Assignment Code: BBCCT-113 / TMA / 2021-2022

Course Code: BBCCT-113

Assignment Name: Metabolism of Amino Acids and Nucleotides

Year: 2021-2022

Verification Status: Verified by Professor

Note: Attempt all questions. The marks for each question are indicated against it. Write all answers in your own words; do not copy from the course material.

PART-(A)

Q1. a) Draw a representative figure of TCA cycle and mark the points of entry of intermediates of amino acid catabolism into TCA cycle. (6)

Q1. b) What are Transaminases? (4)

Ans) Transaminase, also called Aminotransferase, any of a group of enzymes that catalyze the transfer of the amino group (―NH2) of an amino acid to a carbonyl compound, commonly an a-keto acid (an acid with the general formula RCOCOOH). The liver, for example, contains specific transaminases for the transfer of an amino group from glutamic acid to a-keto acids that correspond to most of the other amino acids. Other transaminases catalyze reactions in which an amino group is transferred from glutamic acid to other compounds—e.g., to aldehydes to form amines. Transamination is one of the principal mechanisms for the formation of necessary amino acids in the metabolism of proteins. Vitamin B6 is commonly involved in the action of the transaminases.

Q2. a) Distinguish between Positive and Negative Nitrogen Balance. (5)

Ans) A negative nitrogen balance occurs when more protein is used by the body than is taken in. A positive nitrogen balance implies a net gain of protein in the body.

Negative nitrogen balance

A condition in which protein catabolism (breakdown) exceeds protein anabolism (synthesis) resulting in tissues losing protein faster than it can be replaced. A negative nitrogen balance may occur during physical or emotional stress, starvation, when an individual is on a very low-calorie diet, or when the quality of protein is poor (e.g., when the diet is lacking essential amino acids). Adrenal cortical hormones, such as cortisone, released during stress enhance protein breakdown and the conversion of amino acids to glucose.

Positive Nitrogen Balance

When you eat protein, your body breaks the protein down into amino acids. Those amino acids are then used to repair and grow new muscle fibers. When you consume an adequate amount of protein, your body will experience something called a positive balance of nitrogen.

Positive nitrogen balance means that nitrogen intake is more than nitrogen loss.

It occurs in:

• Growing children

• Pregnancy

• Convalescence from wasting diseases

Negative nitrogen balance means that nitrogen loss is more than nitrogen intake.

It occurs in:

• Diabetes mellitus

• Fever

• Starvation

• Wasting diseases

Nitrogen equilibrium means that nitrogen intake equals nitrogen loss.

It occurs in healthy adults on an adequate diet

Q2. b) What short note on Kwashiorkor? (5)

Ans) Kwashiorkor is a severe form of malnutrition associated with a deficiency in dietary protein.[2] The extreme lack of protein causes an osmotic imbalance in the gastrointestinal system causing swelling of the gut diagnosed as an edema or retention of water.[6]

Extreme fluid retention observed in individuals suffering from kwashiorkor is a direct result of irregularities in the lymphatic system and an indication of capillary exchange. The lymphatic system serves three major purposes: fluid recovery, immunity, and lipid absorption. Victims of kwashiorkor commonly exhibit reduced ability to recover fluids, immune system failure, and low lipid absorption, all of which result from a state of severe undernourishment. Fluid recovery in the lymphatic system is accomplished by re-absorption of water and proteins which are then returned to the blood. Compromised fluid recovery results in the characteristic belly distension observed in highly malnourished children.

3. a) What are Polyamines? (5)

Ans) Polyamines (PAs) are low molecular weight aliphatic nitrogenous bases containing two or more amino groups. They are produced by organisms during metabolism and are present in almost all cells. Because they play important roles in diverse plant growth and developmental processes and in environmental stress responses, they are considered as a new kind of plant biostimulant. With the development of molecular biotechnology techniques, there is increasing evidence that PAs, whether applied exogenously or produced endogenously via genetic engineering, can positively affect plant growth, productivity, and stress tolerance.

