BIOCHEMISTRY

P.03 REMOVAL OF NITROGEN

FROM AMINO ACIDS (PART 2)
Dr. Domantay | January 20, 2017

OVERVIEW: Removal of Nitrogen from Amino Acids

1. Transamination
2. Oxidative deamination

OUTLINE

I. Oxidative deamination
II. Allosteric regulators of glutamate
dehydrogenase
III. Transport of Ammonia to the Liver
IV. Case presentation
A. Biochemical Pathway involved in Portal
Hypertension
B. Regulation of Ammonia in the Body
C. Sources of Ammonia
D. Transport of Ammonia
E. Hyperammonemia and its types

I. OXIDATIVE DEAMINATION

- Liberation of the amino group as free ammonia

(NH3). - Glutamate: only amino acid that undergoes
- It occurs in the liver and kidneys. rapid oxidative deamination and it is the
- Involves the enzyme glutamate dehydrogenase connection between transamination and
which catalyzes the oxidative deamination of oxidative deamination
glutamate to ketoglutarate o glutamate dehydrogenase can use
- Both the transamination and oxidative either NAD or NADP as a coenzyme.
deamination occurs in successively. Niacin (Vitamin B3) is a precursor these
coenzymes.

IF NAD IF NADPH(reverse
process)
Oxidative Reductive amination
deamination

NAD+ NADH
GLUTAMATE KETOGLUTARATE
+NH3
NADP+ NADPH

OXIDATIVE DEAMINATION OF GLUTAMATE WILL Oxidative deamination: simultaneous loss of

PRODUCE: ammonia coupled with the oxidation of the carbon
skeleton
Reductive amination: simultaneous gain of
o Alpha ketoacids: ( ketoglutarate)
ammonia coupled with the reduction of the carbon
can enter the central pathway of
skeleton.
energy metabolism. alpha keto acids
are reffered as carbon skeleton.
- Direction of the reaction depends on:
o Ammonia: source of nitrogen for urea
o relative concentration of glutamate,
synthesis
alpha ketoglutarate and ammonia
- These reactions (transamination and oxidative
o ratio of oxidized to reduced coenzyme
deamination) provide a pathway for amino
** examples: after a meal production of more
groups to be released as ammonia
ammonia

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so after heavy meal with high protein, the reaction
shifts from left to right.

II. ALLOSTERIC REGULATORS OF GLUTAMATE

DEHYDROGENASE
converted to urea. Most tissues
ENHANCES GLUTAMATE INHIBITS GLUTAMATE
use glutamine synthetase but
DEHYDROGENASE DEHYDROGENASE
skeleton muscles use glucose-
ADP ATP alanine cycle.
GDP GTP
2. Glucose alanine cycle: used primarily by
RATIONALE: muscles
LOW energy levels in the cell (depleted ATP, more
ADP)

Amino acid degradation is high

Increase in energy production from carbon skeleton

More energy production from carbon skeleton

( Alpha-KA which go into Krebs cycle)

D-Amino acid oxidase

D-Amino acids are found in plants and in the cell
walls of microorganisms, but are not used in the
synthesis of mammalian proteins. D-Amino acids are,
however, present in the diet, and are efficiently o Glucose is converted to pyruvate as
metabolized by the kidney and liver. D-Amino the end product of aerobic glycolysis
acid oxidase (DAO) is an FAD-dependent o Transamination of pyruvate to form
peroxisomal enzyme that catalyzes the alanine via the enzyme Alanine
oxidative deamination of these amino acid Aminotransferase (ALT)
isomers, producing keto
acids, ammonia, and Transamination transfer of
hydrogen peroxide. The keto
acids can enter the amino group
general pathways of amino acid metabolism, and be o Alanine is transported by the blood to
reaminated to L-isomers, or catabolized for energy. the liver, where it is converted to
Increased DAO activity has been linked to pyruvate by transamination.
increased susceptibility to schizophrenia. L- o Pyruvate is metabolized to form
amino acid oxidases are components of several glucose via the gluconeogenesis
pathway.
III. TRANSPORT OF AMMONIA TO THE LIVER
o Glucose can enter the blood, to be
- transport of ammonia from the peripheral used by the muscle.
tissues to the liver happens via 2 mechanisms:
1. Glutamate combines with ammonia via the IV. CASE PRESENTATION
enzyme glutamine synthetase to form
glutamine. 50 y/o MALE
History of alcoholism
Glutamine nontoxic form Unresponsive to stimuli when brought to the ER
of ammonia and is transported Vomited blood 3 mos. ago but received no
to the liver by the blood. In treatment
the liver, it is cleaved by Drunk as often as 3x a week for 4 years until 1
glutaminase to produce year ago
glutamate and free ammonia.
End product is the ammonia is LAB EXAM:
transported to liver where it is

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Slight thrombocytopenia
Prolonged PT
Macrocytic anemia
Increased bilirubin
High serum transaminases & alkaline
phosphatase
Low serum albumin w/ increased globulins
High levels of blood ammonia

