Amino acid (Nitrogen) metabolism

Introduction I General: digestion, absorption transamination and urea.

Q01. In which form atmospheric nitrogen is used by all organisms?

A01.

• Nitrogen ranks fourth important element after carbon, hydrogen and oxygen, of the mass of living cells.
• Though atmospheric nitrogen N2 is most abundant but is too inert for use in most biochemical processes.
• Only few microorganisms can convert N2 to biological useful form such as NH3, amino groups are used with great economy in biological system.
• Atmospheric nitrogen needs to be converted by some organisms into forms acceptable to the all the organisms.
• Nitrogen fixation is carried out by bacterial nitrogensases forming reduced nitrogen, NH₄⁺ that can then be used by all organisms to form amino acids.

Q02. How nitrogen enters the human body?

A02. Reduced nitrogen enters the human body as dietary free amino acids, protein, and the ammonia produced by intestinal tract bacteria. Amino acids derived from dietary proteins are the main source of amino groups.

Q03. In which metabolic circumstances, amino acids in body can undergo oxidative degradation?

A03. In body, amino acids can undergo oxidative degradation in three different metabolic conditions:

1. During the normal synthesis and degradation of cellular proteins (protein turnover). Some of amino acids released during protein breakdown will undergo oxidative degradation if they are not needed for new protein synthesis.

2. When a diet is rich in protein, the surplus amino acids may be catabolized when they are in excess. Amino acids cannot be stored.

3. During starvation or in diabetes mellitus, when carbohydrates are either unavailable or not properly utilized, body proteins are used as fuel.

Under these different circumstances amino acids lose their amino groups and the alpha-keto acids so formed may undergo oxidation to CO2 and H2O. In addition, the carbon skeleton of amino acids provides three and four carbon units that can be converted to glucose to be used by body.

Q04. Does metabolic energy derived from amino acids varies greatly with the type of organism and with metabolic situation?

A04.

• Carnivores immediately following a meal may obtain up to 90% of their energy requirements from amino acid oxidation.
• Herbivores may obtain a small fraction of their energy needs from amino acids.
• Most microorganisms can scavenge amino acids form their environment and can use for their metabolic need.
• Photosynthetic plants rarely oxidize amino acids for energy purpose. Instead they convert CO2 and H2O into carbohydrates that are mainly used as energy source.
• The amount of amino acids in plant tissues are carefully used for biosynthesis of proteins, nucleic acids and other molecules needed to support the growth.
• Amino acids catabolism does occur in plants and their metabolites are used for other biosynthetic pathways.

Q05. What is gastrin and what is its role in protein digestion?

A05. Entry of protein in to stomach stimulates gastric mucosa to secreate the hormone gastrin. Which stimulates the secretion of hydrochloric acid by the parietal cells of gastric glands and pepsinogen by the chief cells.

Q06. How protein digestion takes place?

A06. Protein digestion begins in the stomach, where a proenzyme called pepsinogen is secreted, autocatalytically converted to Pepsin A, and used for the first step of proteolysis. However, most proteolysis takes place in the duodenum as a consequence of enzyme activities secreted by the pancreas. All of the serine proteases and the zinc peptidases of pancreatic secretions are produced in the form of their respective proenzymes. These proteases are both endopeptidase and exopeptidase, and their combined action in the intestine leads to the production of amino acids, dipeptides, and tripeptides, all of which are taken up by enterocytes of the mucosal wall.

Q07. How preoteolytic enzymes are regulated?

A07. A circuitous regulatory pathway leading to the secretion of proenzymes into the intestine is triggered by the appearance of food in the intestinal lumen.

- Special mucosal endocrine cells secret the peptide hormones cholecystokinin (CCK) and secretin into the circulatory system.

- Together, CCK and secretin cause contraction of the gall bladder and the exocrine secretion of a bicarbonate-rich, alkaline fluid, containing protease proenzymes from the pancreas into the intestine.

- A second, paracrine role of CCK is to stimulate adjacent intestinal cells to secrete enteropeptidase, a protease that cleaves trypsinogen to produce trypsin.

- Trypsin also activates trypsinogen as well as all the other proenzymes in the pancreatic secretion, producing the active proteases and peptidases that hydrolyze dietary polypeptides.

Q08. What is celiac disease?

A08. Celiac disease is a condition in which the intestinal enzymes are unable to digest certain water insoluble proteins of wheat, particularly gliadin, which is injurious to the cells lining the small intestine. Wheat products must be avoided in this condition.

Q09. What is acute pancreatitis?

