Chemistry 301 Introduction to Biochemistry

Study Guide :: UNIT 7

Metabolism II

Overview

Unit 7 continues the subject of Unit 6, as there is too much material to cover in one unit. In Unit 6, we presented metabolic pathways that played important oxidative/reductive roles in cellular energy. In Unit 7, we focus on catabolism and anabolism of amino acids and nucleic acids, as well as pathways involved in nitrogen fixation, carbohydrate storage, and carbohydrate degradation.

Unit 7 is divided into seven lessons:

Learning Objectives

After completing this unit, you should be able to:

1. Explain how glycogen is degraded to form glucose, and how glycogen is synthesized from glucose.
2. Demonstrate an understanding of the primary functions of the pentose phosphate pathway.
3. Outline the purpose of the Calvin Cycle.
4. Describe the outcomes of the urea cycle.
5. Demonstrate an understanding of the process of nitrogen fixation.
6. Explain the principle behind amino acid metabolism and catabolism.
7. Demonstrate an understanding of nucleotide metabolism and biosynthesis.
8. Discuss the connections between different metabolic pathways.

Glossary

Lesson 1: Carbohydrate Storage and Breakdown

Overview/Objectives

After completing this lesson, you should be able to:

1. Explain how glycogen is degraded to form glucose, and how glycogen is synthesized from glucose.
2. Explain why there are separate pathways for glycogen synthesis and breakdown.
3. Explain how covalent modification prevents glycogen synthesis and breakdown from occurring at the same time.
4. Describe the principle of a cyclic cascade.

Readings and Activities

1. In Chapter 7 Metabolism II, read the introductory paragraph, and “Carbohydrate Storage/Breakdown,” “Glycogen Breakdown,” “Regulation of Glycogen Metabolism,” “GPa/GPb Allosteric Regulation,” “GPa/GPb Covalent Regulation,” “Turning Off Glycogen Breakdown,” “Glycogen Synthesis,” “Regulation of Glycogen Synthesis,” and “Maintaining Blood Glucose Levels” (pages 173–179 of the textbook).
2. You can also watch the three video lectures on Glycogen Metabolism:
   • #25 Biochemistry Glycogen Metabolism I Lecture for Kevin Ahern’s BB 450/550
   • #26 Biochemistry Glycogen Metabolism II Lecture for Kevin Ahern’s BB 450/550

   • #27 Biochemistry Glycogen Metabolism III / Metabolic Melodies Lecture for Kevin Ahern’s BB 450/550

     (Links to these are also provided on pages 175 and 178 of the textbook.)

Commentary

Glycogen is the polymeric storage form of glucose in vivo. The balance between its synthesis and degradation ensures a steady supply of glucose for carbohydrate metabolism.

Glycogen Degradation

Glycogen is a glucose α(1 → 4) polysaccharide stored in granules in the cytosol of liver and skeletal muscle cells. During cleavage of a glucose molecule from glycogen, a phosphate residue is attached to the glucose residue at the cleavage site (position 1). Glucose 1‑phosphate (G1P) is charged, while glucose itself is not. This factor has the important effect of trapping glucose within the cell, since charged species cannot pass through a cell membrane. G1P is a temporary, relatively nonreactive form of glucose. When the phosphate group is transferred from the 1 position to the 6 position on glucose, glucose is ready to enter glycolysis (i.e., G1P → G6P).

Skeletal muscle does not possess the cleavage enzyme, G6P‑phosphorylase, so all glycogen stored in skeletal muscle cells is used within these cells. Liver cells do possess G6P‑phosphorylase, and so glucose can be used by liver cells or released from liver cells through the cell membranes into the bloodstream.

Synthesis of Glycogen

Glycogen synthesis is not the reverse of the degradation pathway. Rather, the glucose building blocks are UDP‑glucose. This is a good example of of a “high‑energy” compound (UTP) combining with a lower energy compound (G1P) to energize the lower energy compound.

Regulation of Glycogen Synthesis and Degradation

To prevent simultaneous synthesis and degradation of glycogen from occurring, a clever control mechanism has evolved: the phosphorylation cascade.

1. Glucose is released to the system, and glycogen synthesis is shut down by phosphorylation of key enzymes. Under the control of epinephrine, a sequential set of reactions occurs that adds a phosphate group to two key enzymes—phosphorylase (which controls breakdown of glycogen) and glycogen synthase (which controls synthesis of glycogen):

   • phosphorylase‑P is active, so hydrolysis of glycogen is enhanced.
   • glycogen synthase‑P is inactive, so glycogen synthesis is diminished.

   The reactions leading to glycogen synthase‑P (inactive) are more complex than those leading to the phosphorylase‑P (active), although they are similar.

2. Activation of glycogen synthesis occurs when a group of phosphatases (phosphate removing enzymes) cleave the phosphate group from glycogen synthase‑P, phosphorylase‑P, and from several enzymes in the cascade.

   Blood levels of glucose are held constant by the balance of glycogen synthesis and degradation, in the liver. Remember, in the example given in Lesson 1, that a constant supply of reactants is crucial to the “steady state.” The phosphorylation cascade and dephosphorylating enzymes (under hormonal control) control this balance.

Study Questions

1. What is the purpose of storing glucose in the form of glycogen?
2. What is the significance of glucose‑1‑phosphate in glycogen degradation?
3. What is the building block for glycogen synthesis?
4. Glycogen phosphorylase and glycogen synthase are the main enzymes involved in glycogen breakdown and synthesis respectively. How does the phosphorylation cascade regulate these enzymes and the pathways they are involved in?
5. What advice would you give to a person who has an enzymatic deficiency in either the synthetic or the degradative path of glycogen metabolism?

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.

