Protein metabolism transamination deamination, fate of ammonia, urea cycle I AHS.pdf

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Protein metabolism transamination deamination, fate of ammonia, urea cycle notes for First year Allied Health Science students. RGUHS syllabus

1. Amino acid and Protein
Metabolism
By P. Santosh Kumar

2. AminoacidandProteinMetabolism-syllabus
Introduction,
Digestionandabsorptionproteins
Transamination,deamination,Transdeamination
Fateofammonia,transportofammonia,
Ureacycle

3. Transamination, Deamination,
Fate of Ammonia, Transport of Ammonia,

4. AMINO ACID METABOLISM

5. CATABOLISM OF AMINO ACIDS
•Aminoacidconsistsofaminogroupandcarboxylicgroup
•CarboxylicgroupisremovedasCO
2bydecarboxylationreactions
•Removalofα-aminogroupintheformofammoniaby
•Trans-deaminationmajorprocess
•Deamination(oxidativeornon–oxidative)minorprocess
•Ammoniaisthendetoxifiedtoureaandexcretedinurine,whereas
remainingcarbonskeletonofaminoacidcanbeconvertedto
intermediatesofenergyproducingmetabolicpathwayslikeglycolysisand
citricacidcycleandmetabolizedtocarbondioxideandwatergiving
energyorconvertedtoglucose(glucogenic),fattyacidsorketonebodies
(ketogenic).

6. Decarboxylation
•The amino acids will undergo alpha decarboxylation to form the
corresponding amine by the enzyme decarboxylase
Decarboxylase
•Histidine Histamine
Vasodilator
•Tyrosine Tyramine
Vasoconstrictor
•Tryptophan Tryptamine Neurotransmitter
•Glutamic acid GABA Neurotransmitter
CO
2
CO
2
CO
2
CO
2
Some of the important biogenic amines produced from amino acids and
functions are

7. Transamination
Transaminase
Definition:
•Transamination is the process of transfer of an amino group from one
amino acid to keto acid with the formation of a new amino acid and keto
acid, without liberation of ammonia and takes place in the liver
•Transamination is a reversible process which is catalyzed by enzymes
transaminases also called as amino transferases. Pyridoxal phosphate
(PLP) is the coenzyme of transaminases.

8. Examples of transamination:
Alanine Pyruvic acid
α-ketoglutarate
Glutamate
Serum Glutamate pyruvate
transaminase (SGPT)
Pyridoxal
phosphate (PLP)
Aspartate Oxaloacetate
α-ketoglutarate Glutamate
Serum Glutamate Oxaloacetate
transaminase (SGOT)
Pyridoxal
phosphate (PLP)
Example 1:
Example 2:

9. Salient features of transamination
1.All transaminases require pyridoxal phosphate (PLP), a
coenzyme derived from vitamin B6.
(Transfer of the amino group to the coenzyme PLP to form pyridoxamine phosphate is
then transferred to a keto acid to produce a new amino acid and the enzyme with PLP
is regenerated).
2.Degradation of amino acids: in transamination amino group of
amino acids removed as ammonia and converting them into
corresponding α-ketoacids which will enters into catabolic
pathways.
3.There is no free NH3 liberated, only the transfer of amino
group occurs.
4.It is reversible reaction catalyzed by Transaminases

10. 5.Amino groups of all amino acids undergo transamination are
concentrated as glutamate which will transported to liver and
release of ammonia by glutamate dehydrogenase.
6.Transamination is very important for Synthesis of non-essential
amino acids by the body from keto acid available from other
sources. Ex: pyruvate transaminated to alanine and
α-ketoglutarate to glutamate.
7.Role in Gluconeogenesis- glucogenic amino acids like aspartate,
alanine etc. undergoes transamination to form oxaloacetate, pyruvate
which can form glucose by gluconeogenesis.
8.Provision of energy: transamination produces compounds like
oxaloacetate, pyruvate and α-ketoglutarate etc. which can enter
glycolysis or TCA cycle and provide energy.

