The document summarizes nitrogen fate in the body. It discusses protein turnover and degradation pathways. Amino acids are removed from proteins and enter metabolic pathways where their nitrogen is eventually converted to urea in the urea cycle in the liver. The urea is transported to the kidneys and excreted in urine. Transamination and glutamine transport ammonia from tissues to the liver for urea synthesis. Genetic defects in the urea cycle can cause hyperammonemia, a potentially fatal condition.

1. NITROGEN FATE
IN
THE BODY
Kshema Thakur, PhD
Molecular Biology & Biochemistry

2. SOURCE AND FATE OF AMINO ACIDS

3. OVERVIEW OF AMINO ACID CATABOLISM

4. Protein turnover
Hydrolysis and resynthesis of 300-400 g
protein/day
Protein degradation:
ATP dependent Ubiquitin-proteasome system of
cytosol
ATP independent lysosomal system of protein
hydrolysis
Chemical signals for degradation
• Binding of ubiquitin
• Serine on N terminus : half life as long as 20 hours
• Aspartate on N terminus : 3 minutes
• PEST (Proline, Glutamine, Serine & Threonine)
sequence in protein: intermittent short half lives

5. UBIQUITIN-PROTEASOME DEGRADATION
PATHWAY

6. Degradation and absorption of proteins in the
GIT
ACTION OF PANCREATIC PROTEASES
Acute pancreatitis is a disease caused by obstruction of the
normal pathway by which pancreatic secretions enter the
intestine. The zymogens of the proteolytic enzymes are
converted to their catalytically active forms prematurely, inside
the pancreatic cells, and attack the pancreatic tissue itself.
This causes excruciating pain and damage to the organ that
can prove fatal.

7. • Free amino acids are taken into the enterocytes by a Na-linked secondary transport
system of the apical membrane.
• Di- and tri - peptides, however, are taken up by a H-linked transport system.
• The peptides are hydrolyzed in the cytosol to amino acids that are released into the
portal system by facilitated diffusion.
• Thus, only free amino acids are found in the portal vein after a meal containing
protein.
• These amino acids are either metabolized by the liver or released into the general
circulation.
• [Note: Branched-chain amino acids are important examples of amino acids that are
not metabolized by the liver, but instead are sent from the liver primarily to muscle
via the blood.]
• The small intestine and the proximal tubule of the kidney have common transport
systems for amino acid uptake.
• At least seven different transport systems are known that have overlapping
specificities for different amino acids.
• uptake of cystine and the dibasic amino acids, ornithine, arginine, and lysine
(represented as “COAL”). In the inherited disorder cystinuria, this carrier system is
defective, and all four amino acids appear in the urine
Degradation and absorption of proteins in the
GIT (contd.)

9. REMOVAL OF NITROGEN FROM
AMINO ACIDS
TRANSAMINATION
Glutamate
formed acts as
a collector of
nitrogen
Aspartate
formed acts aa
a source of
nitrogen in urea
cycle

10. Mechanism of action of
transaminases
Removal of nitrogen from amino acids
(contd.)
PLP (red) as prosthetic group to
one of the active sites of dimeric
aspartate aminotransferase

11. Glutamate dehydrogenase: transdeamidation
Transamination + oxidative deamination
Glutamate dehydrogenase operates at an important
intersection of carbon and nitrogen metabolism. An
allosteric enzyme with six identical subunits, its activity is
influenced by a complicated array of allosteric modulators.
The best-studied of these are the positive modulator ADP
and the negative modulator GTP.

12. Glutamine transfers ammonia in the blood
stream
AMMONIA TRANSPORT IN THE
FORM OF GLUTAMINE
• In metabolic acidosis there is an increase in
glutamine processing by the kidneys.
• Not all the excess NH4 thus produced is released
into the bloodstream or converted to urea; some is
excreted directly into the urine.
• In the kidney, the NH4 forms salts with metabolic
acids, facilitating their removal in the urine.
• Bicarbonate produced by the decarboxylation of -
ketoglutarate in the citric acid cycle can also serve
as a buffer in blood plasma.
• Taken together, these effects of glutamine
metabolism in the kidney tend to counteract
acidosis.

13. GLUCOSE ALANINE CYCLE
ALANINE TRANSPORTS AMMONIA FROM
SKELETAL MUSCLES TO THE LIVER

14.  Ammonia is very toxic for the cell.
 The molecular basis for this toxicity is not entirely understood.
 High levels of NH4 lead to increased levels of glutamine, which acts as an
osmotically active solute (osmolyte) in brain astrocytes, star-shaped cells of the
nervous system that provide nutrients, support, and insulation for neurons. This
triggers an uptake of water into the astrocytes to maintain osmotic balance, leading
to swelling and the symptoms noted and increased cranial pressure.
 So research and speculation on ammonia toxicity have focused on this tissue.
Speculation centers on a potential depletion of ATP in brain cells.
 Excess of glutamate can also result in overexcitation of neurons due to
overproduction of GABA.
 α-ketoglutarate to glutamate by glutamate dehydrogenase and conversion of
glutamate to glutamine by glutamine synthetase.
 Both enzymes are present at high levels in the brain, although the glutamine
synthetase reaction is almost certainly the more important pathway for removal of
ammonia.
AMMONIA TOXICITY