Lesson 2: Pentose Phosphate Pathway

Overview/Objectives

After completing this lesson, you should be able to:

1. List the primary functions of the pentose phosphate pathway.
2. List the substrates, products, and intermediates of the pentose phosphate pathway.
3. Discuss the “reversibility” of the pentose phosphate pathway.
4. Demonstrate an understanding of the connections between the pentose phosphate pathway and other metabolic pathways.

Readings and Activities

1. Read “Pentose Phosphate Pathway,” (pages 179–181 of the textbook).

Commentary

The pentose phosphate pathway (PPP) is also known by two other names, the phosphogluconate pathway and the hexose monophosphate shunt. In many ways, this pathway is a metabolic parallel to glycolysis in that it generates NADPH and pentoses (5‑carbon sugars). In the first phase (oxidative) NADPH is produced and in the second phase (non‑oxidative) sugars are synthesized. It is not important to know all of the reactions in this pathway, but you should understand the functions of this pathway, how reversible this pathway is, and how this pathway is regulated.

The primary functions of the pentose phosphate pathway (PPP) are as follows:

• produce NADPH (for anabolic reactions)
• produce ribulose‑5‑phosphate (for making nucleotides)
• produce erythrose‑4‑phosphate (for making aromatic amino acids)

Note the figure on page 179 of the textbook. This figure outlines the PPP and the reactions involved. You are not required to identify each reaction, but note the two phases, the oxidative generation of NADPH and the non‑oxidative synthesis of a number of 3‑, 5‑, and 6‑carbon sugars.

In the oxidative phase, two molecules of NADPH are produced as well as ribulose‑5‑phosphate. The overall reaction is:

Glucose 6‑phosphate + 2 NADP⁺ + H₂O → ribulose 5‑phosphate + 2 NADPH + 2 H⁺ + CO₂

In the non‑oxidative phase, a number of sugars can be synthesized by interconversion of sugar phosphates, including erythrose‑4‑phosphate. Three intermediates of glycolysis can be funnelled into this pathway: glucose‑6‑phosphate (G6P), fructose‑6‑phosphate, and glyceraldehyde‑3‑phosphate. The overall reaction can be stated as:

Net reaction: 3 ribulose‑5‑phosphate → 1 ribose‑5‑phosphate + 2 xylulose‑5‑phosphate → 2 fructose‑6‑phosphate + glyceraldehyde‑3‑phosphate

However, as shown in the figure, the PPP is reversible and different sugars can be produced depending on the needs of the cell. As mentioned, the pathway links to glycolysis but also provides a mechanism to metabolize sugars (Xu5P and ribulose‑5‑phosphate). The pathway can provide ribulose‑5‑phosphate for nucleotide and nucleic acid synthesis and erythrose‑4‑phosphate for aromatic amino acid synthesis. Pentose sugars from digestion of nucleic acids can be utilized through this pathway by being converted to glycolytic and or gluconeogenic intermediates. The PPP occurs in the cytoplasm, which facilitates intermingling of intermediates. NADPH production is also beneficial to the cell in that it can prevent oxidative stress by reducing glutathione so that it converts reactive H₂O₂ into H₂O. The amount of NADPH generated by the PPP in mammals is significant.

The regulation of the PPP is through the enzyme glucose‑6‑phosphate dehydrogenase. This enzyme is involved in the first step of the PPP. Glucose‑6‑phosphate dehydrogenase (G6PD) catalyzes the following reaction:

Glucose‑6‑phosphate + NADP⁺ → 6‑phosphogluconolactone + NADPH

This is a dehydrogenation reaction whereby the hydroxyl on carbon 1 of G6P turns into a carbonyl. This generates a lactone and NADPH.

Regulation of this enzyme (G6PD) is by two methods:

• allosteric regulation by the amount of NADPH. NADP⁺ stimulates the enzyme and NADPH inhibits the enzyme.
• post‑transcriptional regulation by cytoplasmic deacetylase SIRT2. Deacetylation by SIRT2 activates G6PD to stimulate the PPP and produce NADPH to combat oxidative damage in the cell.

Study Questions

1. What are the three primary functions of the pentose phosphate pathway (PPP)?
2. What are the two main phases of the PPP and what happens overall in each phase?
3. Explain the allosteric regulation of the PPP. What enzyme is regulated and how is it allosterically regulated?
4. How is the PPP connected to glycolysis? What does this mean in terms of the metabolic needs of an organism?

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.

Lesson 3: Calvin Cycle

Overview/Objectives

After completing this lesson, you should be able to:

1. Discuss the purpose of the Calvin Cycle.
2. Outline the overall reactants and products of the Calvin Cycle.

Readings and Activities

1. Read “Calvin Cycle” and “C₄ Plants” (pages 181–184 of the textbook).
2. You can also watch the video lecture on Photosynthesis:

   • #34 BB 350 Photosynthesis – Kevin Ahern’s Biochemistry Online

     (A link to this is also provided on page 183 of the textbook.)

Commentary

The Calvin Cycle is part of photosynthesis, and occurs in plants and other photosynthetic organisms. It is the pathway that comprises the “Dark Cycle” of photosynthesis, also referred to as the “Light‑Independent Reactions.” The Calvin Cycle occurs in the chloroplast stroma. If you look back at the last lesson on the pentose phosphate pathway (PPP), you will see that parts of the Calvin Cycle are similar to the PPP, in particular the involvement of the sugars ribulose‑5‑phosphate and glyceraldehyde‑3‑phosphate (G3P).

