this chapter looked at two aspects of nitrogen metabolism; the urea cycle in the liver, and supply of urea / nitrogen from different sources in the body.
the metabolism of amino acids releases carbon which can be used for ATP generation, as well as nitrogen in the form of NH4+/NH3. NH4+ is ammonium ion, which dissociates into NH3 and H+. at physiologic pH, NH4 predominates (it remains undissociated). NH4+/NH3 is toxic to the body and needs to be converted to urea in the liver; this chapter explains how the body transports nitrogen from amino acids to the liver for conversion in the urea cycle.
the most common reactions that frees nitrogen from amino acids are transamination reactions with glutamate. in this reaction, the amino group from the amino acid is transferred to an alpha keto-glutarate, forming glutamate and an alpha keto acid. glutamate can then receive another nitrogen and form glutamine, which is then released into the bloodstream. nitrogen in glutamine is one of the main transport mechanisms for nitrogen in the blood; the other is alanine, which is formed by transamination of pyruvate into alanine by glutamate.
glutamate is a crucial molecule which serves as an intermediate in several different pathways in nitrogen metabolism. in the glutamate dehydrogenase reaction, glutamate is oxidized to alpha-keto glutarate via the enzyme glutamate dehydrogenase, releasing NH4+ (basically the opposite of the transamination reaction without the transfer to an amino acid). the glutaminase reaction was mentioned above- the enzyme glutaminase cleaves glutamine into glutamate and NH4+. the opposite reaction, glutamine synthetase, creates glutamine from glutamate. thus glutamate can serve as both the nitrogen acceptor and the nitrogen donor, aiding in amino acid degradation as well as synthesis.
when the nitrogen in the form of glutamine or alanine reaches the liver, it is cleaved into carbon, which can be used for metabolism or gluconeogenesis, and nitrogen in the form of ammonia. in the mitochondria, the urea cycle begins:
1. ammonium combines with bicarbonate, forming carbamoyl phosphate via carbamoyl phosphate synthetase I
2. carbamoyl phosphate combines with mitochondrial ornithine, forming citrulline via ornithine transcarbamoylase.
3. citrulline is transported out of the mitochondria into the cytoplasm, where it combines with aspartate to form argininosuccinate via argininosuccinate synthetase.
4. argininosuccinate is cleaved by argininosuccinate lyase into arginine and fumarate.
5. arginine is then cleaved into urea and ornithine via arginase.
6. ornithine is transported back into the mitochondria in exchange for citrulline formed in step 2.
in step 4, the fumarate that is created can be acted upon by the same enzymes from the TCA cycle- fumarase catalyzes the hydration reaction to malate, which is then oxidized by malate dehydrogenase into oxaloacetate. oxaloacetate can then be transaminated into aspartate. thus the net result is the recycling of fumarate back into aspartate, where it can be reused in the urea cycle in step 3.
the urea cycle is regulated by a "feed forward" positive feedback mechanism in which the relative activity of the urea cycle is dependent of substrate availability. thus in conditions of high amino acid availability such as fasting or a high protein diet, more NH4 will be transported to the liver, and the urea cycle will be stimulated. another regulatory mechanism is the allosteric activation of CPSI, the enzyme in the first step of the urea cycle, by a protein NAG. NAG is synthesized in response to high arginine levels- when arginine levels are high, this indicates the need for the urea cycle to be activated further.
questions
1. what is the main way by which nitrogen is removed from amino acids in the body?
2. describe the formation of an alpha keto acid from an amino acid.
3. which amino acids do not undergo transamination reactions?
4. what is the cofactor in transamination reactions?
5. what is another way that nitrogen is removed from amino acids?
6. what is the relative concentration of ammonia to ammonium in the body and why?
7. which can cross cell membranes, ammonia or ammonium?
8. which reaction does glutamate dehydrogenase catalyze?
9. how is histidine deaminated?
10. how are serine and threonine deaminated?
11. how are glutamine and asparagine deaminated?
12. why is the deamination of glutamine important in the kidney?
13. what occurs in the brain and muscle that allows NH4+ to be released from amino acids?
14. how does glutamate aid in amino acid synthesis?
15. how does glutamate supply nitrogen to the urea cycle?
16. what are the two transporters of nitrogen in the blood?
17. how is alanine formed in the muscles?
18. what happens to alanine in the liver?
18b. what is the glucose alanine cycle and when is it used?
19. what does the enzyme glutamine synthetase do?
20. describe the production of glutamine in the tissues.
21. what is glutamine used for in the liver, intestines, and kidney?
urea cycle...
