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

CHAPTER II. BIOENERGETICS AND METABOLISM

21. BIOSYNTHESIS OF LIPIDS

Problems and Questions

1. Carbon pathway in fatty acid synthesis.

Using your knowledge of FATTY ACID Biosynthesis, explain the following two experimental observations.

а) Addition of uniformly labeled [¹⁴C]-acetyl-CoA to a soluble Liver fraction yields palmitate uniformly labeled with ¹⁴C.

б) However, addition of trace amounts of uniformly labeled [¹⁴C]-acetyl-CoA in the presence of excess unlabeled malonyl-CoA to a soluble liver fraction yields palmitate labeled with ¹⁴C only at C-15 and C-16.

2. Synthesis of Fatty acids from glucose.

Following a heavy meal rich in sucrose, the excess glucose and fructose that exceed the body's energy demands are converted into fatty acids for triacylglycerol synthesis. The synthesis of fatty acids consumes acetyl-CoA, ATP, and NADPH. How are these substances generated from glucose?

3. Overall equation for fatty acid synthesis.

Write the balanced overall equation for The biosynthesis of palmitate in rat liver, starting from mitochondrial acetyl-CoA and cytosolic NADPH, ATP, and СO₂.

4. Hydrogen pathway in fatty acid synthesis.

Consider a Cell-free preparation containing all the Enzymes and Cofactors required for fatty acid biosynthesis from added acetyl-CoA and malonyl-CoA.

а) If [2-²Н]-acetyl-CoA (labeled with deuterium ²Н, a heavy isotope of hydrogen) and an excess of unlabeled malonyl-CoA are added as substrates, how many deuterium atoms are incorporated into each palmitate molecule? Where are they located? Explain.

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б) If unlabeled acetyl-CoA and [2-²Н]-malonyl-CoA are added as substrates, how many deuterium atoms are incorporated into each palmitate molecule? Where are they located? Explain.

5. Energetics of β-ketoacyl-ACP synthase.

In the Condensation reaction catalyzed by β-ketoacyl-ACP synthase (see Fig. 21-6), a four-carbon unit is synthesized by combining a two-carbon and a three-carbon unit, with the release of СO₂. What is the thermodynamic advantage of this process over the direct combination of two two-carbon units?

6. Modulation of acetyl-CoA carboxylase.

Acetyl-CoA carboxylase is the primary regulatory site in fatty acid biosynthesis. Some of The properties of this enzyme are listed below.

а) Addition of citrate or isocitrate increases the Vm_ах of the enzyme 10-fold.

б) The enzyme exists in two interconvertible forms that differ significantly in activity:

Protomer (inactive) ⇄ Filamentous polymer (active)

Citrate and isocitrate bind preferentially to the protomer.

Explain how these properties are consistent with the regulatory role of acetyl-CoA carboxylase in fatty acid biosynthesis.

7. Shuttle translocation of acetyl groups across The inner mitochondrial membrane.

The acetyl group of acetyl-CoA, formed during The oxidative decarboxylation of Pyruvate in the mitochondrion, is transported into the Cytosol via the acetyl group shuttle schematically illustrated in Fig. 21-10.

a) Write the overall equation for The transport of a single acetyl group from the mitochondrion into the cytosol.

b) What is the "cost" of this process in equivalents of ATP per acetyl group?

c) In Chapter 17, we encountered an acyl shuttle involved in transporting fatty acyl-CoA from the cytosol into the Mitochondria in preparation for β-oxidation (see Fig. 17-6). One consequence of this shuttle transfer was the Separation of the mitochondrial and cytosolic CoA pools. Does the acetyl shuttle perform a similar function? Explain.

8. Oxygen requirement for desaturases.

The biosynthesis of palmitoleate (see Fig. 21-12)—a widely distributed unsaturated fatty acid with a cis-double bond at the Δ⁹ position—uses palmitate as a precursor. Can this process take place under strictly anaerobic conditions? Explain.

9. Energetic "cost" of triacylglycerol synthesis.

Use the overall equation for the biosynthesis of tripalmitoylglycerol (tripalmitin) from glycerol and palmitate to demonstrate how many ATP molecules are required per molecule of tripalmitin formed.

10. Triacylglycerol turnover in adipose tissue.

When [¹⁴C]glucose is added to the balanced diet of adult rats, there is no net increase in the total amount of stored triacylglycerols, yet the triacylglycerols contain the ¹⁴C label. Explain.

11. Energetic "cost" of phosphatidylcholine synthesis.

Write The sequence of steps and the overall reaction for the biosynthesis of phosphatidylcholine from oleate, palmitate, dihydroxyacetone phosphate, and Choline via Salvage Pathways. Based on previous data, calculate the "cost" (in ATP molecules) of synthesizing phosphatidylcholine by this pathway.

12. Synthesis of phosphatidylcholine via salvage pathways.

Young rats fed a Methionine-deficient diet failed to thrive unless choline was included in their diet. Explain.

13. Synthesis of isopentenyl pyrophosphate.

If [¹⁴C]acetyl-CoA is added to a rat liver homogenate synthesizing Cholesterol, where will the ¹⁴C label appear in Δ³-isopentenyl pyrophosphate (the activated form of the isoprene unit)?

14. Activated Donors in lipid synthesis.

During the biosynthesis of Complex Lipids, the assembly of components is accompanied by The transfer of the required group from an activated donor. For example, the activated donor of acetyl groups is acetyl-CoA. For each of the groups listed below, name the form of the activated donor: (a) phosphate; (b) D-glucosyl; (c) phosphoethanolamine; (d) D-galactosyl; (e) fatty acyl; (f) methyl; (g) two-carbon unit in fatty acid biosynthesis; (h) Δ³-isopentyl.