The main purpose of the Calvin Cycle is the eventual production of glucose and other sugars from CO₂. Energy from the light‑dependent reactions of photosynthesis generates NADPH and ATP for the reduction of CO₂ to glucose in these light‑independent reactions (Calvin Cycle) and gluconeogenesis. Glucose is not a direct product of the Calvin Cycle; glucose is subsequently made in gluconeogenesis with one of the 3‑phosphoglycerate molecules (3PG) produced in the Calvin Cycle.

In the Calvin Cycle, the CO₂ molecules are absorbed one at a time with ribulose‑1,5‑bisphosphate (Ru1,5BP). In a carboxylation reaction, the enzyme RuBisCO (ribulose‑1,5‑bisphosphate carboxylase/oxygenase) catalyzes the formation of an unstable 6‑carbon intermediate from Ru1,5BP and CO₂, which is then split into two molecules of 3‑phosphoglycerate (3PG or 3‑PGA). One molecule of the 3PG can go into gluconeogenesis to produce glucose. The other 3PG is converted to 1,3 bisphosphoglycerate using ATP, and then the 1,3 bisphosphoglycerate is reduced using NADPH to glyceraldehyde‑3‑phosphate (G3P). The next phase is to regenerate Ru1,5BP (or RuBP). G3P is converted to ribulose‑5‑phosphate and then Ru1,5BP. If you consult the figure of the Calvin Cycle (on page 181) in the textbook, it outlines the overall steps in the cycle. (You are not required to know the steps involved in the regeneration of RuBP, but you should understand that a number of G3P molecules are required for this to occur.)

Five G3P molecules produce three RuBP molecules, using up three molecules of ATP. Since each CO₂ molecule produces two G3P molecules, three CO₂ molecules produce six G3P molecules, of which five are used to regenerate RuBP, leaving a net gain of one G3P molecule per three CO₂ molecules (as would be expected from the number of carbon atoms involved). The one G3P produced can be converted to a hexose through the PPP. (This information is available in the text and on the Wikipedia page Light‑independent reactions.)

Therefore, the overall reaction for the Calvin Cycle is:

3CO₂ + 6 NADPH + 5 H₂O + 9 ATP → glyceraldehyde‑3‑phosphate (G3P) + 2 H⁺ + 6 NADP⁺ + 9 ADP + 8 P_i

(P_i = inorganic phosphate)

Study Questions

1. What are the overall reactants and products of the Calvin Cycle?
2. What is made in the Calvin Cycle that is used for the production of glucose by gluconeogenesis? What enzyme catalyzes the formation of this product in the Calvin Cycle?
3. What molecule has to be regenerated in the Calvin Cycle and how many G3P molecules are required for this to happen? What does this mean in terms of input of CO₂? What happens to the extra G3P produced?

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.

Lesson 4: Urea Cycle

Overview/Objectives

After completing this lesson, you should be able to:

1. Explain the three chemical strategies used by living organisms in the elimination of amino groups.
2. List the five reactions of the urea cycle, and their subcellular locations.
3. Explain the origin of the nitrogen in urea.
4. Describe the regulation of the urea cycle.

Readings and Activities

1. Read “Urea Cycle,” (pages 184–185 of the textbook).

Commentary

The ammonium ion (NH₄⁺) is the same size and charge as the potassium ion (K⁺). K⁺ is essential for both ion balance in vivo and for regulation of some enzymes. NH₄⁺ can substitute chemically but not functionally for K⁺ in vivo. The brain, in particular, is very sensitive to elevated ammonium levels in the blood. Organisms not living in an aqueous environment have adopted various chemical paths to avoid the toxicity of NH₄⁺. These paths incorporate the ammonium ion into larger complexes for excretion. Urea and uric acid are the most common excreted nitrogen compounds.

Urea, the excretory nitrogen compound of most terrestrial animals, is formed in the liver from the ammonium ions of oxidative deamination. It must pass through the bloodstream before passing out through the kidneys. The formation of one urea molecule (which contains two amino groups) requires three ATP molecules. Thus, elimination of excess nitrogen is an expensive metabolic process.

The overall reaction is as follows:

The process of converting NH₄⁺ to urea is energy‑requiring (three high‑energy phosphate bonds per urea), and it is cyclic. A figure of the urea cycle is on page 184 of the text. The major steps of the urea cycle are:

1. The first committed step in urea synthesis is combination of an ammonium ion with bicarbonate to form carbamoyl phosphate.
2. Then, carbamoyl phosphate combines with ornithine to yield citrulline.
3. Ornithine is regenerated after urea is expelled.

The five chemical reactions of the urea cycle occur variously in the mitochondrion and the cytosol in liver cells. Urea itself is produced in the cytosol.

Regulation of the urea cycle is by allosteric control of the enzyme, which catalyzes the first committed step: carbamoyl phosphate synthetase, and by the concentrations of the cycle intermediates.

Study Questions

1. Why do multicellular organisms require an elaborate mechanism to excrete nitrogen, while unicellular organisms do not?
2. What does NH₄⁺ combine with and what product is formed? What molecule is recycled in the urea cycle?

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.

Lesson 5: Nitrogen Fixation

Overview/Objectives

After completing this lesson, you should be able to:

1. Describe the biochemical process by which elemental nitrogen is reduced to ammonia.
2. Describe the high energetic expense of converting elemental nitrogen to ammonia.
3. Describe the enzyme nitrogenase, and explain how it is “protected” from O₂.

Readings and Activities

1. Read “Nitrogen Fixation,” (pages 185–186 of the textbook).

Commentary

Elemental nitrogen (N₂) is an extremely stable species. To reduce it completely to ammonia, an enzyme with a powerful reducing potential is required. Recall that NADH possesses two high‑energy electrons that can reduce the electron transport chain. NADH cannot reduce N₂. It does not have a high enough electron potential. Nitrogenase is a rare, unstable enzyme that is capable of reducing N₂.