22. describe the first step of the urea cycle. where does it occur?
23. describe the formation of citrulline.
24. describe the transport of citrulline.
25. describe the formation of argininosuccinate.
26. describe the formation of fumarate and arginine.
27. how is fumarate recycled?
28. describe the formation of urea from arginine.
29. how is the urea cycle regulated?
30. describe the regulation of CPSI.
31. describe the regulation of the urea cycle via induction of urea cycle enzymes.
32. why is urinary excretion of urea high during fasting?
33. why does urea excretion decrease during prolonged fasting?
answers
1. through transamination reactions.
2. via a transamination reaction where the amino group from the amino acid is transferred to alpha keto-glutarate, which forms an alpha keto acid from the amino acid, and glutamate from the alpha keto glutarate.
3. lysine and threonine.
4. pyridoxal phosphate.
5. through the release of ammonia and ammonium.
6. about one hundred times more ammonium, because the pKa of the dissociation reaction (NH4+ -> NH3 + H+) is 9.3. this means that at pH 9.3, 50% of the NH4+ is dissociated into ammonia, but at the lower body pH of 7.4, most of the NH4+ is undissociated.
7. ammonia (recall the ammonia buffer system from the kidney)
8. the oxidative deamination of glutamate, producing ammonium and alpha keto glutarate.
9. directly deaminated to form NH4 and urocanate.
10. dehydration reactions that require pyridoxal phosphate as a cofactor and are catalyzed by serine dehydratase, releasing NH4+ in both cases. serine forms pyruvate, threonine forms alpha-ketobutyrate.
11. they both contain R group amides that may be released as NH4+ by deamidation.
12. because it produces ammonium, which is secreted directly into the renal tubules, which forms salts with metabolic acids and aids in their excretion.
13. the purine nucleotide cycle.
14. glutamate supplies nitrogen for amino acid synthesis; either from the glutamate dehydrogenase reaction or the transamination reaction. it then donates the amino group to an alpha keto acid to create an amino acid.
15. it supply nitrogen through NH4 produced in the glutamate dehydrogenase reaction, or it can transaminate oxaloacetate to aspartate, which then enters the urea cycle.
16. alanine and glutamine.
17. the metabolism of glucose in the muscles produces pyruvate, which can be transaminated by glutamate (see question 2) into alanine.
18. alanine is transaminated back to pyruvate, and the nitrogen will be used for urea synthesis. pyruvate can be used for gluconeogenesis and the glucose can be transported back to the muscles.
18b. the glucose/alanine cycle is the production of alanine via transamination of pyruvate in the muscles, and the breakdown of the alanine in the liver to ammonium and glucose. the cycle can take place in exercise, when the muscles "use blood borne glucose".
19. it is a cytoplasmic enzyme present in all cells that catalyzes the conversion of glutamate to glutamine.
20. in conditions of rapid amino acid degradation, glutamine is formed from glutamate via glutamine synthetase.
21. in the liver, glutamine is used in the urea cycle. in the intestines glutamine is used as fuel. in the kidney the NH4 released from glutamine degradations aids in excretion of metabolic salts.
22. in the mitochondria of liver and kidney cells: ammonium combines with bicarbonate and forms carbamoyl phosphate via CPSI and 2 ATP.
23. the carbomoyl phosphate formed in step one combines with ornithine to form citrulline, via ornithine transcarbamoylase.
24. citrulline is transported out of the mitochondria in exchange for cytoplasmic ornithine. =
25. citrulline in the cytoplasm combines with arginine (see question 15) to form argininosuccinate via argininosuccinate synthetase. this reactions costs 1 ATP
26. argininosuccinate is cleaved by argininosuccinate lyase to form fumarate and arginine.
27. fumarate is converted to malate via fumarase, which can be converted to oxaloacetate, which can then be transaminated to reform aspartate.
28. arginine is cleaved by arginine lyase into urea
29. by availability of substrates, regulation of CPSI, regulation of urea cycle enzymes.
30. high arginine levels stimulates NAG production from acetyl CoA, which activates CPSI.
31. during conditions of high protein metabolism (ie. high protein diet or fasting/starvation) causes urea cycle enzymes to be inducted.
32. because during fasting amino acids are used to supply substrates for gluconeogenesis; when an amino acid is converted to pyruvate, nitrogen is released and therefore excreted in the urine as urea.
33. during prolonged fasting ketone bodies are used for metabolism, sparing the need for amino acids as gluconeogenic precursors, which leads to less urea excretion.
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