Dietary polyamines have important implications in human health, mainly in the intestinal maturation and in the differentiation and development of immune system. The antioxidant and anti-inflammatory effect of polyamine can also play an important role in the prevention of chronic diseases such as cardiovascular diseases. In addition to endogenous synthesis, food is an important source of polyamines. Polyamines can be found in all types of foods in a wide range of concentrations. Spermidine and spermine are naturally present in food whereas putrescine could also have a microbial origin. The main polyamine in plant-based products is spermidine, whereas spermine content is generally higher in animal-derived foods

Q3.b) Illustrate briefly about the Porphyrins? (5)

Ans) Porphyrins are a group of heterocyclic macrocycle organic compounds, composed of four modified pyrrole subunits interconnected at their α carbon atoms via methine bridges (=CH−). The parent of porphyrin is porphine, a rare chemical compound of exclusively theoretical interest. Substituted porphines are called porphyrins. With a total of 26 π-electrons, of which 18 π-electrons form a planar, continuous cycle, the porphyrin ring structure is often described as aromatic. One result of the large conjugated system is that porphyrins typically absorb strongly in the visible region of the electromagnetic spectrum, i.e., they are deeply colored. The name "porphyrin" derives from the Greek word (porphyra), meaning purple.

Metal complexes derived from porphyrins occur naturally. One of the best-known families of porphyrin complexes is heme, the pigment in red blood cells, a cofactor of the protein hemoglobin. A porphyrin without a metal-ion in its cavity is a free base. Some iron-containing porphyrins are called hemes. Heme-containing proteins, or hemoproteins, are found extensively in nature. Hemoglobin and myoglobin are two O2-binding proteins that contain iron porphyrins. Various cytochromes are also hemoproteins.

Q4. Explain the following disorders of amino acid metabolism: (5+5=10)

Q4. a) Phenylketonuria

Ans) Phenylketonuria (commonly known as PKU) is an inherited disorder that increases the levels of a substance called phenylalanine in the blood. Phenylalanine is a building block of proteins (an amino acid) that is obtained through the diet. It is found in all proteins and in some artificial sweeteners. If PKU is not treated, phenylalanine can build up to harmful levels in the body, causing intellectual disability and other serious health problems.

The signs and symptoms of PKU vary from mild to severe. The most severe form of this disorder is known as classic PKU. Infants with classic PKU appear normal until they are a few months old. Without treatment, these children develop permanent intellectual disability. Seizures, delayed development, behavioral problems, and psychiatric disorders are also common. Untreated individuals may have a musty or mouse-like odor as a side effect of excess phenylalanine in the body. Children with classic PKU tend to have lighter skin and hair than unaffected family members and are also likely to have skin disorders such as eczema.

Q4. b) Alkaptonuria

Ans) Alkaptonuria is a rare genetic metabolic disorder characterized by the accumulation of homogentisic acid in the body. Affected individuals lack enough functional levels of an enzyme required to breakdown homogentisic acid. Affected individuals may have dark urine or urine that turns black when exposed to air. However, this change may not occur for several hours after urination and often goes unnoticed. Aside from dark urine that is present from infancy, affected individuals generally do not develop symptoms (asymptomatic) during infancy or childhood and often remain unaware of their condition until adulthood.

Affected individuals eventually develop ochronosis, which is the bluish-black discoloration of connective and other tissue within the body. Affected individuals may develop discoloration of the skin overlying cartilage within the body such as over part of the outer ear. In some cases, the whites of the eyes (sclera) may also become discolored. In adulthood, affected individuals also develop progressive arthritis of the spine and large joints. The HGD gene codes for the enzyme required for the breakdown of homogentisic acid. Mutations in the HGD gene cause alkaptonuria.

Q5. Describe biosynthesis of non-essential amino acids of glutamate family. (10)

Ans) All amino acids are derived from intermediates in glycolysis, the citric acid cycle, or the pentose phosphate pathway. Nitrogen enters these pathways by way of glutamate and glutamine. Some pathways are simple, others are not. Ten of the amino acids are only one or a few enzymatic steps removed from their precursors. The pathways for others, such as the aromatic amino acids, are more complex.