NADH    ε^o′ = +0.315

Reduced Nitrogenase    ε^o′ = +0.40

Photosynthesis is usually the source of electrons to reduce nitrogenase (i.e., the ultimate source of the electrons for nitrogen fixation).

Nitrogenase is found only in a few bacterial strains that exist symbiotically within nodules of leguminous plants such as peas, beans, and alfalfa. When these bacteria produce excess reduced nitrogen, they excrete it into the soil; this is the only biochemical source of fixed (i.e., reduced) nitrogen. Nitrogenase contains Fe‑S and Mo‑Fe clusters that can be organized to donate electrons to reduce nitrogen. But note that metal clusters in an enzyme are like elemental metals: they are easily oxidized by oxygen, just as iron rusts easily.

Nitrogenase is protected from inactivation by oxygen in one of two ways:

• by compartmentalization of the enzyme in specialized cells; this occurs in cyanobacteria
• through leghemoglobin, a protein with a greater affinity for oxygen than nitrogenase.

The complete reduction of one mole of N₂ to two moles of ammonia requires 16 moles of ATP.

N₂ + 8H⁺ + 8e⁻ + 16ATP → 2 NH₃ + H₂ + 16ADP + 16P_i

Examine the above reaction carefully. Both nitrogen and hydrogen are reduced in this reaction. This can create a situation where nitrogen is not completely reduced to ammonia, and instead eventually reappears as elemental nitrogen. Consider each reduction separately:

1. $\ce{N\bond{#}N}$ + 6e⁻ + 6H⁺ → 2 HN₃

   This is a three‑step process in which each bond connecting the two nitrogen atoms is reduced sequentially; that is,

   $\ce{$\underset{\text{nitrogen}}{\ce{N\bond{#}N}}$ -> $\underset{\text{diimine}}{\ce{HN\bond{=}NH}}$ -> $\underset{\text{hydrazine}}{\ce{H2N\bond{-}NH2}}$ -> $\underset{\text{ammonia}}{\ce{2HN3}}$}$

2. 2 e − +2 H + → H 2

   Diimine is a reactive molecule that can be reconverted to nitrogen by hydrogen. This cyclic process (H₂ produced by nitrogenase causes diimine to reconvert to nitrogen rather than to ammonia) proceeds when the ATP driving the whole process is in low supply.

Fertilizers contain reduced nitrogen, either decayed biological matter or chemically reduced nitrogen. One of the goals of genetic engineering is to decrease the need for fertilizers by making nitrogenase more widely available in the plant world.

The genes that code for nitrogenase have been identified and isolated. Great efforts are being made to insert these genes into more strains of bacteria or into plant cells themselves. As you will see in the final two units, insertion of the desired genes into a foreign host is only the first step. The foreign host must then produce proteins from the inserted genes; it must produce the proteins in the correct ratio and in the correct three‑dimensional configuration; and, most importantly, the foreign host must continue to function normally!

Study Questions

1. Why is nitrogenase not more widely distributed in nature?
2. Why is it relatively easy to convert diimine back to elemental nitrogen, while hydrazine is more likely to convert into ammonia?

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.

Lesson 6: Amino acid Metabolism and Catabolism

Overview/Objectives

The main use of amino acids in vivo is to build proteins. However, amino acids can also be used (after carbohydrate and lipid reserves have been exhausted) as fuel molecules. In addition, amino acids are the precursors of other nitrogen‑containing biomolecules, such as nucleotides, enzyme cofactors, neurotransmitters, and the heme group.

Unlike carbohydrates and lipids, amino acids contain nitrogen (situated in the amino group). Since it is only the carbon‑hydrogen portion that acts as a fuel, two processes have evolved to handle the nitrogen part of the amino acid. “Transamination”, which we cover in this lesson, transfers the amino group from the amino acid to a keto‑acid “holding molecule.” The amino group can then be recycled or eliminated. As we discussed in Lesson 4, the urea cycle is the mammalian process for eliminating amino groups. The deaminated amino acid (i.e., the carbon‑hydrogen portion) enters the citric acid cycle.

After completing this lesson, you should be able to:

1. Explain the deamination of amino acids, and describe the fate of the removed amino group.
2. Describe the variety of catabolic paths for the non‑nitrogen portions of an amino acid.
3. List some of the uses of amino acids as biosynthetic precursors.
4. Describe the biochemical process that reduces elemental nitrogen to ammonia.
5. List the biological roles of amino acids.
6. Explain how an amino acid is deaminated if the rest of the amino acid is to be metabolized for fuel.
7. Explain how an amino acid is deaminated if the amino group is not to be saved.
8. Describe the role of pyridoxal‑5′‑phosphate in aminotransferase activity.
9. Describe the specificity of aminotransferases for their substrates.
10. Explain the regulation of the enzyme glutamate dehydrogenase, and discuss the physiological significance of this regulation.
11. Summarize the major catabolic paths for the breakdown of the non‑nitrogen portions of the amino acids (i.e., indicate which amino acids are broken down to which metabolic intermediates).
12. Explain why the catabolic paths for the non‑nitrogen portions of some of the amino acids are so complex.
13. Explain the difference between a glucogenic and a ketogenic amino acid.
14. Explain why ketogenic amino acids cannot be used to supply glucose to the body.
15. List the types of biomolecules that are synthesized using amino acids as precursors.
16. Describe the fates of heme derived from degraded red blood cells.
17. Describe the formation of GABA, histamine, and serotonin.
18. Explain how different cells make different amines.
19. Describe the biochemical roles of nitric oxide.
20. Describe the biochemical role of glutathione.