11. 9.All amino acid except threonine, lysine, proline and hydroxy
proline undergo transamination
10.Transamination is not restricted to α-amino groups only. For
instance, δ- amino group of ornithine is transaminated.
11.Serum transaminases are important for diagnostic and
prognostic purposes. Ex: AST and ALT

12. Clinical significance of transaminases
AST and ALT used as diagnostic enzymes
•Alanine aminotransferase(ALT/SGPT) – levels increases in
serum in liver diseases e.g. infective hepatitis due to damage to
the liver.
Normal serum level is 10-35 U/L
•Aspartate aminotransferase(AST/SGOT) – levels increases in
serum in myocardial infarction due to damage to the cardiac
muscle
Normal serum level is 8-20 U/L
By transamination process non essential amino acids can be
synthesized

13. Deamination of amino acids
•Deamination is the direct removal of α amino group from
amino acids and ammonia (NH
4) is released
•Two types – Oxidative and Non-oxidative deamination
•Oxidative deamination by amino acid may be –
•L – amino acid oxidases
•D –amino acid oxidases
•Oxidative deamination of glutamate by glutamate
dehydrogenase

14. •L-amino acid oxidases – it act on all amino acids except
hydroxy amino acids and dicarboxylic amino acids to form α-
ketoacid and ammonia. It uses FMN as co-enzyme.

15. Oxidative deamination by Glutamate dehydrogenase (GDH)
•GDH is an oxidative deamination reaction.
•GDH is mainly associated with transdeamination reactions
•NAD
+
or NADP
+
act as coenzymes
•D-amino acid oxidases – it can act on glycine and any D-
amino acids (found in the plants and micro-organisms) to form
α-ketoacid and ammonia. It uses FAD as co-enzyme.
D-α amino acid α-ketoacid+ NH
3
FMN
D-amino acid oxidase
FMNH
2H
2O

16. Transdeamination
•The amino group from amino acids released by a coupled
reaction called as transdeamination, that is transamination
followed by oxidative deamination of glutamate
•Transamination takes place in the cytoplasm of the cells, In
this the amino group of different amino acids are transferred
to α-ketoglutarate to form glutamate,
•The glutamate is then transported to liver and oxidatively
deaminated to α-ketoglutarate and ammonia in the
mitochondria of hepatocytes.
•Free ammonia is highly toxic

17. Transdeamination
Amino acid α-ketoglutarate
Corresponding
ketoacids
Glutamate
transaminationIn all tissues
Transported to liver
Glutamate α-ketoglutarate+ NH
4
+
NAD
+
Glutamate dehydrogenase
NADH
+
+ H

18. Oxidative deamination by glutamate dehydrogenase

19. Non oxidative deamination
•Hydroxyl (OH) amino acids (serine and threonine), Sulphur
containing amino acid (cysteine), and Histidine are deaminated
by non-oxidative deamination to release ammonia.
1. Serine Pyruvate + NH
3
PLP
Serine dehydratase
2. Threonine α-ketobutyrate + NH
3
PLP
Threonine dehydratase
3. Cysteine Pyruvate + NH
3 + H
2S

PLP
Desulphydratase
4. Histidine Urocanate + NH
3
PLP
Histidase

20. Formation and fate of ammonia
Ammonia formed in the body from following Sources-
−Removal of amino group of amino acids through transamination,
deamination and transdeamination
−Degradation of purine nucleotides
−Degradation of pyrimidine nucleotides
−Degradation of biogenic amines
−Degradation of amino sugars
−Putrefaction by intestinal bacteria
Ammonia is highly toxic, it should be immediately transported to
liver as non toxic form to and detoxified.

21. Disposal/ Detoxification of Ammonia
Ammonia transported to liver in nontoxic form
•from extrahepatic tissues as glutamate (by transamination of
amino acids),
•In the brain and intestine free ammonia immediately trapped
by Glutamate in the presence of Glutamate synthetase to form
Glutamine. Glutamine is carried to the liver where it gives
Glutamate and ammonia
•Alanine transports ammonia from muscles to the liver by
Glucose- Alanine cycle.

22. Transport of ammonia from muscle to liver by glucose alanine cycle
After Ammonia transported to liver as non-toxic forms ammonia is detoxified
by conversion to urea by urea cycle and excreted in urine.

23. Ammonia toxicity:
NH
3 is highly toxic, even minute amounts of NH
3 is toxic to CNS.
Reason for ammonia intoxication:
Accumulated ammonia in brain reacts with α-ketoglutarate to form
glutamate, resulting in depletion of α-ketoglutarate which impairs
TCA cycle and impairs the function in brain
Symptoms of ammonia intoxication:
Slurred speech, blurred vision, tremors, vomiting, lethargy,
aversion to high protein food, disorientation, irritability, mental
retardation, in severe cases coma and death.
Normal range of Ammonia in Blood is <50 μg/dl

24. Fate of ammonia
Ammonia formed in the various sources are transported to liver
where the it is converted to (major fate of ammonia) urea
through urea cycle.

