LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL. 2. BIOENERGETICS AND METABOLISM - 2014

PART II. BIOENERGETICS AND METABOLISM

17. FATTY ACID CATABOLISM

Questions and Problems

1. Energy of triacylglycerols.

On a per-carbon basis, where is the most biologically useful energy located in triacylglycerols: in the fatty acid or the glycerol moiety? Explain how the Chemical Structure of triacylglycerols Supports your answer.

2. Energy reserves in adipose tissue.

Among the major nutrients, triacylglycerols, with their hydrocarbon-like Fatty acids, contain the highest energy density.

(a) If 15% of the body weight of a normal adult (mass 70.0 kg) consists of triacylglycerols, What is the total energy reserve in the form of triacylglycerols, expressed in both kilojoules and kilocalories? 1.00 kcal = 4.18 kJ.

(b) If the daily energy requirement is approximately 8400 kJ/day (2000 kcal/day), how long could a person survive if the Oxidation of Fatty acids stored as triacylglycerols were the sole source of energy?

(c) How much body weight (in pounds) would be lost per day under these starvation conditions? 1 lb = 0.454 kg.

3. Common reactions in Fatty acid oxidation and The Tricarboxylic Acid Cycle.

Cells frequently employ similar enzymatic reaction patterns for analogous metabolic transformations. For example, the steps in the Oxidation of Pyruvate to acetyl-CoA and of α-ketoglutarate to succinyl-CoA are very similar, although catalyzed by different Enzymes. The sequence of reactions in the initial stage of fatty acid oxidation closely resembles a sequence in the tricarboxylic acid cycle. Using equations, illustrate the analogous reaction sequences of these two metabolic pathways.

4. β-Oxidation: how many cycles?

How many cycles of β-oxidation are required for the Complete oxidation of activated oleic acid (18:1 (∆⁹))?

5. Acyl-CoA synthetase reactions.

Fatty acids are converted into their coenzyme A esters in a reversible reaction catalyzed by acyl-CoA synthetase:

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(a) The enzyme-bound intermediate in this reaction has been shown to be a mixed anhydride of a Fatty acid and adenosine monophosphate (AMP), an acyl-AMP:

Write two equations representing the two steps of the reaction catalyzed by acyl-CoA synthetase.

(b) The acyl-CoA synthetase reaction is readily reversible, with an Equilibrium Constant close to 1. How can the reaction be driven forward toward The formation of the fatty acyl-CoA derivative?

6. Intermediates in oleic acid oxidation.

What is The structure of the partially oxidized acyl group formed after three cycles of oxidation of oleic acid, 18:1(∆⁹)? What are the next two steps in The oxidation of this compound?

7. β-Oxidation of a fatty acid with an odd number of carbon atoms.

What are the direct products of β-oxidation of a fully saturated straight-chain fatty acid consisting of 11 carbon atoms?

8. Oxidation of tritium-labeled palmitate.

Palmitate uniformly labeled with tritium (³H) to a specific activity of 2.48 • 10⁸ cpm/μmol of palmitate is added to a mitochondrial preparation, where it is oxidized to acetyl-CoA. The acetyl-CoA is isolated and hydrolyzed to acetate. The specific activity of the isolated acetate is 1.00 • 10⁷ cpm/(min • mmol). Is this result related to β-oxidation? Explain. What is the ultimate fate of the removed tritium?

9. Compartmentation in β-oxidation.

Before being oxidized in Cell/35.html">Mitochondria, free palmitate is activated in the Cytosol to its coenzyme A derivative (palmitoyl-CoA). If palmitate and [¹⁴C] coenzyme A are added to a Liver homogenate, palmitoyl-CoA isolated from the cytosolic fraction is radioactive, whereas palmitoyl-CoA from the mitochondrial fraction is not. Explain this result.