Readings and Activities

1. Read “Amino Acid Metabolism” and “Amino Acid Catabolism” (pages 186–189 of the textbook).
2. You can also watch two video lectures on Nitrogen Metabolism (2 links, page 186 of the textbook).
   • #35 BB 350 Nitrogen Metabolism – Kevin Ahern’s Biochemistry Online

   • #36 BB 350 Nitrogen Metabolism – Kevin Ahern’s Biochemistry Online

     (Links to these are also provided on page 186 of the textbook.)

Commentary

Deamination of Amino Acids

Nitrogen is carefully conserved in the body. Thus, excess amino acids are never excreted as such. They are deaminated or cleaved to an amino group and an α‑ketoacid. How the amino group is cleaved depends on whether the nitrogen is to be recycled (transamination) or excreted (oxidative deamination).

Transamination. Transamination usually involves α‑ketoglutaric acid as the acceptor of amino groups, but other α‑ketoacids can also be used.

Note that this is an exchange reaction: aa₁ + k₂ → k₁ + aa₂.

There are three important points here:

• The “amino carrier” above can interact with another α‑ketoacid to transfer the amino group again.
• All amino acids without their amino groups are α‑ketoacids.
• Many of the amino acid precursor α‑ketoacids are synthesized in vivo.

Therefore, transamination allows the body to maintain a dynamic pool of amino acids ready for use as protein building blocks. Transamination can be used both as the first step in the elimination of excess nitrogen and as the final step in biosynthesis of amino acids. Amino acids that can be synthesized this way are called “nonessential” amino acids. The amino acids whose α‑ketoacid framework cannot be synthesized by an organism are called “essential amino acids.” Which amino acids are “essential” and which are “nonessential” varies with the organism in question. The essential and nonessential amino acids in humans are listed below:

Essential and nonessential amino acids in humans (adapted from “Essential amino acid,” https://en.wikipedia.org/wiki/Essential_amino_acid):

*essential in certain cases.

Oxidative Deamination. If the amino group is not to be recycled, but rather eliminated, it is cleaved from the “amino carrier” (above) in the liver. The cleavage product is the ammonium ion (NH₄⁺). This process is called “oxidative deamination.” The only amino acid from which the amino group can be cleaved directly (as opposed to being transaminated) is glutamate. Therefore, an amino group to be discarded from other excess amino acids must be transaminated (through α‑ketoglutaric → acid glutamate) first. This two‑step process protects rare, essential amino acids, such as methionine and tryptophan, when other metabolic fuels are low. In oxidative deamination:

glutamate → GDH α−ketoglutaricacid+ NH 4 +

Note: GDH is glutamate dehydrogenase.

Deaminated Amino Acids as Metabolic Fuels

There are 20 common amino acids, all of which can be oxidatively deaminated. The resulting carbon skeletons can then be funnelled into metabolism. The amino acids are classified as glucogenic or ketogenic, depending on whether their carbon skeletons are converted to citric acid cycle intermediates or to acetyl‑CoA or acetoacetyl‑CoA. Remember that glucogenic amino acids form intermediates of the citric acid cycle, and ketogenic amino acids are broken down to acetyl‑CoA or acetoacetyl‑CoA. The following table is adapted from page 187 of the textbook.

The carbon skeletons of the amino acids are converted to one of seven metabolites: pyruvate, acetyl‑CoA, acetoacetyl‑CoA, α‑ketoglutarate, succinyl CoA, oxaloacetate, or fumarate. For some of the glucogenic amino acids, the size of the carbon skeleton determines which citric acid cycle intermediate it will become.

Conversion of phenylalanine to acetoacetate and fumarate is difficult, because this amino acid contains a very stable aromatic ring. Molecular oxygen, an unusual biochemical “reactant,” is used to convert phenylalanine to tyrosine, where the aromatic ring can be more readily cleaved. Lack of phenylalanine hydroxylase, the enzyme catalyzing the conversion of phe to tyr, leads to phenylketonuria (PKU). Infants born with PKU can suffer from a variety of medical problems, including intellectual disability and seizures, if untreated because of the toxicity of the accumulated phenylalanine. This disease is treated by removal of phenylalanine from the diet.

The complexity of the degradation pathways for some of the amino acid carbon skeletons (e.g., tryptophan) is caused by their intricate chemical structures; but a side benefit is that these complex, relatively rare amino acids will not enter metabolism as readily as will the more common, nonessential amino acids (e.g., glycine and alanine). They will be preferentially conserved, even when the body has been forced to use amino acids as biochemical fuel. In addition, intermediate products of the degradation reactions may be tapped off for use in non‑degradative processes in the body.

The carbon skeletons of glucogenic amino acids can serve, via the citric acid cycle intermediates, as precursors for glucose synthesis. The ketogenic amino acids cannot do so; this limitation occurs because mammals cannot synthesize glucose using only acetyl‑CoA or acetoacetyl‑CoA as starting materials.

Amino Acids as Biosynthetic Precursors

Besides acting as the building blocks for proteins and peptides, amino acids are the precursors of other biomolecules. Some important examples are

• purines and pyrimidines: bases for DNA and RNA
• sphingosine: a lipid found in brain tissue
• histamine: a vasodilator
• tetrapyrrole: organic portion of the heme and chlorophyll rings
• thyroxine and epinephrine: hormones
• melanin: skin pigment
• serotonin: a neurotransmitter
• glutathione: a major cellular sulphhydryl reducing agent
• tetrahydrofolate: cofactor in transfer of C₁ units NAD⁺ FAD, Coenzyme‑A

Tetrapyrroles. Tetrapyrroles with various side groups are a dramatically coloured group of biomolecules. (Most biomolecules are colourless.) Tetrapyrroles are derived from succinyl‑CoA and glycine. The tetrapyrrole ring is a large aromatic structure that can bind iron or magnesium. With iron in the centre and with various side groups, it is called a heme ring (the oxygen binding site in hemoglobin and the cofactor in many redox enzymes). With magnesium in place, it is called chlorophyll.

