glucokinase?
17. Write the net reaction of the metabolic step catalyzed by pyruvate dehydrogenase. Why is
this reaction inhibited by a diet deficient in vitamin B1?
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18. When radioactive H-NADH is added to a cell extract containing mitochondria, radioactivity
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quickly appears in the mitochondrial matrix. However, when radioactive C-NADH is added to
the same cell extract, no radioactivity is found in the mitochondrial matrix. Explain this
observation.
19. What are the two primary reasons why a suspension of chloroplasts are unable to
synthesize glucose for an extended period of time when they are shifted from the light to the
dark, even in the presence of the Calvin Cycle substrates CO2 and H2O?
20. Phosphofructokinase (PFK) catalyzes the conversion of Fructose 6-phosphate to Fructose
1,6-bisphosphate and is a key regulatory enzyme in carbohydrate metabolism. Name one
allosteric regulator of PFK activity that activates its activity and one allosteric regulator that
inhibits PFK activity.
21. What is the primary reason that the rate citrate cycle reactions decreases sharply following
addition of the ATP synthase inhibitor oligomycin to isolated mitochondria?
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Dr. Roger Miesfeld Bioc 460 Spring 2005
22. What is the effect on the rate of CO2 production when dinitrophenol (DNP) is added to a
mitochondrial suspension that has been pre-treated with the ATP synthase inhibitor oligomycin?
Explain.
23. The ATP synthase complex is capable of synthesizing 1 mole of ATP for every 3 moles of
H+ that flow through F0 subunit. Based on the observation that FADH2 oxidation results in the
pumping of 6 moles of H+ across the innermembrane, explain why FADH2 oxidation produces
only 1.5 moles of ATP in mitochondria, rather 2 moles of ATP as would be expected?
24. A continual supply of reduced glutathione is required to protect red blood cells against the
toxic effects of pamaquine. Why would individuals with a defect in the enzyme glucose 6phosphate dehydrogenase be susceptible to pamaquine-induced hemolytic anemia?
25. What do photosystem I and II have in common with the electron transport chain?
26. Why do plant cells require mitochondria considering that chloroplasts produce ATP and
glucose, and plants metabolize glucose by the glycolytic pathway?
27. Glycolytic enzymes are allosterically-regulated in response to the Energy Charge of the cell.
Label the graph by circling the best choice at (a), (b), (c) and (d).
28. Early biochemists used an in vitro system containing acetyl CoA with radioactively labeled
carbon to show that the citrate cycle was indeed a cycle.
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a. How did the pattern of C-labeled cycle intermediates demonstrate that this was a
cyclic pathway and not a linear pathway?
b. Explain why no radioactive carbon was found in oxaloacetate when inorganic
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phosphate (Pi) was removed from the in vitro system prior to adding C-Acetyl CoA?
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Dr. Roger Miesfeld Bioc 460 Spring 2005
29. A student set-up an in vitro respiration system using cell extracts in which the rate of
carbohydrate metabolism could be measured by monitoring the conversion of radioactive
glucose to CO2 . The biochemist found that the addition of citrate to this system led to a rapid
decrease in the level of glucose, however, the addition of acetyl CoA had little effect on the rate
of glucose metabolism. Explain this observation.
30. Explain why the energy yield from glucose metabolism is 32 ATP in heart cells that use
primarily the malate-aspartate shuttle, but only 30 ATP in muscle cells that rely on the glycerol
phosphate shuttle to perform the same function .
31. Explain why adding cyanide to a suspension of mitochondria blocks ATP synthesis.
32. Thermogenin is an Electron Transport System (ETS) uncoupling protein that has the same
effect on mitochondria as the chemical uncoupler dinitrophenol (DNP). Why is it advantageous
for hibernating animals to have thermogenin in the mitochondria of special fat cells that surround
arteries bringing blood to the brain?
33. What explains the observation that people born with a deficiency in the enzyme glucose 6phosphate dehydrogenase become clinically anemic if they have a diet rich in fava beans?
34. How many ATP are produced from glucose catabolism in humans under aerobic and
anaerobic conditions? Explain.
35. Infants express the enzyme lactase in their intestines to metabolize lactose. What are the
two products of the lactase reaction? How many pyruvate molecules can be generated from the
metabolism of one molecule of lactose?
36. Explain why it makes sense that the enzyme pyruvate kinase is activated by fructose 1,6bisphosphate when blood glucose levels are high.
37. The liver mitochondrial enzyme pyruvate carboxylase catalyzes the reaction:
Pyruvate + CO2 + ATP + H2O  Oxaloacetate + ADP + Pi + H+
What is most of the oxaloacetate converted into under the following conditions;
a) when blood glucose levels are low and the energy charge is high?
b) when mitochondrial acetyl CoA levels are high and energy charge is low?