- Patient is suffering from PORTAL

HYPERTENSION
due to Alcoholic Liver Cirrhosis

Portal hypertension
- elevated pressure within the portal system
(portal vein and tributaries)
- Usually caused by liver disease/failure
Cirrhosis
- slowly progressing disease
- healthy liver tissue is replaced with scar tissue
- Slows the processing of nutrients, hormones,
drugs, and naturally produced toxins.
- Sustained excessive alcohol consumption, viral
hepatitis B and C, and fatty liver disease

A. BIOCHEMICAL PATHWAY INVOLVED

Urea cycle: major disposal form of amino groups

derived from amino acids degradation
- accounts for about 90% of the nitrogen-
containing components of urine.
- Produced by the liver, then transported in UREA CYCLE
the blood to the kidneys for excretion in the
urine FIRST TWO REACTIONS OCCUR IN THE
MITOCHONDRIA

1. FORMATION OF CARBAMOYL PHOSPHATE

- substrates:
o Carbon Dioxide ( provides carbon
atom of
urea)
o Ammonia (provides one of the
nitrogen
atoms of urea)
Ammonia (NH3) comes from the
oxidative deamination of
glutamate where NH3 as one of
the products
o 2 ATP (cleavage will drive the reaction)
- enzyme:
o Carbamoyl phosphate synthetase I
(CPS I) which is rate limiting
enzyme of the cycle.
o N-acetyl glutamate (acts as a
positive allosteric regulator and it
is combination of acetyl co-enzyme
A and glutamate and acted upon
by N-acetyl glutamate synthase) is
needed by CPS I for activation, this
CPS-I produce bicarbonate and amino
acid to produce carbamoyl phosphate.

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2. FORMATION OF CITRULLINE - substrate:
- substrate: o Arginine
o Carbamoyl ( from Carbamoyl o Water
phosphate) - Enzyme:
o L- Ornithine o Arginase
- Enzyme: - Yields:
o Ornithine transcarbamoylase o Urea
o Ornithine
- Carbamoyl is transferred to L-ornithine, - Only the liver can cleave arginine, thereby
with the release of the phosphate group (Pi) via synthesizing urea.
the enzyme ornithine transcarbamoylase to - Ornithine is regenerated and transported ino
form Citrulline the mitochondrion.
- Citrulline is transported to the cytosol.
FATE OF UREA
Note: Ornithine and citrulline are basic 1. Diffuses from liver
amino acids that will need a cotransporter to 2. Transported to kidneys to be filtered and
move across the inner mitochondrial excreted via urine.
membrane. They are not incorporated in the 3. A portion diffuses from the blood and into
cellular proteins because there are no codons the intestine. It is cleaved by bacterial
for these amino acids. urease to CO2 and NH3.
THE FOLLOWING REACTIONS OCCUR IN THE
CYTOSOL Clinical Correlation:
(Where remaining enzymes are located) In patients with kidney failure, plasma urea
levels are elevated. More urea can be
3. SYNTHESIS OF ARGININOSUCCINATE reabsorbed from blood to the gut. The action of
- substrate: the bacterial urease enzyme in the intestine
o Citrulline becomes the source of excess ammonia resulting
o Aspartate* to HYPERAMMONEMIA.
o ATP Treatment: Neomycin (oral administration)
- Enzyme: reduces the number of intestinal bacteria
o Argininosuccinate synthetase responsible for NH production.
- Note:
- Once citrulline is transported to cytosol it The 1st nitrogen comes from: free
reacts with aspartate. ammonia (NH3)
- Citrulline + Aspartate Arginosuccinate The 2nd nitrogen: Aspartate
via the enzyme argininosuccinate synthetase The Carbon atom comes from: CO2
- Alpha amino group of aspartate will
provide the 2nd nitrogen incorporated into
urea. B. REGULATION OF UREA CYCLE
- This pathway is regulated through N-acetyl
4. CLEAVAGE OF ARGININOSUCCINATE TO FORM glutamate: an essential activator of carbamoyl
ARGININE AND FUMARATE phosphatase synthetase I
- substrate:
o Argininosuccinate
- Enzyme:
o Argininosuccinate lyase
- Yields:
o Arginine (immediate precursor of urea)
o Fumarate
Hydrated to malate, that
provides a link to several
metabolic pathways
Malate can reenter TCA and
oxidized to oxaloacetate
Oxaloacetate
o Can be used for
- Glutamate + Acetyl CoA N-acetyl glutamate
gluconeogenesis
via N-acetylglutamate synthase enzyme
o converted to aspartate
- Ingestion of protein-rich meal, will provide as a
via transamination to substrate of glutamate (proteinsamino acid
enter urea cycle glutamate), increase in glutamate will result to
5. CLEAVAGE OF ARGININE TO ORNITHINE AND UREA