25. Urea cycle /Kreb’s Hanseleit cycle /Ornithine Cycle
Nitrogen of amino acids is removed in the form of ammonia, that’s
why urea is the end product of protein or amino acid
metabolism.
Urea cycle: Converts the toxic ammonia into less toxic and more
soluble urea, which is excreted in the urine
It is the first metabolic pathway to be elucidated in 1932, by
Hans Krebs and Kurt Henseleit Hence, the cycle is known as
Krebs-Henseleit urea cycle.
As ornithine is the first member of the reaction, it is also called as
Ornithine cycle.

26. Urea cycle /Kreb’s Hanseleit cycle /Ornithine Cycle
Salient features:
•Starting material: CO
2 and NH
3
•Site: Liver
•Subcellular site: Enzymes are distributed in the mitochondria
and the cytosol of the liver
•The molecular formula of urea is NH2-CO-NH2.
•Nitrogen atom donors in cycle: urea formed from ammonia and
α-amino nitrogen of aspartate
•The two nitrogen atoms of urea are derived from two different
sources, one from ammonia and the other directly from the α-
amino group of aspartic acid.

27. Importance or Significance of urea cycle
•Urea cycle Converts the toxic ammonia into less toxic and
more soluble urea, which is excreted in the urine
•Disposes ammonia and CO
2
•Forms semi essential amino acid, arginine
•Ornithine which is formed from fumarate can form non
essential amino acid- proline,
•Ornithine is a precursor for the formation of polyamines like
putrescine, spermidine and spermine
•Also called as urea bicycle it is linked with TCA cycle through
the fumarate

28. Mitochondria
Cytosol
Arginosuccinate
synthetase
PPi
Mg
2+
1
2
3
4
5
N-acetyl glutamine
(NAG)
Mg++
Reactions of Urea cycle

29. Steps of Urea cycle
1. Formation of carbamoyl phosphate
•Condensation of CO
2, ammonia and ATP forms carbamoyl
phosphate
•Catalyzed by carbamoyl phosphate synthetase – I (CPS-I)
•N-acetyl glutamine (NAG) formed from glutamine and acetyl
CoA with the help of an enzyme synthase allosterically activates
the activity of CPS-I.
•Requires 2 ATP molecules, one as source of phosphate and the
second is converted to AMP and PPi

30. Comparison of CPS –I and CPS –II enzymes
CPS - I CPS - II
Site Mitochondria Cytosol
Pathway of Urea Pyrimidine
Positive effectorNAG Nil
Source for n Ammonia Glutamine
Inhibitor Nil CTP

31. 2. Formation of citrulline
•Carbamoyl phosphate donates its carbamoyl group to ornithine
to citrulline and releases the phosphate
•Catylsed by ornithine transcarbamoylase
•Citrulline formed leaves the mitochondria and enters the
cytosol of the liver

32. Arginosuccinate
synthetase
Mg
2+
PPi
3. Formation of arginosuccinate
•Transfer of second amino group (from
aspartate) to citrulline occurs by condensation
between amino group of aspartate and
citrulline in the presence of ATP to form
arginosuccinate
•Catalyzed by arginosuccinate synthetase
dependent on Mg
2+

33. 4. Formation of arginine and fumarate
Then arginosuccinate is cleaved by arginosuccinate lyase to form
arginine and fumarate. Fumarate formed in this reaction enters
into TCA cycle.
In the TCA cycle Fumarate converts to malate with the help of an
enzyme fumarase. The malate undergoes dehydrogenation with the
help of an enzyme malate dehydrogenase to form Oxaloacetate.
The NAD
+
converts NADH
+
+ H
+
enters into ETC to produce 2.5
ATPs. The oxaloacetate undergoes transamination to form
aspartate. This is the link between urea cycle and TCA cycle
hence called as urea bicycle.

34. Urea bicycle

35. 5. Formation of Urea and Ornithine
•Liver hydrolytic enzyme arginase
cleaves arginine to give urea and
ornithine
•Ornithine is regenerated and enters
the mitochondria again to initiate
another round of urea cycle
•Urea thus formed is excreted in the
urine

36. Mitochondria
Cytosol
Arginosuccinate
synthetase
PPi
Mg
2+

37. Energetics of urea cycle
•The overall reaction may be summarized as:
NH
3 + CO
2 + Aspartate → Urea + Fumarate
Four ATPs are consumed in the synthesis of each molecule of urea as follows:
•Two ATP are needed to make carbamoyl phosphate.
−One ATP serves as a source of phosphate
−Second ATP is converted to ADP + Pi.
•One ATP is converted to AMP + PPi which equivalent to 2 ATP required to make
arginosuccinate.
•Fumarate formed in the 4th step may be converted to malate in the TCA cycle.
•Malate when oxidized to oxaloacetate produces 1 NADH equivalent to 2.5 ATP
•So, net energy expenditure is only 1.5 high energy phosphates
•The urea cycle and TCA cycle are interlinked, and so, it is called as “Urea bicycle”