10. Comparative biochemistry: energy-yielding pathways in birds.

One indicator of the relative importance of various ATP-producing pathways is the V_max of the enzymatic pathways. The V_max values for several enzymes from the pectoral Muscles (the breast muscles used in flight) of the pigeon and the pheasant are listed below.

a) Compare the roles of glycogen and Fat METABOLISM in ATP production within the pectoral muscles of these two birds.

b) Compare the oxygen consumption of the two birds.

c) Based on the data in the table, deduce which of the birds is capable of long-distance flight. Justify your answer.

d) Why were these specific enzymes chosen for comparison? Are the activities of triose phosphate isomerase and malate dehydrogenase equally suitable for comparison? Explain.

11. Carnitine acyltransferase mutation.

What metabolic alterations would result from a mutation in Muscle carnitine acyltransferase I in which the mutant protein loses its affinity for malonyl-CoA while retaining its catalytic activity?

12. Effect of carnitine deficiency.

A patient developed a condition characterized by progressive muscle weakness and painful muscle cramps. Fasting, exercise, and a high-fat diet exacerbated the symptoms. A homogenate prepared from a Skeletal Muscle biopsy of the patient oxidized added oleate more slowly than control homogenates from healthy individuals. Upon addition of carnitine to the patient's muscle homogenate, the oxidation rate matched that of the control homogenate. The patient was diagnosed with carnitine deficiency.

a) Why does The addition of carnitine increase The rate of oleate oxidation in the patient's muscle homogenate?

b) Why did fasting, exercise, and high-fat foods worsen the patient's symptoms?

c) Propose two possible causes for carnitine deficiency in such a person's muscles.

13. Fatty acids as a source of metabolic Water.

Contrary to popular belief, camels do not store water in their humps; rather, the humps contain large fat reserves. How can this fat serve as a source of water? Calculate the volume of water (in liters) that can be produced in a camel's body from 1.0 kg of fat. For simplicity, assume that the fat consists entirely of tripalmitoylglycerol.

14. Petroleum as a food source for microbes.

Certain microorganisms of the genera Nocardia and Pseudomonas are able to grow in environments where Hydrocarbons serve as the sole food source. These Bacteria oxidize straight-chain aliphatic hydrocarbons, such as octane, into the corresponding carboxylic acids:

СН₃(СН₂)₆СН₃ + NAD⁺ + O₂ ⇄ СН₃(СН₂)₆СООН + NADH + Н⁺

How can these bacteria be utilized for cleaning up oil spills? What factors might limit the efficiency of this process?

15. Metabolism of a straight-chain fatty acid with a phenyl substituent.

A crystalline metabolite was isolated from the urine of a rabbit fed a straight-chain fatty acid with a terminal phenyl group.

A 302 mg sample of the metabolite in aqueous solution was completely neutralized by the addition of 22.2 mL of 0.100 M NaOH.

a) What are the possible molecular mass and STRUCTURE OF THE metabolite?

b) Does the straight-chain fatty acid contain an even or odd number of methylene groups (-СН₂^-)n (where n is even or odd)? Explain.

16. Fatty acid oxidation and uncontrolled diabetes.

When The amount of acetyl-CoA produced via hepatic fatty acid $\beta$-oxidation exceeds the capacity of the tricarboxylic acid cycle, the excess is converted into Ketone Bodies—acetone, acetoacetate, and D-$\beta$-hydroxybutyrate. This occurs during starvation and uncontrolled diabetes: because Tissues cannot utilize glucose, they instead oxidize fatty acids in large amounts. Although acetyl-CoA itself is not toxic, mitochondria must convert it into ketone bodies. What problem would arise if acetyl-CoA could not be converted into ketone bodies? How does this conversion solve the problem?

17. Consequences of a high-fat, carbohydrate-free diet.

Suppose you are forced to subsist on a diet consisting entirely of seal and whale blubber, with little or no CARBOHYDRATES.

a) How would the lack of carbohydrates affect the utilization of fats for energy?

b) If your diet were entirely devoid of carbohydrates, would it be better to consume fatty acids with an even or an odd number of carbon atoms? Explain.