15. Essential importance of dietary fats.

When young rats are fed a diet completely devoid of fats, they fail to grow, develop a scaly dermatitis, lose their Hair, and soon die. These symptoms can be prevented by including linoleate or plant Materials in the diet. What makes linoleate an essential fatty acid? Why can plant material serve as a substitute?

16. Regulation of Cholesterol Biosynthesis.

Humans can obtain cholesterol from their diet or synthesize it de novo. An adult consuming a low-cholesterol diet typically synthesizes 600 mg of cholesterol per day in the liver. If The amount of dietary cholesterol is high, de novo cholesterol synthesis drops sharply. How is this regulation accomplished?

17. Lowering of serum cholesterol levels by statins.

Patients taking statins typically experience a sharp decrease in serum cholesterol levels. However, this Treatment can lead to a marked increase in the amount of HMG-CoA reductase within their Cells. Explain this observation.

18. The Role of thioesters in cholesterol biosynthesis.

Propose a mechanism for each of the three reactions shown in Fig. 21-34, indicating the pathway for The formation of mevalonate from acetyl-CoA.

19. Potential side effects of statin therapy.

Some physicians suggest that patients taking statins should also be prescribed coenzyme Q (although clinical trials confirming the benefits or adverse side effects of such a regimen have not yet been conducted). Explain the rationale behind this recommendation.

Assessing Experimental Data

20. Engineering E. coli cells for high-level isoprenoid production.

Nature produces a vast array of Isoprenoids, some of which hold significant medical or commercial value and are manufactured on an industrial scale. One approach to obtaining isoprenoids is in vitro enzymatic synthesis; however, this method is expensive and suffers from low productivity. In 1999, Wang, Oh, and Liao reported the creation of an E. coli strain capable of producing large quantities of astaxanthin, a commercially important isoprenoid.

Astaxanthin is a red-orange pigment and antioxidant produced by marine Algae. Certain marine animals (such as shrimp, lobsters, and some fish) that feed on algae acquire an orange hue due to the accumulation of astaxanthin in their Tissues. This compound has the empirical formula C40H52O4 and is built from eight isoprene units:

a) Outline the eight isoprene units within the astaxanthin molecule. Hint: Look for the protruding methyl groups.

The astaxanthin biosynthetic pathway is illustrated on the following page, beginning with Δ³-isopentenyl pyrophosphate (IPP). Steps (1) and (2) are depicted in Fig. 21-36, and the reaction catalyzed by IPP isomerase is shown in Fig. 21-35.

b) In step (4), two molecules of geranylgeranyl pyrophosphate couple to form phytoene. Does this condensation occur via a "HEAD-to-head" or "head-to-tail" mechanism? (See Fig. 21-36 for details.)

c) Explain the chemical transformation that takes place in step (5).

d) Cholesterol synthesis (Fig. 21-37) involves a cyclization (ring-closure) step driven by O₂ oxidation. Does the substrate (lycopene) undergo oxidation during the cyclization in step (6) of astaxanthin synthesis? Explain your reasoning.

E. coli does not naturally produce high levels of isoprenoids nor does it synthesize astaxanthin. Nevertheless, the bacterium is known to synthesize small amounts of isopentenyl phosphate, dimethylallyl pyrophosphate, geranyl pyrophosphate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate. Wang and coworkers cloned several E. coli genes encoding enzymes required for astaxanthin synthesis into Plasmids designed to achieve high-level Gene Expression. Specifically, they cloned the idi gene (encoding IPP isomerase) and the ispA gene (encoding the prenyltransferase that catalyzes steps (1) and (2)).

To engineer E. coli cells capable of complete astaxanthin biosynthesis, Wang et al. cloned selected genes from other Bacteria into plasmids for high-level expression in E. coli. These included the crtE gene from Erwinia uredovora, which encodes the enzyme

responsible for step (3), as well as the crtB, crtI, crtY, crtZ, and crtW genes from Agrobacterium aurantiacum, which encode the enzymes for steps (4), (5), (6), (7), and (8), respectively.

The researchers also cloned the gps gene from Archaeoglobus fulgidus, achieved its high-level expression in E. coli, and isolated the resulting product. When this cell extract was incubated with [¹⁴C]isopentenyl phosphate, [¹⁴C]dimethylallyl pyrophosphate, [¹⁴C]geranyl pyrophosphate, or farnesyl pyrophosphate, the sole product formed in every case was [¹⁴C]geranylgeranyl pyrophosphate.

e) Based on these findings, determine which step(s) of the metabolic pathway are catalyzed by the enzyme encoded by the gps gene. Explain your answer.

Next, Wang and coworkers constructed several E. coli variants with overexpressed genes of interest, assessing both the orange coloration of the colonies (wild-type E. coli colonies lack orange pigmentation) and the amount of astaxanthin produced. Their results are summarized below.

ND — not detected.

e) Compare the results for strains 1–4 with those for strains 5–8 and draw a Conclusion about the expression level of The enzyme catalyzing step (3) in astaxanthin synthesis in the wild-type E. coli strain. Explain your answer.

f) Based on the presented data, conclude which of the enzymes—IPP isomerase or the enzyme encoded by the idi gene—rate-limits this metabolic pathway. Explain your answer.

g) What would the expression level of astaxanthin be in a strain with overexpressed crtBIZYW, gps, and crtE (low (+), moderate (++), or high (+++), as estimated by the intensity of the orange coloration)? Explain your answer.