While the colour of blood and muscle is caused by heme, the green colour of plants is caused by chlorophyll. When your eye is “blackened,” hemoglobin has escaped from damaged blood vessels under the skin. The heme ring is then degraded to various open tetrapyrroles, called bile pigments. The resulting colours demonstrate the full range of colours available to the tetrapyrroles.

Tetrapyrrole degradation compounds are insoluble. Two enzyme systems in the liver, however, will attach glucosyl moieties to tetrapyrrole degradation products to increase their solubility before elimination. The colour of feces is due, in part, to degraded tetrapyrroles eliminated as glucosyl derivatives. This liver glycosylation system can be brought into play to increase the solubility of (and enhance the elimination of) foreign, nonsoluble toxins as well.

Nitric Oxide. Nitric oxide is a reactive nitrogen intermediate formed from arginine and synthesized by NO‑synthase. It is predominantly formed in cells of the body (macrophages and neutrophils). As one of the first‑line defence mechanisms in the body, nitric oxide is excreted by the cells and dilates blood vessels by relaxing smooth muscle cells in the vessels. The pharmaceutical agent nitroglycerin (used to increase blood flow to the heart) is metabolized to form nitric oxide, and so promote the cellular effects seen in the body.

Glutathione. Glutathione is formed from glutamate, glycine, and cysteine. This unusual tripeptide is found in high concentration in the cytosol (~4mM). It is a reactant in leukotriene biosynthesis. Leukotrienes are C₂₀ hormone‑like substances that help to mediate inflammatory and allergic reactions.

Glutathione is also the major part of the cellular defence against uncontrolled oxidation. Glutathione acts principally to protect free sulphhydryl groups. The outer world is an oxidizing one (which is why iron rusts spontaneously), and the molecules in the intracellular space are generally reduced. Thus, it is necessary to have recyclable, oxidizable molecules. These molecules, of which glutathione is the most important, can act as barriers between the oxidized outside and the reduced inside. Glutathione is the major buffer protecting free sulphhydryl groups. Vitamins C and E are also intracellular buffers against oxidation.

The physiologically active amines, epinephrine, norepinephrine, dopamine, serotonin, γ‑aminobutyric acid, and histamine, are derived from amino acids by processes that contrast with the degradation of amino acids for fuel in which the amino functional group is removed. During synthesis of the physiologically active amines, the precursor amino acid must be decarboxylated.

Study Questions

1. What is the purpose of transamination? Of oxidative deamination?
2. Is lysine a ketogenic or glucogenic amino acid? What about serine? What about isoleucine?
3. Name three compounds that are derived from amino acid precursors.
4. How could a person adjust their diet to compensate for a partial deficiency in GDH?
5. What is the difference between a vegetarian diet and an omnivorous diet in terms of transamination and deamination?
6. Would a dietary deficiency of glutamate or α‑ketoglutarate cause problems in efficient transamination?
7. Why doesn’t a tyr deficiency necessarily develop in PKU sufferers who are on a phe‑free diet?
8. What is the biochemical basis for the change in the colour of leaves in the fall?

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.

Lesson 7: Nucleotide Metabolism and de novo Biosynthesis

Overview/Objectives

After completing this lesson, you should be able to:

1. Demonstrate an understanding of ribonucleotide and deoxyribonucleotide synthesis.
2. Discuss the overall process for purine and pyrimidine de novo biosynthesis.
3. Describe the regulation of ATCase in pyrimidine de novo biosynthesis.
4. Describe the regulation of PRPP amidotransferase in purine de novo biosynthesis.
5. Explain the function of ribonucleotide reductase.

Readings and Activities

1. In Chapter 7 of the textbook, read “Nucleotide Metabolism,” “Pyrimidine de novo Biosynthesis,” “Purine de novo Biosynthesis,” and “Deoxyribonucleotide de novo Synthesis” (pages 189–194).
2. You can also watch two video lectures on Nucleotide Metabolism:
   • #39 Biochemistry Nucleotide Metabolism I Lecture for Kevin Ahern’s BB 451/551

   • #40 Biochemistry Nucleotide Metabolism II Lecture for Kevin Ahern’s BB 451/551

     (Links to these are also provided on page 191 of the textbook.)

Commentary

Pyrimidine and Purine de novo Biosynthesis and Regulation

The initial substrates for pyrimidine de novo biosynthesis are bicarbonate, amine from glutamine, and phosphate from ATP to make carbamoyl‑phosphate. The enzyme aspartate transcarbamoylase (ATCase) catalyzes the formation of carbamoyl aspartate by joining carbamoyl‑phosphate to aspartic acid. Following a number of reactions, the final product CTP is formed from UTP after the formation of UMP and then UDP. UDP is a branch point to deoxyribonuceloside diphosphates. Excess CTP inhibits the formation of CTP. The pyrimidine synthesis pathway is illustrated on page 190 of the textbook. (You are not required to know all of the steps in these pathways, but to understand the major reactions highlighted here and understand the regulation of these pathways.)

