1. Ammonia and amino acid
metabolism

3. AMMONIA METABOLISM
The ways of ammonia formation
1. Oxidative deamination of amino acids
2. Deamination of physiologically active amines and nitrogenous
bases.
3. Absorption of ammonia from intestine (degradation of
proteins by intestinal microorganisms results in the ammonia
formation).
4. Hydrolytic deamination of AMP in the brain (enzyme –
adenosine deaminase)

4. Ammonia is a toxic substance to plants and animals (especially for
brain)
Normal concentration: 25-40 µmol/l (0.4-0.7 mg/l)
Ammonia must be removed from the organism
Terrestrial vertebrates synthesize
urea (excreted by the kidneys) -
ureotelic organisms
Urea formation takes place in the
liver
Birds, reptiles synthesize uric acid

5. Peripheral Tissues Transport Nitrogen to the Liver
Two ways of nitrogen transport from peripheral
tissues (muscle) to the liver:
Glutamate is not
1. Alanine cycle. Glutamate is deaminated in
formed by transamination reactions peripheral
tissues

6. Nitrogen is then transferred to pyruvate to
form alanine, which is released into the blood.
The liver takes up the alanine and converts it back
into pyruvate by transamination.
The glutamate formed in the liver is deaminated
and ammonia is utilized in urea cycle.

7. Reaction catalyzed by
glutamate dehydrogenase.
The glutamate dehydrogenase
of mammalian liver has the
unusual capacity to use either
NAD or NADP as cofactor.
The mammalian enzyme is
allosterically regulated by GTP
and ADP.

8. 2. Nitrogen can be
transported as glutamine.
Glutamine synthetase
catalyzes the synthesis of
glutamine from glutamate and
NH4+ in an ATP-dependent
reaction:
Ammonia transport in the form of
glutamine.
Excess ammonia in tissues is added
to glutamate to form glutamine, a
process catalyzed by glutamine
synthetase. After transport in the
bloodstream, the glutamine enters
the liver and NH4 is liberated in
mitochondria by the enzyme
glutaminase.

11. Hypotheses toxicity of ammonia
A. The binding of ammonia in the synthesis of glutamate causes an outflow of α-
ketoglutarate from the tricarboxylic acid cycle, with decreased formation of ATP
energy and deteriorates the activity of cells.
B. Ammonium ions NH4 + caused alkalization of blood plasma. This increases the
affinity of hemoglobin for oxygen (Bohr effect), the hemoglobin does not release
oxygen to the capillaries, resulting the cells hypoxia occurs.
C. The accumulation of free NH4 + ion in the cytosol affects the membrane
potential and intracellular enzymes work - it competes with ion pumps, Na + and K
+.
D. The producing ammonia tramsform glutamic acid - glutamine - an osmotically
active substance. This leads to water retention in the cells and the swelling that
causes swelling of tissues. In the case of nervous tissue it can cause brain swelling,
coma and death.
E. The use of α-ketoglutarate and glutamate to neutralize the ammonia causes a
decrease in the synthesis of γ-aminobutyric acid (GABA) inhibitory
neurotransmitter of the nervous system.

12. THE UREA CYCLE
Urea cycle - a cyclic pathway of urea synthesis
first postulated by H.Krebs
The sources of
nitrogen atoms in
urea molecule:
- aspartate;
- NH4+.
Carbon atom
comes from CO2.

13. SYNTHESIS OF UREA
Carbamoilphosphate synthase
O OH
NH 3 + CO 2 + 2 АТФ + H 2O
ATP H 2N C O~P O + 2 АДФ + H 3PO 4
ADP
OH
carbamilphosphate

14. The carbamoyl phosphate generated in the
mitochondria now donates its carbamoyl group to
ornithine, which is formed in the cytosol but enters the
mitochondrion via a spe­cific inner­membrane transport
system. The product is citruiline:
Ornitine-carbomoil transferasa
NH 2
NH 2 C O
O OH
(CH 2)3 NH
H 2N C O ~ P O + + H 3PO 4
HC NH 2 (CH 2)3
OH
COOH HC NH 2
carbamilphosphate
COOH
ornitine
citrulline

15. The second amino group required for urea synthesis
now arrives in the form of aspartate, which in turn
acquired it from glutamate by the action of aspartate
transaminase in the cytosol. The amino group of
aspartate condenses reversibly with the carbamoyl
carbon atom of citrulline in the presence of ATP to
form argininosuccinate; this reaction is catalyzed by
argminosuccinate synthetase:

16. SYNTHESIS OF UREA
Arginino-succinate synthase
NH2 NH2 COOH
C O COOH C N CH
NH H2 N CH NH CH2
+ + АТФ COOH
(CH2)3 CH2 (CH2)3
HC NH2 COOH HC NH2
citrulline COOH aspartate COOH
NH COOH arginino­succinate
C NH CH
NH CH2
+ АMФ + H4P2O7
COOH
(CH2)3
HC NH2
COOH

17. In the next reaction argininosuccinate
undergoes a elimination reaction by the
action of argininosuccinate lyase to form free
arginine and fumarate:
Arginino-succinate lyase
NH COOH NH2
COOH
C NH CH C NH
CH
NH CH2 NH +
CH
(CH2)3 COOH (CH2)3
COOH
HC NH2 HC NH2
COOH COOH fumarate
arginino­succinate arginine

18. SYNTHESIS OF UREA
arginase
NH2
NH2
C NH NH2
(CH2)3
NH + H2O + C O
HC NH2
(CH2)3 NH2
COOH
HC NH2
urea
COOH ornitine
arginine

19. The Linkage between Urea Cycle, Citric Acid Cycle
and Transamination of Oxaloacetate
Fumarate formed in urea cycle enters citric acid cycle
and is converted to oxaloacetate.
Fates of oxaloacetate:
(3) transamination to aspartate,
(4) conversion into glucose,
(5) condensation with acetyl CoA to form citrate,
(6) conversion into pyruvate.

20. Diagnostic significance of the
determination of urea in urine.
25­30 g/day of urea is excreted in normal
conditions.
The increase of urea in urine occurs in
high fever, malignant anemia, poisoning
by phosphorus, intensive decomposition
of protein in organism. The decrease of
urea in urine occurs in liver diseases,
kidney unsufficiency, acidosis.

21. Fates of carbon skeleton of amino acids

22. Transformation of AA carbone backbone
Serine Glycine
Phenylalanine Tyrosine
Cystine
Homogentizinic acid
Cysteine Fumarate
Treonine Acetoacetate
Alanine Leucine Oxaloacetate SuccinylCoA
Pyruvate AcetylCoA
Serine Ketoglutarate Lysine
Glucose Isoleucine
Glutamate Urocainic acid

23. epinephrine
horepinephrine
Triiodtyronine
Melanine Dopamine
D
F
Dioxyindol DOPA Tyroxine
E
A E
Phenylalanine Tyrosine Mono­, Diiodtyrosine
B B C
Phenylpyruvate oxyphenylpyruvate Homogentisinic acid
Maleiloacetate
Benzoic acid Phenylacetate Phenyllactate Fumariloacetate
A – PKU D – Albinosis
B – Tyrosinosis, scurvy E – Cretinism, Graves' disease Fumarate Acetoacetate
C - Alkaptonuria F – Parkinsons’ disease

25. INBORN ERRORS OF AMINO ACIDS METABOLISM
Alcaptonuria - inherited disorder of the
tyrosine metabolism caused by the
absence of homogentisate oxidase.
 homogentisic acid is accumulated and
excreted in the urine
 turns a black color upon exposure to air
 In children:
 urine in diaper may
darken
 In adults:
 darkening of the ear
 dark spots on the on the
sclera and cornea
 arthritis

27. Phenylketonuria is caused by an absence or deficiency
of phenylalanine hydroxylase or of its
tetrahydrobiopterin cofactor.
Phenylalanine accumulates in all body fluids and converts
to phenylpyruvate.
Defect in myelination of nerves
The brain weight is below normal.
Mental and physical retardations.
The life expectancy is drastically
shortened.
Diagnostic criteria:
 phenylalanine level in
the blood
 FeCl3 test
 DNA probes
(prenatal)

28. NH3 CO2
Glutamate
Glutamine
Aminobutaric acid
methylaspartate
TRANSAMINASE carbamylglutamate
Pyruvate Acetate
ketoglutarate

29. NH3 CO2
ASPARTATE
Asparagine
Alanine
Aspartylphosphate
TRANSAMINASE Fumarate
Metyonine Lisine
Oxaloacetate

30. Maple syrup urine disease - the disorder of the
oxidative decarboxylation of α-ketoacids derived
from valine, isoleucine, and leucine caused by the
missing or defect of branched-chain dehydrogenase.
The levels of branched-chain amino
acids and corresponding α-ketoacids
are markedly elevated in both blood
and urine.
The urine has the odor of maple syrup
The early symptoms:
 lethargy
 ketoacidosis
 unrecognized disease leads to
seizures, coma, and death
 mental and physical retardation