38. Why do individuals with the neurological disorder beriberi contain a large amount of pyruvate
in the blood after eating a high carbohydrate meal?
39. Why does it make sense that a dramatic increase in cytosolic levels of cytochrome c
functions as an initiating signal for cell death (apoptosis) in eukaryotes?
40. An ATP synthase complex has been characterized that contains a c ring with 12 identical c
subunits. Experiments have shown that 1 ATP is synthesized for every 4 H+ that move through
the complex into the matrix. Explain this 1:4 ratio in terms of the structure and function of the
gamma and beta subunits of ATP synthase.
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Dr. Roger Miesfeld Bioc 460 Spring 2005
41. What happens to the rate of ATP synthesis when dinitrophenol (DNP) is added to a
suspension of mitochondria that have been pretreated with cyanide? Explain.
42. The diagram below illustrates the photosynthetic process in plants showing two different
labels at five places, only one of each label is correct. Draw a circle around each of the correct
labels in A through E that accurately depicts photosynthesis. There should only be five circles
when you are done.
43. Ribulose 1,5 bisphosphate is a C5 substrate in the Calvin Cycle reaction catalyzed by the
enzyme Rubisco. What is the one C1 substrate and two C3 products of this reaction? The mass
of Rubisco on planet earth is higher than that of any other enzyme, why?
44. Explain why a C4 plant such as crabgrass has a growth advantage in the middle of
summer, whereas, a typical C3 plant such as turf grass has a growth advantage in the spring
and fall when temperatures are moderate.
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Dr. Roger Miesfeld Bioc 460 Spring 2005
Answers
1.
Glycolysis
1,3-BPG + ADP ---> 3-PG + ATP
PEP + ADP ---> Pyruvate + ATP
Citrate cycle
Succinyl-CoA + GDP + Pi ---> Succinate + GTP + CoA
2. No. The two reactions in glycolysis can occur under anaerobic conditions when pyruvate is
converted to lactate.
3.
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H20 --->
2 CO2 + 3 NADH + FADH2 + GTP + 2 H+ + CoA
4. Hexokinase is in all cells, glucokinase is liver-specific
Glucokinase has a much higher Km for glucose than hexokinase
Hexokinase is feedback inhibited by glucose 6-P, glucokinase is not.
5. Because NAD+ is not being regenerated by the lactate dehydrogenase reaction which will
lead to the inhibition of Gly 3-P dehydrogenase and block ATP generation by substrate level
phosphorylation.
6. Pyruvate + 4 NAD+ + FAD + GDP + Pi + 2 H2O --->
3 CO2 + 4 NADH + FADH2 + GTP + 2 H+
Since there is no functional electron transport chain under these conditions, the NADH and
FADH2 accumulate. In intact mitochondria, the NAD/NADH and FAD/FADH2 cancel out and the
net reaction includes ATP ---> ADP + Pi.
7. The rate of oxygen consumption is limited by the rate acetyl CoA metabolism in the citrate
cycle. However, by increasing the concentration of citrate cycle intermediates through the
addition of fumarate, it increased the capacity of the cycle to oxidize acetyl CoA, resulting in a
large increase in oxygen consumption.
8. Oxidation of NADH by the electron transport chain results in proton pumping across the inner
mitochondrial membrane. This proton gradient is utilized by ATP synthase to drive the synthesis
of ATP in response to protons channeling through the enzyme complex back into the matrix.
9. There are only 1.5 net ATP synthesized from the reducing energy in cytosolic NADH in
muscle cells because NADH donates 2 e- to FADH2 by the glycerol phosphate shuttle (other
cells use the malate-aspartate shuttle). FADH2 oxidation results in the production of 1.5 ATP.
10. H2O + CO2 ---> (CH2O) + O2
Photosynthesis takes place in chloroplast organelles of special cells found in plants and
microorganisms.
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Dr. Roger Miesfeld Bioc 460 Spring 2005
11. 6 CO2 + 18 ATP + 12 NADPH + 12 H2O --->
C6H12O6 + 18 ADP + 18 Pi + 12 NADP + 6 H+
Rubisco is the key enzyme in the Calvin Cycle and it is activated by light.
12. The three primary mechanisms are:
a. The amount of rate-limiting enzyme
- changes in gene transcription (hormonal control).
- changes in protein synthesis (amino acid pathways).
b. The catalytic activity of rate-limiting enzymes.
- covalent modifications (phosphorylation).
- non-covalent modifications (cAMP as an allosteric regulator).
c. The accessibility of substrates.
- nutritional supplies (diet).