Different organisms vary greatly in their ability to synthesize the 20 amino acids. Whereas most bacteria and plants can synthesize all 20, mammals can synthesize only about half of them.

Those that are synthesized in mammals are generally those with simple pathways. These are called the nonessential amino acids to denote the fact that they are not needed in the diet. The remainder, the essential amino acids, must be obtained from food. Unless otherwise indicated, the pathways presented below are those operative in bacteria.

PART-(B)

6. a) How purine nucleotide, inosine monophosphate is assembled? Describe the steps. (10)

Ans) Purines are one of three components of nucleotides; phosphate esters of a pentose sugar (either ribose or deoxyribose) in which a purine or pyrimidine base is linked to C1 of the sugar

Purine synthesis yields inosine monophosphate

In the first step of purine biosynthesis, ribose phosphate pyrophosphokinase activates the ribose by reacting it with ATP to form 5-phosphoribosyl-alpha-pyrophosphate (PRPP).

Step 2 is the committed step of purine biosynthesis. In this reaction amidophosphoribosyl transferase catalyses the displacement of PRPP’s pyrophosphate group by glutamine’s amide nitrogen. This reaction is the pathway’s flux-controlling step i.e., the rate at which the biosynthetic pathway outputs product. It is shown in figure below:

Figure above:  (A) Step 1 - Activation of ribose-5-phosphate. The starting material for purine biosynthesis ribose-5-phosphate, a product of the pentose phosphate Pathway. In the first step of purine biosynthesis, ribose phosphate pyrophosphokinase activates the ribose by reacting it with ATP, which drives the reaction, to form 5-phosphoribosyl-alpha-pyrophosphate (PRPP). (B) Step 2 – Flux-controlling step. Amidophosphoribosyl transferase catalyses the displacement of PRPP’s pyrophosphate group by glutamine’s amide nitrogen forming Beta-5-phosphoribosylamine. This step is also driven by ATP.

Following the remaining 9 steps, the first purine derivative that is synthesised is inosine monophosphate (IMP). This can be seen in figure below.

Q7. a) How is DNA chemically different from RNA?  (4)

Ans) DNA stands for deoxyribonucleic acid, while RNA is ribonucleic acid. Although DNA and RNA both carry genetic information, there are quite a few differences between them. This is a comparison of the differences between DNA versus RNA, including a quick summary and a detailed table of the differences.

Summary of Differences Between DNA and RNA

1. DNA contains the sugar deoxyribose, while RNA contains the sugar ribose. The only difference between ribose and deoxyribose is that ribose has one more -OH group than deoxyribose, which has -H attached to the second (2') carbon in the ring.

2. DNA is a double-stranded molecule, while RNA is a single-stranded molecule.

3. DNA is stable under alkaline conditions, while RNA is not stable.

4. DNA and RNA perform different functions in humans. DNA is responsible for storing and transferring genetic information, while RNA directly codes for amino acids and acts as a messenger between DNA and ribosomes to make proteins.

5. DNA and RNA base pairing is slightly different since DNA uses the bases adenine, thymine, cytosine, and guanine; RNA uses adenine, uracil, cytosine, and guanine. Uracil differs from thymine in that it lacks a methyl group on its ring.

Q7. b) Explain the salvage pathway of Pyrimidine bases and Nucleosides. (6)

Ans) Nucleotides and nucleosides can be supplied to an organism by either a salvage reaction or by synthesis from smaller precursors. Salvage reactions convert free purine and pyrimidine bases into nucleotides. Additionally, free purines and pyrimidines can be degraded, the purines to the oxidized ring compound uric acid and the pyrimidines to smaller compounds (β‐amino acids, not the α‐amino acids found in proteins). Finally, purines and pyrimidines can be synthesized from smaller precursors ( de novo synthesis). Thus, three interacting pathways for nucleotides, nucleosides, and the free bases exist: salvage, degradation, and biosynthesis.