18. Nutritional role of even- and odd-chain fatty acids.

In a laboratory experiment, two groups of rats were fed two Different types of fatty acids as their sole carbon source for a month. The first group received enanthic (heptanoic) acid (7:0), while the second group received caprylic (octanoic) acid (8:0). At the end of the experiment, a striking difference in the condition of the two groups was observed: the animals in the first group were healthy and had gained weight, whereas those In the second group were weak and emaciated due to loss of muscle mass. What is the biochemical basis for this difference?

19. Metabolic consequences of $\omega$-fluorooleate ingestion.

The shrub Dichapetalum toxicarium, native to Sierra Leone, produces $\omega$-fluorooleate, which is highly toxic to warm-blooded animals.

This compound has been used as an arrowhead poison, and the crushed fruits of the plant are sometimes employed as a rat poison. Why is this substance so toxic? (Hint: See Chapter 16, Problem 22.)

20. Mutant acetyl-CoA carboxylase.

How would fat metabolism be affected in a mutant acetyl-CoA carboxylase in which the Ser residue normally phosphorylated by AMPK is replaced by an Ala residue? What would happen if the same Ser residue were replaced by an Asp residue? (Hint: See Fig. 17-12.)

21. Effect of a PDE inhibitor on adipocytes.

How would the response of adipocytes to epinephrine be altered in the presence of a cAMP phosphodiesterase (PDE) inhibitor? (Hint: See Fig. 12-4.)

22. FAD as an electron acceptor.

Acyl-CoA dehydrogenase uses an enzyme-bound FAD as a prosthetic group to dehydrogenate the $\alpha$- and $\beta$-carbons of fatty acid acyl-CoA derivatives. What is the advantage of using FAD rather than NAD⁺ as the electron acceptor? Explain based on the standard reduction potentials of the half-reactions Enz-FAD/FADH₂ ($E^{\prime\circ} = -0.219$ V) and NAD⁺/NADH ($E^{\prime\circ} = -0.320$ V).

23. $\beta$-Oxidation of arachidonic acid.

How many times must the fatty acid oxidation cycle be repeated for the complete oxidation of arachidonic acid (see Table 10-1) to acetyl-CoA?

24. The pathway of Labeled Propionate.

If [3-¹⁴C]propionate (with ¹⁴C in the methyl group) is added to a liver homogenate, ¹⁴C-labeled oxaloacetate is rapidly formed. Outline the pathway by which propionate is converted to oxaloacetate, and indicate THE POSITION OF the ¹⁴C isotope in the resulting oxaloacetate.

25. Phytanic Acid Metabolism.

If mice are fed phytanic acid uniformly labeled with the ¹⁴C isotope, the radioactive label appears within a few minutes in malate, an intermediate of The Citric Acid Cycle. Draw a scheme of this metabolic pathway. Which carbon atoms in malate will contain the ¹⁴C label?

26. Sources of Water Formed during β-Oxidation.

The complete oxidation of palmitoyl-CoA to carbon dioxide and water is described by the overall equation

Пальмитоил-СоА + 23 O₂ + 10 P_i + 108 ADP —> СоА + 16 СO₂ + 108 АТР + 23 Н₂O

Water is also formed in the reaction

ADP + P_i —> АТР + Н₂O

yet it is not included as a product in the overall equation. Why?

27. Biological Significance of Cobalt.

In the rumen of cattle, deer, sheep, and other ruminants, vast amounts of propionate are produced through the bacterial Fermentation of ingested plant material. For these animals, propionate serves as the primary source of glucose, which is synthesized via the pathway: propionate —> oxaloacetate —> glucose. In certain Regions of the world, particularly in Australia, ruminants sometimes exhibit symptoms of anemia accompanied by loss of appetite and stunted growth, which stem from an inability to convert propionate to oxaloacetate. This condition is caused by a cobalt deficiency resulting from extremely low levels of cobalt in the soil and, consequently, in the plant material. Explain.

28. Fat Loss during Hibernation.

During periods of hibernation, which can last for seven months, bears expend approximately 25 • 10⁶ J/day. The energy required to sustain life is provided by the oxidation of fatty acids. What will be the animal's loss of body mass (in kilograms) after seven months? How can Ketosis be minimized during hibernation? (Assume that fat oxidation yields 38 kJ/g.)