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increase intrahepatic concentration of N-
acetylglutamate. A. Transport of ammonia in the circulation
1. IN THE FORM OF GLUTAMINE:
C. SOURCES OF AMMONIA - Glutamate + ammonia (NH3) Glutamine
via glutamine synthethase
a. GLUTAMINE - Formation of glutamine occurs in the liver,
- Important source of plasma glutamine: skeletal muscles and brain
catabolism of branchedchain AA in skeletal - Major mechanism for removal of ammonia in
muscles the brain
- Kidneys: - Circulating glutamine is removed by the
o glutamine ammonia (NH3) + liver and kidney and deaminated by
glutamate via renal glutaminase glutaminase, to become a source of
enzyme ammonia
o ammonia is excreted into the urine as
NH4+ for bodys acid-base balance.
- Liver: ammonia is detoxified to urea and
excreted
- Intestines: Ammonia is generated by intestinal
glutaminase

b. BACTERIAL ACTION IN THE INTESTINE

- Ammonia is formed from urea by bacterial
urease in the lumen of the intestine
- Ammonia is absorbed from the intestine via the
portal vein and removed by the liver via
conversion to urea.

2. UREA
- Formation of urea in the liver, most
important disposal route for ammonia.
- Oxidative deamination of glutamate by
glutamate dehydrogenase to yield ammonia
that can enter the urea cycle
- Incorporation of ammonia occurs during the
first step of the urea cycle
c. AMINES AND MONOAMINES
- Amines are obtained from diet
- Monoamines serves as
hormones/neurotransmitters
- Both will give rise to ammonia by the action of
the enzyme monoamine oxidase

d. PURINES AND PYRIMIDINE

- Amino groups attached to the rings =
released as ammonia

B. Transport to the liver ( Refer to III. Transport of

Ammonia To The Liver)

D.TRANSPORT OF AMMONIA
Ammonia has very low levels in the blood due to the E. HYPERAMMONEMIA AND ITS TYPES
rapid removal of blood ammonia by the liver, and many
tissues will release amino acid nitrogen in the form of 1. ACQUIRED HYPERAMMONEMIA
glutamine or alanine rather as free ammonia.

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- Liver disease is a common cause of - There is a failure to synthesize urea
hyperammonemia in adults, and may be due, hyperammonemia (first weeks following birth)
for example, to viral hepatitis or to
hepatotoxins such as alcohol. o ORNITHINE TRANSCARBAMOYLASE
- Cirrhosis of the liver portal blood is shunted DEFICIENCY
directly into the systemic circulation, resulting X-linked
to having no access to the liver impaired most common of these
conversion of ammonia to urea disorders
predominantly affect
2. CONGENITAL HYPERAMMONEMIA males
- An accumulation of excess ammonia in the
body due to a congenital deficienct of enzyme, o ARGINASE DEFICIENCY
either carbamoylphosphate synthetase or less severe
ornithine transcarbamoylase, essential to arginine contains two
the metabolism of ammonia. waste nitrogens
excreted in the urine
CHECKPOINT!!!
Identification:
1. In urea cycle, what compound moves from
the cytosol into the mitochondria?
2. Oxidative deamination of glutamate will
result to _________ and ________.
3. The coenzymes of glutamate dehydrogenase
are ______ or ______.
4. Give one positive allosteric regulator of
glutamate dehydrogenase.
5. Ammonia can be transported in the
circulation in the form of _________.
6. The rate limiting step of urea cycle.
ANSWER KEY:
7. The immediate precursor of Urea. Identification:
8. An abnormal excess of ammonia is called 1.
2.
Citrulline
Alpha ketoglutarate and
ammonia
True or False: 3. NAD or NADP
4. ADP/low energy level
5. Glutamine
9. After heavy meal with high protein content 6. Carbamoyl phosphate
synthethase I
there is less production of ammonia. 7. Argnine
10. Oxidative deamination simultaneous 8. Hyperammonemia
loss of ammonia with reduction of the carbon True or False:
skeleteon. 9. F
11. ATP is a positive allosteric regulator 10. False/ oxidation of carbon
skeleton
of glutamate dehydrogenase. 11. False/ negative
12. Glutamate dehydrogenase is an 12. False / D-amino oxidase
13. True
FAD-dependent peroxisomal enzyme that 14. True
catalyzes the oxidative deamination of these 15. True
amino acid isomers, producing keto acids, 16. True
17. False / Glutaminase enzyme
ammonia, and hydrogen peroxide.
13. First two reactions of the urea cycle
occur in the mitochondria.
14. The second nitrogen of Urea is from
Aspartate.
15. Urea is diffused from the liver, to be
transported to the kidneys and excreted via
urine.
16. N-acetyl glutamate is an essential
activator of carbamoyl-phosphate
synthetase I.
17. Glutamine via the enzyme glutamine
Transcribers:
synthetaseDIO, MEKALA,
will produve SOGUE
glutamate and Page 6 of 6
ammonia.