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

PART II. BIOENERGETICS AND METABOLISM

20. BIOSYNTHESIS OF CARBOHYDRATES IN PLANTS AND BACTERIA

Questions and Problems

1. Compartmentation of METABOLISM in Organelles.

What are the advantages to a plant Cell of having different organelles that house distinct reaction sequences sharing common intermediates?

2. Stages of Photosynthesis.

When a suspension of green Algae is illuminated in the absence of CO₂ and then incubated in the dark with ¹⁴CO₂, the ¹⁴CO₂ is rapidly converted into [¹⁴C]glucose. What is The Significance of these observations for understanding CO₂ assimilation processes? How does this relate to the light reactions of photosynthesis? Why does The conversion of ¹⁴CO₂ into [¹⁴C]glucose stop after a short period of time?

3. Identification of Key Intermediates in CO₂ Assimilation.

Calvin and his colleagues used the unicellular green alga Chlorella to study photosynthetic carbon assimilation reactions. They incubated ¹⁴CO₂ with an illuminated suspension of algae and monitored the time course of ¹⁴C appearance in two compounds, X and Y, under two different sets of conditions. Based on The Calvin Cycle, predict what compounds X and Y might be.

a) Chlorella was grown in the light in the presence of unlabeled CO₂. The light was then turned off and ¹⁴CO₂ was added (vertical dashed line on the graph). Under these conditions, X was the first compound to become labeled; Y remained unlabeled.

Class="center">

b) Chlorella Cells were grown in the presence of ¹⁴CO₂. The cells were illuminated until all the ¹⁴CO₂ was exhausted (vertical dashed line on the graph). Under these conditions, X became labeled but rapidly lost its radioactivity, whereas Y became progressively more radioactive over time.

4. Regulation of the Calvin Cycle.

Iodoacetate reversibly reacts with free -SH groups of Cysteine residues in Proteins. Which Calvin cycle Enzymes

are inhibited by iodoacetate? Explain why.

5. Thioredoxin in The regulation of Calvin Cycle Enzymes.

Motohashi and colleagues used thioredoxin as a trap to identify thioredoxin-activated proteins in plant extracts. To do this, they synthesized a modified thioredoxin in which one of the Cys residues was replaced by Ser. Explain why this modification was necessary for their experiments.

6. Comparison of the Reductive and Oxidative Pentose Phosphate Pathways.

The reductive Pentose Phosphate Pathway shares only some intermediates with the oxidative pentose phosphate pathway (Chapter 14). What is The Physiological Role of each pathway in the cells where it occurs?

7. Photorespiration and Mitochondrial Respiration.

Compare the photosynthetic carbon oxidation cycle (C₂ cycle), also known as photorespiration, with mitochondrial respiration—the driving force for ATP synthesis. Why do both processes share the term "respiration"? Where do they occur within The Cell, and under what conditions? What is The pathway of electron flow in each case?

8. Rubisco and Atmospheric Composition.

Tolbert argued that the dual Specificity of RuBisCO for CO₂ and O₂ did not simply arise as a byproduct of enzyme evolution under low oxygen conditions. Instead, he proposed that The ratio of the carboxylation and oxygenation activities of RuBisCO actually regulated, and now maintains, the ratio of CO₂ and O₂ in Earth's atmosphere. Discuss the pros and cons of this hypothesis in both molecular and general terms. How does the existence of C₄ plants follow from this hypothesis?

9. The Role of Sedoheptulose-1,7-Bisphosphatase.

What impact would a defect in sedoheptulose-1,7-bisphosphatase have on a human cell and Organism (a) in a hepatocyte? (b) In a green plant leaf?

10. The CO₂ Assimilation Pathway in Maize.

When corn (maize) plants are illuminated in the presence of ¹⁴CO₂, more than 90% of the radioactivity incorporated into the cell appears within about a second in the C-4 positions of malate, aspartate, and oxaloacetate. Only after 60 s does ¹⁴C appear in the C-1 position of 3-phosphoglycerate. Explain why.