The substrate or starting point for purine de novo biosynthesis is ribose‑5′‑phosphate. This substrate is phosphorylated by phosphoribosylpyrophosphate (PRPP) synthetase to form PRPP using two phosphates from ATP. PRPP amidotransferase catalyzes the transfer of an amine group to PRPP, which replaces the pyrophosphate on carbon 1 to start the synthesis of the purine ring (phosphoribosylamine). Through a series of reactions the two‑ringed structure inosine monophosphate (IMP), the final product, is formed. IMP is a branch point for the synthesis of adenine and guanosine. The general pathway for purine biosynthesis is shown on page 191 of the textbook. AMP is formed from IMP and the addition of an amine from aspartate and requires energy from GTP. The formation of GMP from IMP involves an addition of an amine from glutamine and requires energy from ATP. These pathways are shown on page 192. Logically, formation of GMP is inhibited by accumulation of GMP, and the formation of AMP is inhibited by accumulation of AMP. This achieves a balance in nucleotides.

Regulation of pyrimidine biosynthesis is primarily achieved through the activity of ATCase, which synthesizes carbamoyl aspartate. This enzyme is regulated by three compounds. The enzyme is activated by aspartate and ATP, and as mentioned previously, inhibited by CTP.

Regulation of purine biosynthesis is through the enzyme PRPP amidotransferase, which is responsible for the formation of the purine ring (phosphoribosylamine). This enzyme is inhibited by GMP and AMP. If both are present, the enzyme is fully inhibited, but otherwise the activity is merely slowed if there is more of one purine than the other. As mentioned above, the nucleotide in excess causes feedback inhibition and stops formation of that particular nucleotide. Therefore, if there is a lot of GMP, then synthesis of AMP proceeds from IMP and vice versa. This maintains an appropriate balance of nucleotides.

Deoxyribonucleotide de novo Synthesis

Deoxyribonucleotides are synthesized from ribonucleotides by the enzyme ribonucleotide reductase (RNR). This enzyme catalyzes the formation of dADP, dGDP, dCDP, and dUDP from ADP, GDP, CDP, and UDP. The formation of dATP, dGTP, dCTP, and dUTP is then catalyzed by the enzyme nucleoside diphosphokinase (NDPK). The name of this enzyme makes sense, as kinases are responsible for the addition of a phosphate residue. The de novo synthesis of dNTPs is outlined on page 192 of the text. As mentioned in the text, thymidine nucleotides are synthesized from dUDP via a more elaborate pathway, which you are not required to know.

Regulation of deoxyribonucleotide de novo synthesis is through RNR. This is important, as this enzyme is responsible for the synthesis of all four deoxyribonucleotides, and a balance of nucleotides is required. This is achieved by having separate sites on the enzyme, one that determines which substrate will be acted on (specificity binding site that binds dNTPs), and one that controls the catalytic activity of the enzyme (activity binding site which controls whether the enzyme is active, ATP activates and dATP inactivates). If a deoxypyrimidine triphosphate is abundant, it will inhibit formation of pyrimidine diphosphates and will stimulate the formation of purine diphosphates.

Study Questions

1. What is the function of ATCase? Do the following molecules inhibit or stimulate this enzyme?
   • aspartate
   • ATP
   • CTP
2. How is AMP formed from IMP? Does this process require energy?
3. If the amount of GTP is high in the cell, what will be the effect on the enzyme RNR and what nucleotides will be produced?

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.

Answers to Unit 7 Study Questions

Lesson 1: Carbohydrate Storage and Breakdown

1. The purpose of storing glucose as glycogen is to have a source of glucose as a backup, and to not rely on simply obtaining glucose from gluconeogenesis.
2. The significance of glucose‑1‑phosphate(G1P) in glycogen degradation is that, when glucose is released from glycogen, it is attached to a phosphate. The advantage of this is that G1P is charged and glucose is not. This prevents the glucose from crossing the membrane and keeps it in the cell for use.
3. The building block for glycogen synthesis is UDP‑glucose, a combination of a high energy compound (UTP) with a low energy compound (G1P).
4. Glycogen phosphorylase is needed for glycogen breakdown, and glycogen synthase is needed for glycogen synthesis. When glucose is released into the system, epinephrine signals a cascade of reactions and these enzymes are both phosphorylated. Phosphorylase‑P is active so glycogen breakdown is enhanced. Glycogen synthase‑P is inactive so glycogen synthesis is halted. The process is switched by the activity of phosphatases, which remove the P groups from these key enzymes. Phosphorylase in now inactive so glycogen breakdown stops, and glycogen synthase is active so glycogen synthesis proceeds.
5. Such a person should eat many small meals. Glycogen storage diseases are a serious medical problem. Glycogen is the shock absorber of metabolism. Without an efficient metabolism of glycogen, there must be constant, controlled eating to supply metabolic needs without overburdening the system.

Lesson 2: Pentose Phosphate Pathway

1. The three functions of the PPP are to produce: NADPH for use in anabolic reductions, ribose‑5‑phosphate for making nucleotides, and erythrose‑4‑phosphate for making aromatic amino acids.
2. The two main phases of the PPP are:
   • oxidative phase: two molecules of NADPH are produced as well as ribulose‑5‑phosphate.
   • Non‑oxidative phase: a number of sugars are synthesized including erythrose‑4‑phosphate.
3. Regulation of the PPP is through the enzyme glucose‑6‑phosphate dehydrogenase. This enzyme is allosterically regulated by the concentration of NADPH. NADP⁺ stimulates the enzyme and NADPH inhibits the enzyme.
4. The PPP is connected to glycolysis because three glycolytic intermediates, G6P, fructose‑6‑P, and G3P, can be funnelled into the PPP for the synthesis of different sugars depending on the energy needs of the cell. In addition, the PPP can provide a mechanism to metabolize sugars (Xu5P and ribulose‑5‑phosphate). The PPP can provide ribulose‑5‑phosphate for nucleotide and nucleic acid synthesis, and erythrose‑4‑phosphate for aromatic amino acid synthesis. Pentose sugars from digestion of nucleic acids can be utilized through this pathway by being converted to glycolytic and or gluconeogenic intermediates.