A09. In this condition the normal pathway of secretion of pancreatic juice into the intestine is obstructed. Thus the zymogens of the proteolytic enzymes are converted to the active forms inside the pancreatic cells, prematurely. This active proteolytic enzymes act on the pancreatic tissue itself, causing serious destruction of pancreas, which is very painful and can be fatal.

Q10. How intestinal bacterial activity contributes to nitrogen metabolism?

A10. Many other nitrogenous compounds are formed in the intestine as a result of intestinal bacterial activity. Some have powerful pharmacological (vasopressor) effects. Intestinal bacteria convert lysine, arginine, tyrosine, ornithine and histidine to their vasopressor amines such as cadaverene, agmatine, tyramine, putrescine and histamine respectively.

Q11. What are essential amino acids?

A11.

Prokaryotes such as E. coli can make the carbon skeletons of all 20 amino acids and transaminate those carbon skeletons with nitrogen from glutamine or glutamate to complete the amino acid structures.

Humans cannot synthesize the branched carbon chains found in branched chain amino acids or the ring systems found in phenylalanine and the aromatic amino acids; nor can we incorporate sulfur into covalently bonded structures.

Therefore, the 9 so-called essential amino acids must be supplied from the diet. They are:

Branched chain amino acids: leucine, Isoleucine, valine,

Aromatic amino acids: Phenylalanine, tryptophan,

Sulphur containing amino acid: methionine

Basic amino acids: Histidine and lysine.

And threonine.

Q12. What are semi-essential amino acids?

A12.

• Depending on the composition of the diet and physiological state of an individual, one or another of the non- essential amino acids may also become a required dietary component forming a group of semi-essential amino acids.
• For example, arginine is not usually considered to be essential, because enough for adult needs is made by the urea cycle. However, the urea cycle generally does not provide sufficient arginine for the needs of a growing child.
• To take a different type of example, cysteine and tyrosine are considered non-essential but are formed from the essential amino acids methionine and phenylalanine, respectively.
• If sufficient cysteine and tyrosine are present in the diet, the requirements for methionine and phenylalanine are markedly reduced; conversely, if methionine and phenylalanine are present in only limited quantities, cysteine and tyrosine can become essential dietary components.

Finally, it should be recognized that if the α-keto acids corresponding to the carbon skeleton of the essential amino acids are supplied in the diet, aminotransferases in the body will convert the keto acids to their respective amino acids, largely supplying the basic needs.

Q13. What happens during the degradation of amino acids?

A13. Amino acids can undergo oxidative degradation as a consequence of protein turnover; when the diet is particularly rich in protein or when carbohydrates are not available like in starvation or in diabetes mellitus.

The degradative pathway of every amino acid requires the separation of the amino group from the carbon skeleton. The carbon skeletons enter the Krebs cycle or are channeled into gluconeogenesis. Part of the ammonia is reused for biosynthetic purpose; part is excreted directly and the rest is excreted as urea.

Q14. How removal of nitrogen from amino acids takes place?

A14. Most of the amino acids are metabolized in liver. Some of the ammonia that is generated is recycled and used in a variety of biosynthetic processes. The excess ammonia is either excreted directly or converted to uric acid or urea for excretion depending on the organism. Excess ammonia generated in extrahepatic tissues is transported to the liver for excretion after converting to a proper form. Nitrogen elimination begins intracellularly with protein degradation. There are two main routes for converting intracellular proteins to free amino acids: a lysosomal pathway, by which extracellular and some intracellular proteins are degraded, and cytosolic pathways that are important in degrading proteins of intracellular origin.

Q15. What happens in cystosolic pathway?

Q15. In one cytosolic pathway:

- A protein known as ubiquitin is activated by conversion to an AMP derivative.

- And cytosolic proteins that are damaged or otherwise destined for degradation are enzymically tagged with the activated ubiquitin.

- Ubiquitin-tagged proteins are then attacked by cytosolic ATP-dependent proteases that hydrolyze the targeted protein, releasing the ubiquitin for further rounds of protein targeting.

Q16. How amino acid nitrogen is removed?

A16. The dominant reactions involved in removing amino acid nitrogen from the body are known as transaminations. This class of reactions funnels nitrogen from all free amino acids into a small number of compounds; then, either they are oxidatively deaminated, producing ammonia, or their amine groups are converted to urea by the urea cycle.

Q17. How transaminations take place?

A17. Transaminations involve moving a α-amino group from a donor α-amino acid to the keto carbon of an acceptor α-keto acid. These reversible reactions are catalyzed by a group of intracellular enzymes known as transaminases (aminotransferases), which employ covalently bound pyridoxal phosphate as a cofactor.