- cell compartmentalization (mitochondria vs. cytosol).
13.
Glucose + 2 ADP + 2 Pi + 2 NAD+ ----> 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O
No, this is not a reversible pathway (due to coupled ATP hydrolysis reactions).
14. a) The lack of phosphoglycerate mutase blocks production of pyruvate from glucose. During
intense exercise, lactate is normally produced from pyruvate which replenishes the necessary
NAD+ required for the energy production phase of anaerobic glycolysis.
b) No. Lactate only accumulates in the muscle under anaerobic conditions and this individual
cannot carry out intense exercise.
15. Curve A is hexokinase and curve B is glucokinase.
16. Because liver cells can obtain glucose from the blood under all conditions.
17.
Pyruvate + CoA + NAD+ -----> acetyl CoA + CO2 + NADH
Because thiamine pyrophosphate (TPP) is derived from B1 and it is a required cofactor in the
pyruvate dehydrogenase reaction.
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18. The radioactive H is transported inside the mitochondria by shuttle systems (either glycerol
3-P shuttle or malate shuttle), whereas NAD cannot cross the inner mitochondrial membrane
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and therefore the C label does not get into the matrix.
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Dr. Roger Miesfeld Bioc 460 Spring 2005
19. The two reasons are that 1) both ATP and NADPH reserves will be quickly depleted and 2)
the Calvin cycle enzymes, most importantly Rubisco, are activated by the light.
20. Allosteric activators of PFK activity are AMP and F-2,6-BP, and allosteric inhibitors are ATP
and citrate.
21. Oligomycin shuts down ATP synthesis and thus proton movement into the mitochondrial
matrix is inhibited. This leads to a build-up of protons in the innermembrane space and electron
transport decreases to the point that NADH and FADH2 are no longer being oxidized. The
citrate cycle requires NAD and FAD as substrates for the dehydrogenase reactions.
22. The rate of CO2 production would increase. DNP is an uncoupler that permits H+ to enter
the matrix even though ATP synthase is inhibited. Oxidation of NADH and FADH2 following
DNP addition replenishes the supply of the NAD and FAD required for the production of CO2 by
the citrate cycle.
23. It actually requires 4 protons to synthesize ATP in mitochondria since the phosphate
translocase requires 1 proton for every Pi brought into the matrix. Therefore, FADH2 oxidation
only yields 6/4 = 1.5 net ATP by mitochondria.
24. NADPH is required to sustain a high level of reduced glutathione in red blood cells. Glucose
6-phosphate dehydrogenase is the required enzyme for the first step in the Pentose Phosphate
Pathway, and therefore these individuals are unable to produce sufficient levels of NADPH to
protect against pamaquine-induced toxicity.
25. Both photosystems I/II and electron transport capture redox energy and convert it to protonmotive force.
26. Plants need to generate high levels of ATP at night to sustain metabolic activity.
Mitochondria produce 30 ATPs for every glucose metabolized, whereas, glycolysis only
produces a net of 2 ATPs.
27 The correct answers are shown below in bold.
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Dr. Roger Miesfeld Bioc 460 Spring 2005
28. a) The radioactive carbon became incorporated into all of the cycle intermediates at atom
positions that could only be explained by products also serving as substrates. The metabolism
of oxaloacetate in one round of the cycle produces a different radioactively labeled product
depending on the number of cycles. The products of linear pathways are not substrates of other
enzymatic reactions within the same pathway. If CAC were a linear pathway, only one type of
radioactively labeled intermediate would be observed. b) Inorganic phosphate is required by one
of the CAC reaction steps [succinyl CoA synthetase] that couples GTP synthesis to thioester
bond cleavage. Without Pi, this enzyme reaction is inhibited and radioactive carbon would only
be found in cycle intermediates that precede this reaction step.
29. The addition of citrate increased the capacity of the citrate cycle to metabolize acetyl CoA by
increasing the concentration of all cycle intermediates, most importantly, the concentration of
oxaloacetate. The addition of acetyl CoA to this system had little effect because oxaloacetate
levels were limiting and glucose metabolism was already at a maximum rate. The cycle was
working at full capacity and having more substrate available for the citrate synthase reaction had
little effect on the rate of glucose metabolism.
30. The malate-aspartate shuttle results in an even exchange of reducing equivalents in the
form of NADH between both sides of the innermitochondrial membrane. In contrast, the glycerol
phosphate shuttle converts the reducing equivalents of NADH in the cytosol into FADH2 inside
the matrix. Since the glyceraldehyde 3-phosphate dehydrogenase reaction in] glycolysis
generates 2 NADH/glucose metabolized, and FADH2 oxidation only produces 1.5 ATPs,
whereas, NADH oxidation produces 2.5 ATPs, the energy yield from glucose metabolism is 2
ATP/glucose higher in heart cells than in muscle cells.