Salvage pathways

The nucleotide and nucleosides of a cell are continually in flux. For example, DNA and RNA chains are being synthesized in the cell. Even though the overall DNA content of a cell is constant, small stretches are continually being repaired. Part of the repair process is the breakdown of one strand of the DNA double helix into nucleotides, nucleosides, and free bases. Free purines and pyrimidines are converted back into nucleoside triphosphate monomers to be reincorporated into DNA. A common step in this pathway is the reaction of free bases with phosphoribosyl pyrophosphate (PRPP) to yield nucleotides. PRPP is a general activator of nitrogen ring compounds. For example, PRPP is added to anthranilate during the biosynthesis of tryptophan in bacteria. PRPP is made by the activation of ribose‐5‐phosphate. Ribose‐5‐phosphate can be made through the pentose phosphate pathway. Apparently, two enzymes exist in all systems—one for purines and one for pyrimidines. The synthesis of the glycosidic bond uses the 1′‐pyrophosphate of PRPP as an energy source, and either enzyme transfers the free base to the 1′ position of the ribose, making a nucleotide.

8. a) Draw a schematic representation of electron flow from NADPH to ribonucleotide reductase. (5)

Ans) Protein R2, the small subunit of ribonucleotide reductase of Escherichia coli, contains an essential free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged by hydroxyurea, the enzyme is transformed into an inactive form, metR2. E. coli contains a NAD(P) H:flavin oxidoreductase, named Fre, absolutely required for the regeneration of the radical and the activation of metR2 into R2. Consequently, an E. coli mutant strain lacking an active fre gene is more sensitive to hydroxyurea during growth, demonstrating the physiological protective function of Fre from the loss of the radical. However, this gene is not essential, and we found that E. coli contains a second tyrosyl radical generating activity, also residing in a flavin reductase. The enzyme has been purified 200-fold to homogeneity and found to be identical to sulfite reductase. Pure sulfite reductase has the ability to catalyze the reduction of free riboflavin, FMN, or FAD by NADPH and thus, as Fre, to transfer electrons to the iron center of metR2, a key step during the activation reaction.

Schematic Representation of flow from NADPH to ribonucleotide:

Q8. b) Elaborate role of inhibitors in nucleotide synthesis(5)

Ans) Potent inhibitors of enzymes catalyzing reactions in the de novo pathways for biosynthesis of purine and pyrimidine nucleotides are synthetic or natural-product analogues of pathway intermediates or, more recently, inhibitors rationally designed from a knowledge of the catalytic mechanism. Such inhibitors may be effective drugs against cancer, inflammatory disorders, or various infections. For human cancer, the purine pathway may be a better target for inhibition than the pyrimidine pathway, where toxic side effects are more apparent. Drugs such as methotrexate and 6-mercaptopurine have multiple sites of action, making it difficult to quantitatively predict their effects upon cells. Rational design of inhibitors based upon the X-ray structure of the target enzyme has the prospect of yielding drugs with only one site of action in human cells. Such a drug is VX-497, a potent inhibitor of the purine enzyme, IMP dehydrogenase.

Q9. Discuss catabolism of pyrimidine nucleotides: (10)

Ans) The catabolism of pyrimidine nucleotides, like that of purine nucleotides, involves dephosphorylation, deamination, and glycosidic bond cleavage. However, in contrast to purine catabolism, the pyrimidine bases in most organisms are subjected to reduction rather than oxidation. Reductive pyrimidine base catabolism occurs in most microorganisms, plants, and animals. In plants, the pyrimidine bases, uracil, and thymine, derived from uridine monophosphate and deoxythymidine-5'-monophosphate are directly catabolized by a reductive degradation pathway. The catabolism of cytidine-5'-monophosphate must take place after conversion of cytidine to uridine by cytidine deaminase, which is followed by uracil formation. In plants, a dual function of pyrimidine metabolism has been proposed. In addition to the pyrimidine salvage for nucleotides and nucleic acid synthesis, a degradation product of uracil, p-alanine, is used for pantothenic acid (vitamin B5) synthesis. Hence, a portion of pyrimidine ring catabolites is recovered as amino acid-related compounds.