11. Identification of CAM Plants.

Armed with a small amount of ¹⁴CO₂ and standard biochemical research laboratory equipment, what simple experiment would you design to determine whether a given plant is a typical C₄ or CAM plant?

12. Malate Dehydrogenase Chemistry.

The malate dehydrogenase found in the bundle-sheath cells of C₄ plants catalyzes a reaction that has a direct counterpart in The Tricarboxylic Acid Cycle. What is this reaction? Justify your choice.

13. The Energetic Cost of Storing Glucose as Starch.

Write out the reaction sequence and the overall equation required to calculate the "cost" (in ATP molecules) of converting cytosolic glucose-6-phosphate into starch and back into glucose-6-phosphate. What fraction of the maximum number of ATP molecules obtainable from the complete Catabolism of glucose-6-phosphate to CO₂ and H₂O does this "cost" represent?

14. Inorganic Pyrophosphatase.

The enzyme inorganic pyrophosphatase renders many biosynthetic reactions that produce inorganic pyrophosphate virtually irreversible. By maintaining a very low PP_i concentration, the enzyme "pulls" the reaction in the direction of PP_i formation. The synthesis of ADP-glucose in Chloroplasts is one reaction driven in this direction by such a mechanism. However, the synthesis of UDP-glucose in The plant cell Cytosol, which also yields PP_i, is readily reversible in vivo. How would you account for this apparent contradiction?

15. Regulation of Starch and Sucrose Synthesis.

Sucrose synthesis takes place in the cytosol, whereas starch synthesis occurs in the chloroplast stroma, yet these two processes are intricately balanced. What factors drive the reactions toward (a) starch synthesis and (b) sucrose synthesis?

16. Regulation of Sucrose Synthesis.

3-Phosphoglycerate and P_i play key roles in regulating sucrose synthesis from the triose phosphates produced during photosynthesis (see Fig. 20-26). Explain why the concentrations of these two regulators serve as an indicator of photosynthetic rate.

17. Sucrose and Dental Caries.

Dental caries is the most common human infection, caused by the colonization and erosion of tooth enamel by various acid-producing microorganisms. These microbes inhabit dental plaque, forming Water-insoluble, net-like dextran polymers on the Teeth that consist of α(1—>6)-linked glucose units with numerous α(1—>3) branch points. Dextran polymerization requires dietary sucrose and is catalyzed by the bacterial enzyme dextransucrase (glucosyltransferase).

a) Write the overall reaction for dextran polymerization.

b) Aside from serving as a substrate for plaque formation, how does dietary sucrose provide oral Bacteria with an abundant source of metabolic energy?

18. Differences Between C₃ and C₄ Plants.

The plant genus Atriplex includes several C₃ and C₄ species. Using the graphs (species 1, black curve; species 2, red curve), identify which is the C₃ plant and which is the C₄ plant. Justify your answer by correlating your reasoning with the graphical data.

19. The Single-Cell C₄ Pathway.

In typical C₄ plants, the initial CO₂ capture occurs in one cell type, while the Calvin cycle takes place in another (see Figs. 20–23). Voznesenskaya and colleagues described Bienertia cycloptera, a plant growing in mildly saline soils of the Central Asian semideserts. This plant exhibits the Biochemical characteristics of a C₄ plant, yet unlike typical C₄ plants, it does not compartmentalize the CO₂-fixation reactions into two different cell types. Both PEP carboxylase and rubisco are present within the same cell. However, microscopic examination reveals two distinct types of chloroplasts localized in different Regions of the cell. One type contains relatively few grana (thylakoids) and is located peripherally, whereas typical chloroplasts are separated from the peripheral ones by large vacuoles. Thin cytosolic bridges traverse these vacuoles, connecting the peripheral and central cytosol.

Where would you expect to find (a) PEP carboxylase, (b) rubisco, and (c) starch granules in this plant? Explain your Answers based on The Mechanism of CO₂ fixation in these C₄ cells.