Analyzing Experimental Data

29. β-Oxidation of trans-Fatty Acids.

Unsaturated fats with trans double bonds are often referred to simply as "trans fats." The health effects of dietary trans fats have been a subject of considerable debate. In a study investigating The impact of trans fat metabolism on health, Yu and coworkers (2004) demonstrated that model trans- and cis-fatty acids undergo different transformations in the body. The researchers used three related 18-carbon fatty acids to demonstrate the differences in the β-oxidation pathways of cis- and trans-isomers of fatty acids of the same size.

The researchers incubated the CoA derivatives of each acid with rat liver mitochondria for 5 min, after which the remaining CoA derivatives from each mixture were separated by HPLC. The experimental results for all three cases are presented below.

Following the reaction, an internal standard (IS)—pentadecanoyl-CoA—was added to the mixture. Other Abbreviations: C₁₈-CoA — stearoyl-CoA; cis-∆⁵C₁₄-CoA — cis-∆⁵-tetradecenoyl-CoA; cis-∆⁹C₁₈-CoA — oleoyl-CoA; trans-∆⁵C₁₄-CoA — trans-∆⁵-tetradecenoyl-CoA; and elaidoyl-CoA, trans-∆⁹C₁₈-CoA.

a) Why did Yu and coworkers have to use CoA derivatives of fatty acids rather than free fatty acids for their experiments?

b) Why were no low-molecular-weight CoA derivatives detected in the reaction with stearoyl-CoA?

c) How many cycles of β-oxidation are required to convert oleoyl-CoA and elaidoyl-CoA into cis-Δ⁵-tetradecenoyl-CoA and trans-∆⁵-tetradecenoyl-CoA, respectively?

There are two forms of the enzyme acyl-CoA dehydrogenase (see Fig. 17-8, a): long-chain acyl-CoA dehydrogenase (LCAD) and very long-chain acyl-CoA dehydrogenase (VLCAD). Yu and co-workers determined the kinetic parameters for both enzymes. They used CoA derivatives of three fatty acids: tetradecenoyl-CoA (C₁₄-CoA), cis-∆⁵-tetradecenoyl-CoA (cis-∆⁵C₁₄-CoA), and trans-∆⁵-tetradecenoyl-CoA (trans-∆⁵C₁₄-CoA). The experimental results are presented below. (For Definitions of kinetic parameters, see Chapter 6.)

d) For LCAD, the K_m values differ significantly between the cis- and trans-substrates. How can this observation be explained given the molecular structure of each substrate? (Hint: You may refer to Fig. 10-2.)

e) Do the kinetic parameters of the two enzymes describe the conversions of the respective fatty acids only if the Reactions Catalyzed by LCAD and MCAD (or both enzymes) are the rate-limiting steps of the process? Is this the case here, and why?

f) How do the differences in kinetic parameters explain the differences in the levels of CoA derivatives found after incubating rat liver mitochondria with stearoyl-CoA, oleoyl-CoA, and elaidoyl-CoA (see figure)?

Yu et al. determined the substrate Specificity of rat liver mitochondrial thioesterase, which catalyzes the Hydrolysis of acyl-CoA to CoA and free fatty acid (see Chapter 21). This enzyme was found to be approximately twice as active toward C₁₄-CoA thioesters compared to C₁₈-CoA thioesters.

g) Other researchers have suggested that free fatty acids are capable of crossing membranes. Yu and co-workers detected trans-∆⁵-tetradecenoic acid outside the mitochondria (i.e., in the reaction medium) when mitochondria were incubated with elaidoyl-CoA. Describe the pathway by which this extramitochondrial trans-∆⁵-tetradecenoic acid appears. Indicate in which cellular compartments these transformations occur and which specific enzymes catalyze them.

h) Popular science literature often states that "trans fats are not broken down by the body's cells, but instead accumulate in them." In what sense is this statement valid, and in what sense is it an oversimplification?