Lesson 3: Calvin Cycle

1. The overall reactants and products for the Calvin Cycle are:

   3CO₂ + 6 NADPH + 5 H₂O + 9 ATP → glyceraldehyde‑3‑phosphate (G3P) + 2 H⁺ + 6 NADP⁺ + 9 ADP + 8 P_i

2. The Calvin Cycle makes 3‑phosphoglycerate molecules (3PG), used for the production of glucose by gluconeogenesis. The enzyme that catalyzes the formation of 3PG in the Calvin Cycle is RuBisCO (ribulose‑1,5‑bisphosphate carboxylase/oxygenase).
3. Ribulose‑5‑phosphate or Ru1,5BP (or RuBP) is regenerated in the Calvin Cycle. Five G3P molecules produce three RuBP molecules. Since each CO₂ molecule produces two G3P molecules, three CO₂ molecules produce six G3P molecules; of these, five are used to regenerate RuBP, leaving a net gain of one G3P molecule per three CO₂ molecules, which is converted to a hexose through the PPP.

Lesson 4: Urea Cycle

1. Geography is the key. In multicellular organisms, excess NH₄⁺ must travel from the liver through the bloodstream to the kidneys before being eliminated. A unicellular organism exists (usually) in water as fish do, so NH₄⁺ can be eliminated directly through the cell membrane into the immediate environment.
2. NH₄⁺ combines with bicarbonate to form carbamoyl phosphate. Ornithine is recycled after urea is expelled.

Lesson 5: Nitrogen Fixation

1. The ultimate source of the high‑energy electrons to reduce nitrogenase is photosynthesis. Therefore, only plants or species symbiotic with plants can reduce nitrogenase. Because the enzyme is so easily oxidized, only species with elaborate means to protect it (e.g., bacteria in a relatively O₂‑free soil) will contain this enzyme.
2. The octet rule provides the explanation. Nitrogen is stable because it possesses a stable octet of electrons around each nitrogen. Nitrogen is more electronegative than hydrogen, so the N$\ce{-}$H bonding electrons in diimine will be closer to nitrogen than to hydrogen. Therefore, diimine has an almost‑but‑not‑quite‑stable octet structure, as elemental nitrogen does, and can be easily reduced to N₂. The same arguments can be put forward for hydrazine, but in this case there are two hydrogen atoms on each nitrogen. NH₃ is also stable. It is easier for hydrazine to proceed to ammonia than to reconvert to diimine and then to elemental nitrogen.

Lesson 6: Amino Acid Metabolism and Catabolism

1. The purpose of transamination is to recycle nitrogen in the body by transferring the amino group (NH₃) between α‑ketoacids (amino acids without their amino group). This allows the body to maintain a dynamic pool of amino acids ready for use as protein building blocks. Transamination can be used both as the first step in the elimination of excess nitrogen and as the final step in biosynthesis of amino acids.

   Oxidative deamination is to eliminate amino acids. This occurs through cleavage of the amino group from the α‑ketoacid (carrier), which produces NH₄⁺. The only amino acid from which the amino group can be cleaved directly is glutamate. For other amino acids transamination must occur first.

2. Lysine is a ketogenic amino acid. Serine is glucogenic. Isoleucine is both.
3. The compounds that are derived from amino acid precursors are nucleotide bases (purines and pyrimidines), sphingosine, histamine, (also tetrapyrrole, thyroxine, epinephrine, melanin, serotonin, glutathione, tetrahydrofolate).
4. To compensate for a partial deficiency in GDH, one could reduce protein consumption so that elimination of nitrogen is held to a minimum. GDH is the first enzyme involved in the elimination of nitrogen in vivo.
5. Meat proteins more closely resemble human proteins in amino acid composition than do vegetable proteins. Therefore, more transamination activity would be expected on a vegetarian diet. Deamination involves the elimination of excess nitrogen. A well‑balanced vegetarian diet should not involve any more elimination than a well‑balanced diet that includes meat.
6. No, a dietary deficiency of glutamate or α‑ketoglutarate would not cause problems in efficient transamination. Glutamate is not an essential amino acid, since α‑ketoglutarate can be synthesized by the central metabolic pathway in the body: the citric acid cycle.
7. Since the only way humans can synthesize tyr is from phe, a tyr deficiency is possible in PKU sufferers who are on a phe‑free diet. However tyr can also be obtained from the diet, so a tyr deficiency is not a necessary consequence of a phe‑free diet.
8. Plant proteins (including those associated with chlorophyll) are destroyed by sub‑zero temperatures. The porphyrin ring of chlorophyll will be degraded, as heme is.

Lesson 7: Nucleotide Metabolism and de novo Biosynthesis

1. The function of ATCase is to synthesize carbamoyl aspartate. Aspartate and ATP activate ATCase, and CTP inhibits ATCase.
2. The substrate for purine de novo biosynthesis is ribose‑5′‑phosphate. This is phosphorylated by phosphoribosylpyrophosphate (PRPP) synthetase to form PRPP using two phosphates from ATP. PRPP amidotransferase catalyzes the transfer of an amine group to PRPP, which replaces the pyrophosphate on carbon 1, to start the synthesis of the purine ring (phosphoribosylamine). Through a series of reactions the two‑ringed structure inosine monophosphate (IMP), the final product, is formed. IMP is a branch point for the synthesis of adenine and guanosine. AMP is formed from IMP and the addition of an amine from aspartate. Yes, this process requires energy from GTP.
3. If GTP is abundant, RNR will stimulate the production of more pyrimidine nucleotides such as TTP, UTP, and CTP. It will inhibit production of purines such as GTP and ATP.

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.