SEATWORK 7 BIOCHEMISTRY GLUCONEOGENESIS TO OXIDATIVE PHOSPHORYLATION

Please accomplish this as a group. Members of the same group should work together, members of different
groups should not. You are not allowed to consult with members of other groups – that is considered cheating.

1. Researchers isolated a yeast phosphofructokinase (PFK) mutant in which a serine at the fructose-2,6-
bisphosphate binding site was replaced with an aspartate residue. The amino acid substitution
completely abolished the binding of fructose-2,6-bisphosphate to PFK. There was a dramatic decline
in glucose consumption and ethanol production in the mutant compared to control yeast. (a) Propose a
hypothesis that explains why the mutant PFK cannot bind fructose-2,6-bisphosphate. (b) What does the
decline of glucose consumption and ethanol production in the yeast reveal about the role of fructose-
2,6-bisphosphate in glycolysis.

2. Trypanosomes living in the bloodstream obtain all their free energy from glycolysis. They take up
glucose from the host’s blood and excrete pyruvate as a waste product. In this part of their life cycle,
trypanosomes do not carry out any oxidative phosphorylation, but they do use another oxygen-
dependent pathway, which is absent in mammals, to oxidize NADH. (a) Why is this other pathway
necessary? (b) Would the pathway be necessary if the trypanosome excreted lactate rather than
pyruvate? (c) Why would this pathway be a good target for antiparasitic drugs?

3. 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:

Explain how this reaction inhibits the transformation of lactate to pyruvate. Why does this lead to
hypoglycemia?

4. In the first bypass step of gluconeogenesis, the conversion of pyruvate to phosphoenolpyruvate,

pyruvate is carboxylated by pyruvate carboxylase to oxaloacetate, which is subsequently
decarboxylated by PEP carboxykinase to yield phosphoenolpyruvate. The observation that the addition
of CO2 is directly followed by the loss of CO2 suggests that 14C of 14CO2 would not be incorporated
into PEP, glucose, or any intermediates in gluconeogenesis. However, when a rat liver preparation
synthesizes glucose in the presence of 14CO2, 14C slowly appears in PEP and eventually at C-3 and C-
4 of glucose. How does the 14C label get into PEP and glucose? (Hint: During gluconeogenesis in the
presence of 14CO2, several of the four carbon citric acid cycle intermediates also become labeled.)

5. Metabolites in rat muscle were measured before and after exercising. After exercise, the rat muscle
showed an increase in oxaloacetate concentration, a decrease in phosphoenolpyruvate concentration
and no change in pyruvate concentration. Explain.

6. Fluoroacetate, prepared commercially for rodent control, is also produced by a South African plant.
After entering a cell, fluoroacetate is converted to fluoroacetyl-CoA in a reaction catalyzed by the
enzyme acetate thiokinase:

The toxic effect of fluoroacetate was studied in an experiment using intact isolated rat heart. After the
heart was perfused with 0.22 mM fluoroacetate, the measured rate of glucose uptake and glycolysis
decreased, and glucose 6-phosphate and fructose 6-phosphate accumulated. Examination of the citric
acid cycle intermediates revealed that their concentrations were below normal, except for citrate, with
a concentration 10 times higher than normal. (a) Where did the block in the citric acid cycle occur?
What caused citrate to accumulate and the other cycle intermediates to be depleted? (b) Fluoroacetyl-
CoA is enzymatically transformed in the citric acid cycle. What is the structure of the end product of
fluoroacetate metabolism? Why does it block the citric acid cycle? How might the inhibition be

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 overcome? (c) In the heart perfusion experiments, why did glucose uptake and glycolysis decrease?
Why did hexose monophosphates accumulate? (d) Why is fluoroacetate poisoning fatal?

7. The carboxylation of pyruvate by pyruvate carboxylase occurs at a very low rate unless acetyl-CoA, a
positive allosteric modulator, is present. If you have just eaten a meal rich in fatty acids
(triacylglycerols) but low in carbohydrates (glucose), how does this regulatory property shut down the
oxidation of glucose to CO2 and H2O but increase the oxidation of acetyl-CoA derived from fatty acids?
(Hint: A lot of acetyl CoA results from the oxidation of fatty acids.)