31. Cyanide blocks electron transfer in the ETS resulting in the loss of the proton motive force
that is required to drive ATP synthesis.
32. Thermogenin uncouples the electron transport system from oxidative phosphorylation.
Therefore, more heat is generated by the ETS in thermogenin containing fat cells since none of
the energy is used for bond energy by the ATP synthase complex. Redox heat created by an
uncoupled ETS in these special cells keeps the animal alive even in the middle of winter.
Normally, animals eat every day which provides fuel for thermoregulation and ATP synthesis.
Hibernating animals are dependent on stored fat for energy to keep cells alive and on
thermongenin-mediated futile cycling to create sufficient thermoregulation.
33. Glucose 6-phosphate dehydrogenase is required in the Pentose Phosphate Pathway to
generate reducing equivalents in the form of NADPH. Fava beans contain toxic compounds that
must be reduced by glutathione in red blood cells. Since NADPH is required to keep glutathione
in the reduced state, the red blood cells in these individuals are highly susceptible to favism, a
diet-induced form of anemia.
34. Under aerobic conditions glucose metabolism produces 30 ATP as a result of glycolysis, the
citrate cycle and oxidative phosphorylation. Under anaerobic conditions glucose is metabolized
to pyruvate which is converted to lactate; the net ATP yield from glycolysis alone is 2 ATP.
35. The enzyme lactase converts lactose to glucose and galactose. Lactose metabolism results
in the production of 4 pyruvate molecules.
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Dr. Roger Miesfeld Bioc 460 Spring 2005
36. Pyruvate kinase converts phosphoenolpyruvate and ADP to pyruvate and ATP in the last
step of the glycolytic pathway. When blood glucose levels are high, cells metabolize glucose to
form glycolytic intermediates such as fructose 1,6-bisphosphate, which in turn, activates
pyruvate kinase to stimulate metabolic flux in the direction of pyruvate formation.
37. a) When blood glucose levels are low the oxaloacetate is converted to PEP and used in the
gluconeogenic pathway to produce glucose for release into the blood. b) When mitochondrial
acetyl CoA levels are high, the oxaloacetate is converted to citrate in the citrate cycle to meet
the energy needs of the cell.
38. Beriberi is nutritional deficiency in thiamine pyrophosphate (vitamin B1) which is a required
cofactor in the enzyme pyruvate dehydrogenase. Individuals with beriberi cannot readily
metabolize pyruvate to acetyl CoA because pyruvate dehydrogenase is inactive without
thiamine pyrophosphate. This leads to a build-up of pyruvate in the blood under conditions
when glycolysis is stimulated.
39. Cytochrome c is required for electron transport and is localized to the inner mitochondrial
membrane, it is not supposed to be in the cytosol. The presence of high levels of cytochrome c
in the cytosol signals that the mitochondria are not functioning properly and induces apoptosis.
40. When the H+ binds to a single c subunit, it causes a clockwise rotation of the c ring 30
degrees (1/12 of a circle). The three-sided gamma subunit rotates along with the c ring and
interacts directly with the three beta subunits. For every 120 degree rotation of the c ring, the
gamma subunit induces the O, T or L conformation in each of the beta subunits. Since an ATP
is synthesized every time the gamma subunit completes a turn of 120 degrees (T to O
conformational change in a beta subunit), it requires 4 H+ moving through the complex in order
to synthesize 1 ATP (4 x 30 degree rotation).
41. Cyanide blocks the electron transport system causing ATP synthesis to stop. Neither the
rates of ATP synthesis, nor oxygen consumption, will change (increase) when the uncoupler
dinitrophenol (DNP) is added because the electron transport chain is blocked by cyanide.
42. The correct answers are circled in the diagram below.
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Dr. Roger Miesfeld Bioc 460 Spring 2005
43. The C1 substrate is CO2 and the C3 products are two moles of 3-phosphoglcerate. Rubisco
is a very large multisubunit enzyme that is found in all plants, most notably, marine
photosynthetic organisms.
44. Photorespiration is a "wasteful" reaction because 2-phosphoglycerate must be salvaged by
the glycolate pathway at the expense of ATP hydrolysis, therefore, the C4 plant has an
advantage in the heat of summer because it can minimize loss of carbon to photorespiration.
However, in the spring and fall when temperatures are mild (and O2 solubility is low), then the
energy cost of running the C4 pathway (two high energy phosphate bonds are needed to
convert pyruvate to phosphoenolpyruvate in mesophyll cells) becomes a disadvantage and C3
plants thrive.
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