38. Regulation of urea cycle
Coarse Regulation:
•During starvation the activity of urea cycle enzymes is elevated
due to increase rate of protein catabolism
Fine Regulation
•Carbamoyl phosphate synthase - I (CPS-I) is allosteric key
regulatory enzyme of urea cycle, activated by N-acetylglutamate
(NAG)
•NAG is synthesized from acetyl CoA and glutamate by NAG –
synthase. Synthesis of NAG increases after intake of protein rich
diet and starvation.

39. •Compartmentalization: Urea cycle enzymes are located in such
a way that the first two enzymes are in the mitochondrial matrix
•Inhibitory effect of fumarate on its own formation is minimized
because arginosuccinate lyase is in the cytoplasm, while fumarase
is in mitochondria

40. Disorders of urea cycle/ Inborn errors of urea cycle /
Urea cycle defects
Deficiency of any urea cycle enzymes would result in
hyperammonemia.
Hyperammonemia: means increased ammonia levels in the blood
more than normal level (Blood <50μg/dl) is called as
hyperammonemia.
Clinical symptoms of urea cycle disorders are
Vomiting, irritability, lethargy and severe mental retardation.

41. Disorders of urea cycle/ Inborn errors of urea cycle /
Urea cycle defects
Disorders in the conversion of ammonia to urea results in a clinical condition
called as Hyperammonemia. Increased blood ammonia concentration more
than normal (50 μg/dl) results in ammonia toxicity.
Hyperammonemia may be due to congenital or acquired causes
Congenital Hyperammonemia- due to inherited disorders of ammonia, due to
deficiency/ absence of an enzyme involved in urea cycle. The deficiency of first
two enzymes of urea cycle results in more severe form of Hyperammonemia,
because of ammonia itself accumulates where as deficiency of other three two
enzymes of urea cycle results in less severe form of Hyperammonemia because
of accumulation of other intermediates of urea cycle. These includes

42. Inborn errors of urea cycle
Disorders Defective enzyme Features / Products
accumulated
Hyperammonemia Type - I Carbomyl phosphate synthase - IAmmonia
Hyperammonemia Type -IIOrnithine transcarbamoylaseAmmonia
Citrullinemia Arginosuccinate synthase Citrulline
Argininosuccinic aciduriaArginosuccinate lyase Argininosuccinate
Argininemia Arginase Arginine
Urea cycle defects: are due to deficiency or lack of any enzyme in urea
cycle

43. Acquired hyperammonemia / Hepatic coma
•Normally ammonia and other toxic compounds are transported by the
portal circulation to the liver and detoxified
•When there is portal systemic shunting of blood the toxins bypass the liver
and concentration increases in the systemic circulation
•Signs and symptoms are altered sensorium, convulsions, ascites,
jaundice, hepatomegaly, edema, hemorrhage,
Management of Hyperammonemia-
•Restricted protein intake
•Lactulose used to lower blood ammonia,
•Hemodialysis and blood transfusion
•Bowel disinfectant, avoiding hepatotoxic drugs and maintenance of acid
base and electrolyte balance

44. Uremia: defined as increased urea levels in the blood more than normal
level (20-40 mg/dl) is called as Uremia.
In clinical practice blood urea level is taken as indicator of renal
functions.
Uremia occurs in following conditions
Types of
uremia
Causes
Prerenal High protein diet
Increased protein catabolism: trauma, surgery, starvation, diabetes
mellitus
Renal Nephritis, Acute Glomerulonephritis, Renal tuberculosis
Mercurial poisoning
Postrenal Obstruction to urine outflow: benign prostatic hypertrophy, malignant
stricture, obstruction, stone

45. Normal levels
•Urea
•Blood – 20-40mg/dl
•Urinary excretion – 15-30gms/day
•Ammonia
•Blood - <50μg/dl

46. Thank you
References:
1.Vasudevan DM, Sreekumari S, Vaidyanathan K. Textbook of biochemistry for
medical students. JP Medical Ltd; 2013.
2.Satyanarayana U. Biochemistry. Elsevier Health Sciences; 2013 Jun 15.
3.Krishnananda Prabhu. Jeevan K Shetty. Quick Review of Biochemistry for
Undergraduates. Jaypee Brothers Medical; 2014.