8. Cellular respiration can be studied in isolated mitochondria by measuring oxygen consumption under
different conditions. If 0.01 M sodium malonate is added to actively respiring mitochondria that are
using pyruvate as fuel source, respiration soon stops and a metabolic intermediate accumulates. (a)
What is the structure of this intermediate? (b) Explain why it accumulates. (c) Explain why oxygen
consumption stops. (d) Aside from removal of the malonate, how can this inhibition of respiration be
overcome? Explain.

9. When the antibiotic valinomycin is added to actively respiring mitochondria, several things happen:
the yield of ATP decreases, the rate of O2 consumption increases, heat is released, and the pH gradient
across the inner mitochondrial membrane increases. Does valinomycin act as an uncoupler or an
inhibitor of oxidative phosphorylation? Explain the experimental observations in terms of the
antibiotic’s ability to transfer K+ ions across the inner mitochondrial membrane.

10. UCP1 is an uncoupling protein in brown fat. Experiments using UCP1-knockout mice (animals missing
the gene for UCP1) resulted in the discovery of a second uncoupling protein named UCP2. (a) Oxygen
consumption increased over twofold when a β-3 adrenergenic agonist that stimulates UCP1 was
injected into normal mice. This was not observed when the agonist was injected into the knockout mice.
Explain these results. (b) The UCP1-knockout mice were essentially normal except for increased lipid
deposition in their adipose tissue. Explain why. (c) In one experiment, normal mice and UCP1-
knockout mice were placed in a cold (5 °C) room overnight. The normal mice were able to maintain
their body temperature at 37 °C even after 24 hours in the cold. But the body temperatures of the cold-
exposed knockout mice decreased 10 °C or more. Explain. (d) UCP1-knockout mice did not become
obese when fed a high-fat diet. Propose a hypothesis that explains this observation.

11. Dicyclohexylcarbodiimide (DCCD) is a reagent that reacts with Asp or Glu residues. Explain why the
reaction of DCCD with just one c subunit completely blocks both the ATP-synthesizing and ATP-
hydrolyzing activity of ATP synthase.

12. A patient seeks treatment because her metabolic rate is twice normal and her temperature is elevated.
A biopsy reveals that her muscle mitochondria are structurally unusual and not subject to normal
respiratory controls. Electron transport takes place regardless of the concentration of ADP. (a) What is
the P:O ratio (compared to normal) of NADH that enters the electron transport chain in the
mitochondria of this patient? (b) Why are the patient’s metabolic rate and temperature elevated? (c)
Will this patient be able to carry out strenuous exercise?

13. When O2 is added to an anaerobic suspension of cells consuming glucose at a high rate, the rate of
glucose consumption declines greatly as the O2 is used up, and accumulation of lactate ceases. This
effect, first observed by Louis Pasteur in the 1860s, is characteristic of most cells capable of both
aerobic and anaerobic glucose catabolism. (a) Why does the accumulation of lactate cease after O2 is
added? (b) Why does the presence of O2 decrease the rate of glucose consumption? (c) How does the
onset of O2 consumption slow down the rate of glucose consumption? Explain in terms of specific
enzymes.

14. Muscle biopsy and enzyme assays of a carnitine-deficient individual show that medium-chain (C8–C10)
fatty acids can be metabolized normally, despite the carnitine deficiency. What does this tell you about
the role of carnitine in fatty acid transport across the inner mitochondrial membrane?

15. The β oxidation pathway was elucidated in part by Franz Knoop in 1904. He fed dogs fatty acid phenyl
derivatives and then analyzed their urine for the resulting metabolites. What metabolite was produced
when the dogs were fed with (a) phenylpropionate and (b) phenylbutyrate?

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16. How many molecules of ATP are generated when a fully saturated 17-carbon fatty acid is oxidized via
the β oxidation pathway?

17. Two siblings born years apart were separately diagnosed with pyruvate carboxylase deficiency. Both
neonates died less than a month after they were born. Blood samples taken before the death of the
infants showed a high concentration of ketone bodies in the blood. Explain why the ketone body
concentration was elevated.

*****NOTHING FOLLOWS*****

“Thank you for making me so wonderfully complex! Your workmanship is marvelous — how well I
know it.”

—Psalm 139:14