Transaminases exist for all amino acids except threonine and lysine.

Q18. Which compounds are most commonly involved in transamination?

A18. The most common compounds involved as a donor/acceptor pair in transamination reactions are glutamic acid and α-ketoglutaric acid, which participate in reactions with many different aminotransferases.

Q19. Which aminotransferase is clinically important?

A19. Serum aminotransferases such as serum glutamate-oxaloacetate-aminotransferase (SGOT) have been used as clinical markers of tissue damage, with increasing serum levels indicating an increased extent of damage.

Q20. How creatinine is formed and what is its clinical significance?

A20.

• The first reaction in creatinine formation is the transfer of the amido (or amidine) group of arginine to glycine, forming guanidinoacetate.
• Subsequently, a methyl group is transferred from the ubiquitous 1-carbon-donor S-adenosylmethionine to guanidinoacetate to produce creatine (from which phosphocreatine is formed), some of which spontaneously cyclizes to creatinine, and is eliminated in the urine.
• The quantity of urine creatinine is generally constant for an individual and approximately proportional to muscle mass.
• In individuals with damaged muscle cells, creatine leaks out of the damaged tissue and is rapidly cyclized, greatly increasing the quantity of circulating and urinary creatinine.
• A small but clinically important amount of creatinine is excreted in the urine daily, and the creatinine clearance rate is often used as an indicator of kidney function.

Q21. How glutamete is a prominent intermediate in nitrogen elimination?

A21.

• Because of the participation of α-ketoglutarate in numerous transaminations, glutamate is a prominent intermediate in nitrogen elimination as well as in anabolic pathways.
• Glutamate formed in the course of nitrogen elimination is either oxidatively deaminated by liver glutamate dehydrogenase, forming ammonia.
• The ammonia thus formed is converted to glutamine by glutamine synthase and transported to kidney tubule cells.
• There the glutamine is sequentially deamidated by glutaminase and deaminated by kidney glutamate dehydrogenase.
• The ammonia produced in the latter two reactions is excreted as NH₄⁺ in the urine, where it helps maintain urine pH in the normal range of pH 4 to pH 8.
• The extensive production of ammonia by peripheral or liver glutamate dehydrogenase is not feasible because of the highly toxic effects of circulating ammonia.
• Normal serum ammonium concentrations are in the range of 20-40 mmol, and an increase in circulating ammonia to about 400 mmol causes alkalosis and neurotoxicity.
  
  

Q22. Which amino acid related reaction is therapeutically significant?

A22.

• A therapeutically useful amino acid-related reaction is the amidation of aspartic acid to produce asparagine.
• The enzyme asparagine synthase catalyzes the ATP, requiring the transamidation reaction shown below:
  aspartate + glutamine + ATP --> glutamate + asparagine + AMP + PP_i
• Most cells perform this reaction well enough to produce all the asparagine they need.
• However, some leukemia cells require exogenous asparagine, which they obtain from the plasma. Chemotherapy using the enzyme asparaginase takes advantage of this property of leukemic cells by hydrolyzing serum asparagine to ammonia and aspartic acid, thus depriving the neoplastic cells of the asparagine that is essential for their characteristic rapid growth.
  
  

Q23. How clinically important are sterospecific amino acid oxidases?

A23.

• In the peroxisomes of mammalian tissues, especially liver, there are 2 stereospecific amino acid oxidases involved in elimination of amino acid nitrogen.
• D-amino acid oxidase is an FAD-linked enzyme, and while there are few D-amino acids that enter the human body the activity of this enzyme in liver is quite high. L-amino acid oxidase is FMN-linked and has broad specificity for the L amino acids.
• A number of substances, including oxygen, can act as electron acceptors from the flavoproteins. If oxygen is the acceptor the product is hydrogen peroxide, which is then rapidly degraded by the catalases found in liver and other tissues.
• Missing or defective biogenesis of peroxisomes or L-amino acid oxidase causes generalized hyper-aminoacidemia and hyper-aminoaciduria, generally leading to neurotoxicity and early death.

Q24. Discribe the role and significance of glutamate dehydrogenase?

A24.

The reaction catalyzed by glutamate dehydrogenase is:

NH₄⁺ + α -ketoglutarate + NAD (P) H + H⁺ <----> glutamate + NAD (P)⁺ + H₂O

Glutamate dehydrogenase can utilize either NAD orNADP as cofactor.