Analysis of Experimental Data

20. Rubisco from Endosymbiotic Bacteria Isolated from Hydrothermal Vent Animals.

Submarine hydrothermal vents are unique habitats that support remarkable ecosystems. At great depths, devoid of the sunlight required for photosynthesis, thriving biological communities have been discovered. Their existence is sustained by Chemosynthesis carried out by symbiotic bacteria that inhabit specialized Organs (trophosomes) found in certain deep-sea animals.

The chemosynthesis performed by these bacteria is virtually identical to photosynthesis. Carbon dioxide, CO₂, is fixed by the enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase) and reduced to glucose, while the required amounts of ATP and NADPH are generated through an electron transport process similar to the light reactions of photosynthesis. The primary difference is that in chemosynthesis, the energy required to drive Electron transport is derived from exergonic Chemical Reactions rather than light. Different chemosynthetic bacteria utilize various reactions for this purpose. The bacteria found in hydrothermal vent animals typically utilize The oxidation of H₂S (abundant in deep-sea vents) using O₂, which yields elemental sulfur. These bacteria also harness the conversion of H₂S to sulfur as a source of electrons for the reduction of CO₂.

a) What is the overall reaction of chemosynthesis in these bacteria? There is no need to balance this equation; simply list the reactants and products.

b) Ultimately, the energy utilized by these endosymbiotic bacteria originates from sunlight. Explain this statement.

Robinson and colleagues (2003) studied The properties of Rubisco from the symbiotic bacterium residing within the giant tube worm Riftia pachyptila. Rubisco from any source catalyzes the reaction of either CO₂ (Fig. 20-7) or O₂ (Fig. 20-20) with ribulose-1,5-bisphosphate. Typically, Rubisco reacts much more readily with CO₂ than with O₂. The degree of selectivity (Ω) can be expressed as follows:

V_{carboxylation} / V_oxygenation = Ω ([CO₂]/[O₂])

where V is the reaction rate.

Robinson and coworkers determined the value of Ω for rubisco from bacterial endosymbionts. They purified the enzyme from vestimentiferan tubeworm trophosomes, carried out reactions with O₂ and CO₂ mixtures at various ratios in the presence of [1-³H] ribulose 1,5-bisphosphate, and measured the concentration ratio of [³H] phosphoglycerate to [³H] phosphoglycolate.

c) The measured concentration ratio of [³H] phosphoglycerate to [³H] phosphoglycolate is equal to the ratio of V_{carboxylation} / V_oxygenation.

Explain why.

d) Why is [5-³H] ribulose 1,5-bisphosphate an unsuitable substrate for this study?

For the endosymbiont rubisco, Ω = 8.6 ± 0.9.

e) In atmospheric air, the O₂ content is 20%, and the CO₂ content is about 380 ppm (parts per million). Assuming the endosymbionts could carry out carbon fixation under atmospheric conditions, determine the ratio of reaction rates V_{carboxylation}/V_oxygenation.

f) Based on your answer to the previous question, estimate Ω for terrestrial plant rubisco: Ω > 8.6, Ω < 8.6, or Ω = 8.6. Explain your reasoning.

Two stable carbon isotopes exist in nature: ¹²C is predominant, while ¹³C is less abundant. Rubisco-catalyzed fixation of ¹²CO₂ proceeds faster than that of ¹³CO₂. As a result, the glucose molecule becomes slightly enriched in ¹²C compared to the isotopic composition of environmental CO₂. This preferential use of ¹²CO₂ is explained by several factors, notably the Physical Properties of gases. The Temperature of a gas is related to the kinetic energy of its molecules. Kinetic energy equals 1/₂mv², where m is the molecular mass and v is the molecular velocity. Thus, at the same temperature (equal kinetic energy), lighter gas molecules move faster than heavier ones.

g) How does this fact relate to rubisco "preferring" ¹²CO₂ over ¹³CO₂?

One of the earliest proofs that tubeworms obtain fixed carbon from endosymbionts was the fact that the ¹³C/¹²C ratio in these animals is much closer to that of bacteria than to that of other marine animals.

h) Why is this more compelling evidence of Symbiosis than earlier studies that simply demonstrated the presence of rubisco in bacteria from trophosomes?