15. UREA CYCLE

16. Highlights of urea cycle:
 Urea is synthesised in liver and transported to kidneys for excretion
 First metabolic pathway described by Krebs and Hanseleit
 The first component, carbamoyl phosphate which enters the Urea cycle is synthesised in a rate
limiting step by enzyme CPS–I. This enzyme is different from CPS–II which is involved in
pyrimidine synthesis in cytosol.
 Citrulline and ornithine are basic amino acids and are not incorporated in proteins as there are
no codons for these.
 Regeneration of ornithine is parallel to oxaloacetate formation of TCA cycle
 Aspartate combines with citrulline to give argininosuccinate bringing second amino group of
urea molecule in an ATP driven reaction forming AMP. This is the third and final ATP molecule
required for urea synthesis.
 Breakdown of argininosuccinate into arginine and fumarate provides a link between urea cycle
and TCA.
 Arginase is present exclusively in liver. Thus only liver can synthesise urea while other tissues
use these reactions for arginine synthesis

17. Link between citric acid cycle and urea cycle: krebs bicycle

18. Fate of urea:
 Urea diffuses from the liver, and is transported in the blood to the kidneys, where it
is filtered and excreted in the urine.
 A portion of the urea diffuses from the blood into the intestine, and is cleaved to
CO2 and NH3 by bacterial urease. This ammonia is partly lost in the feces, and is
partly reabsorbed into the blood.
 In patients with kidney failure, plasma urea levels are elevated, promoting a greater
transfer of urea from blood into the gut.
 The intestinal action of urease on this urea becomes a clinically important source
of ammonia, contributing to the hyperammonemia often seen in these patients.
 Oral administration of neomycin reduces the number of intestinal bacteria
responsible for this NH3 production.

19.  Long term regulation: variation in the rates of synthesis
of the four urea cycle enzymes and carbamoyl
phosphate synthetase I in the liver.
 Short term regulation: Allosteric regulation of carbamoyl
phosphate synthetase I
 N-acetylglutamate is the allosteric activator of CPS-I
 Synthesized from acetyl-CoA and glutamate by N-
acetylglutamate synthase, for which Arginine is an
activator
 Thus, N-acetylglutamate and Arginine are both
activators of urea cycle
Regulation of urea cycle
Synthesis of N-acetyl glutamate and
its activation of carbamoyl
phosphate synthetase I

20. Hyperammonemia
 The capacity of the hepatic urea cycle exceeds the normal rates of ammonia
generation, and the levels of serum ammonia are normally low (5–35 μmol/L).
 Blood ammonia levels can rise above 1,000 μmol/L in case of genetic defects of
urea cycle or liver disease.
 Hyperammonemia thus created is a medical emergency due to neurotoxic effect
of ammonia on the CNS.
 The symptoms of ammonia intoxication include tremors, slurring of speech,
somnolence, vomiting, cerebral edema, and blurring of vision.
 At higher concentrations, ammonia can cause coma and death.
 There are two major types of hyperammonemia are:
Genetic defects in any of the urea cycle enzymes:
cannot take protein rich diet

21. Acquired hyperammonemia:
• Liver disease due to viral hepatitis or due to hepatotoxins such as alcohol.
• Cirrhosis of the liver may result in formation of collateral circulation around the liver.
As a result, portal blood is shunted directly into the systemic circulation and does
not have access to the liver. The conversion of ammonia to urea is, therefore,
severely impaired, leading to elevated levels of ammonia.
Congenital hyperammonemia:
• Genetic deficiencies of each of the five enzymes of the urea cycle have been
described, with an overall prevalence estimated to be 1:25,000 live births.
• Ornithine transcarbamoylase deficiency, which is X-linked, is the most common of
these disorders, predominantly affecting males, although female carriers may
become symptomatic.
• All of the other urea cycle disorders follow an autosomal recessive inheritance
pattern.
• In each case, the failure to synthesize urea leads to hyperammonemia during the
first weeks following birth. [Note: The hyperammonemia seen with arginase
deficiency is less severe because arginine contains two waste nitrogens and can be
excreted in the urine.]
Genetic defects in any of the urea cycle enzymes
(contd.)

22.  Aromatic acids benzoate or phenylbutyrate in the diet
can help lower the level of ammonia in the blood.
 Benzoate is converted to benzoyl-CoA, which combines
with glycine to form hippurate.
 Phenylbutyrate is converted to phenylacetate by
oxidation. The phenylacetate is then converted to
phenylacetyl-CoA, which combines with glutamine to
form phenylacetylglutamine.
 Both hippurate and phenylacetylglutamine are nontoxic
and are excreted in the urine.
 Other therapies are more specific to a particular
enzyme deficiency.
 Deficiency of N-acetylglutamate synthase is treated by
administering carbamoyl glutamate, an analog of N-
acetylglutamate that is effective in activating carbamoyl
phosphate synthetase I.
Treatments for urea cycle defects.

25. Sources:
1. Lehninger – Principles of Biochemistry
David L. Nelson & Michael M. Cox
2. Lipincott’s Illustrated Reviews
Richard A. Harvey & Denise A. Ferrier
3. Harper’s Illustrated Biochemistry
Robert K. Murray, Daryl K. Granner, Peter A. Mayes & Victor W. Rodwell
4. Biochemistry
U. Satyanarayan & U. Chakrapani

26. Thank you