• In the forward reaction as shown above glutamate dehydrogenase is important in converting free ammonia and α-ketoglutarate (α-KG) to glutamate, forming one of the 20 amino acids required for protein synthesis.
• However, it should be recognized that the reverse reaction is a key anapleurotic process linking amino acid metabolism with TCA cycle activity.
• In the backward reaction, glutamate dehydrogenase provides an oxidizable carbon source used for the production of energy.
• As expected for a branch point enzyme with an important link to energy metabolism, glutamate dehydrogenase is regulated by the cell energy charge.
• ATP and GTP are negative effectors, whereas ADP and GDP are positive allosteric effectors. Thus, when the level of ATP is high, conversion of glutamate to α-KG and other TCA cycle intermediates is limited; when the cellular energy charge is low, glutamate is converted to ammonia and oxidizable TCA cycle intermediates.

Q25. Describe the role and significance of glutamine synthase.

A25.

The reaction catalyzed by glutamine synthase is:

glutamate + NH₄⁺ + ATP -------> glutamine + ADP + P_i + H⁺

The glutamine synthatase reaction is also important in several respects. First it produces glutamine, one of the 20 major amino acids. Second, in animals, glutamine is the major amino acid found in the circulatory system.

Its role there is to carry ammonia to and from various tissues but principally from peripheral tissues to the kidney, where the amide nitrogen is hydrolyzed by the enzyme glutaminase (reaction below); this process regenerates glutamate and free ammonium ion, which is excreted in the urine.

glutamine + H₂O -------> glutamate + NH₃

Note that, in this function, ammonia arising in peripheral tissue is carried in a nonionizable form, which has none of the neurotoxic or alkalosis-generating properties of free ammonia.

Q26. What is Urea Cycle?

A26. About 80% of the excreted nitrogen is in the form of urea, which is also largely made in the liver, in a series of reactions that are distributed between the mitochondrial matrix and the cytosol. The series of reactions that form urea is known as the Urea Cycle or the Krebs-Henseleit Cycle.

Q27. What are essential features of the urea cycle?

A27. The essential features of the urea cycle reactions and their metabolic regulation are as follows:

1. Arginine from the diet or from protein breakdown is cleaved by the cytosolic enzyme arginase, generating urea and ornithine.

2. Ornithine arising in the cytosol is transported to the mitochondrial matrix, where ornithine transcabamoylase catalyzes the condensation of ornithine with carbamoyl phosphate, producing citrulline. The energy for the reaction is provided by the high-energy anhydride of carbamoyl phosphate.

3. The product, citrulline, is then transported to the cytosol, where the remaining reactions of the cycle take place.

4. In a 2-step reaction, catalyzed by cytosolic argininosuccinate synthetase, citrulline is converted to argininosuccinate. The reaction involves the addition of AMP (from ATP) to the amido carbonyl of citrulline, forming an activated intermediate on the enzyme surface (AMP-citrulline), and the subsequent addition of aspartate to form argininosuccinate.

5. Arginine and fumarate are produced from argininosuccinate by the cytosolic enzyme argininosuccinate lyase. In the final step of the cycle arginase cleaves urea from aspartate, regenerating cytosolic ornithine, which can be transported to the mitochondrial matrix for another round of urea synthesis.

Beginning and ending with ornithine, the reactions of the cycle consumes 3 equivalents of ATP and a total of 4 high-energy nucleotide phosphates. Urea is the only new compound generated by the cycle; all other intermediates and reactants are recycled.

Q28. How the regulation of the Urea Cycle takes place in the body?

A28.

The urea cycle operates only to eliminate excess nitrogen.

On high-protein diets the carbon skeletons of the amino acids are oxidized for energy or stored as fat and glycogen, but the amino nitrogen must be excreted.

To facilitate this process, enzymes of the urea cycle are controlled at the gene level.

When dietary proteins increase significantly, enzyme concentrations rise. On return to a balanced diet, enzyme levels decline. Under conditions of starvation, enzyme levels rise as proteins are degraded and amino acid carbon skeletons are used to provide energy, thus increasing the quantity of nitrogen that must be excreted.

Q29. What happens when excretion of ammonia is deranged?

A29. Built up of ammonia is neurotoxic. Marked brain damage is seen in cases of failure to make urea via the urea cycle or to eliminate urea through the kidneys. The result of either of these events is a buildup of circulating levels of ammonium ion. Aside from its effect on blood pH, ammonia readily traverses the brain blood barrier and in the brain is converted to glutamate via glutamate dehydrogenase, depleting the brain of α-ketoglutarate. As the α-ketoglutarate is depleted oxaloacetate falls correspondingly, and ultimately TCA cycle activity comes to a halt. In the absence of aerobic oxidative phosphorylation and TCA cycle activity, irreparable cell damage and neural cell death ensue.