Q10. Describe briefly the signs and symptoms of:

Q10. i) Gout

Ans) Gout is a common and complex form of arthritis that can affect anyone. It's characterized by sudden, severe attacks of pain, swelling, redness and tenderness in one or more joints, most often in the big toe.

An attack of gout can occur suddenly, often waking you up in the middle of the night with the sensation that your big toe is on fire. The affected joint is hot, swollen and so tender that even the weight of the bedsheet on it may seem intolerable.

Gout symptoms may come and go, but there are ways to manage symptoms and prevent flares.

Signs and Symptoms

The signs and symptoms of gout almost always occur suddenly, and often at night. They include:

Intense joint pain.

Gout usually affects the big toe, but it can occur in any joint. Other commonly affected joints include the ankles, knees, elbows, wrists and fingers. The pain is likely to be most severe within the first four to 12 hours after it begins.

Lingering discomfort.

After the most severe pain subsides, some joint discomfort may last from a few days to a few weeks. Later attacks are likely to last longer and affect more joints.

Inflammation and redness. The affected joint or joints become swollen, tender, warm and red.

Limited range of motion.

As gout progresses, you may not be able to move your joints normally.

Q10. ii) Lesch-Nyhan syndrome

Ans) Lesch–Nyhan syndrome (LNS) is a rare inherited disorder caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). This deficiency occurs due to mutations in the HPRT1 gene located on the X chromosome.

Signs and Symptoms

Signs of Lesch-Nyhan syndrome are usually first seen when a child is as young as six months old, and female carriers tend to not display symptoms. These include:

Orange sand: In infants with the condition, the excess production of uric acid can lead to orange deposits often called “orange sand” in the diapers.

Urate stones: Infants may also develop crystals in the kidneys, which can lead to blood in the urine and raises the risk of urinary tract infection.

Pain and swelling in the joints: In teens and adults, urate crystals can form in the joints of untreated individuals, leading to gout-like pain and swelling.

Cartilage deposits: Older children with the disorder often have uric deposits that collect in cartilage. When these cause bulges in the ears, the condition is called tophi.

Dystonia: Many of those with this condition experience dystonia—characterized by involuntary writhing in the arms and legs.

Chorea: Another issue that arises is chorea—involuntary, purposeless, and repetitive body movements. These can include grimacing, shoulder raising and lowering, as well as finger flexing.

Hypotonia: In infants, Lesch-Nyhan syndrome can lead to under-development of certain muscle groups, sometimes leading to an inability to hold the head up.

Developmental delay: Infants and toddlers with the condition may also experience delayed developmental milestones, such as sitting up, crawling, and walking.

Hypertonia: Children with this condition may develop hypertonia, defined as over-developed musculature. Often, this is paired with spasticity—increased rigidity of muscles.

Spasticity: Rigidity of the tendons, termed hyperreflexia, is also seen in these cases.

Intellectual disability: Moderate intellectual disability often occurs with this condition, though many with it do not display this symptom.

Dysarthia: The ability to articulate speech and pronounce words—dysarthia—is also a common hallmark.

Self-Mutilation: Approximately 85% of those with Lesch-Nyhan syndrome display self-mutilating behaviors, such as compulsive lip, hand, or finger biting as well as head banging.2 These symptoms typically arise between the ages of 2 and 3.

Dysphagia: Children and infants often also have dysphagia, defined as an inability to swallow effectively.

Behavioral abnormalities: Some children with the condition may find it difficult to control behaviors and are prone to screaming and irritability.

Muscle spasm: Lesch-Nyhan syndrome is often characterized by strong muscle spasms that lead to severe arching of the back. The head and heels in these cases can also bend backward.