Biochemistry from Dr. Dhiraj J Trivedi SDM College of Medical Sciences and Hospital, Dharwad, India

1. Happiness is something
comes into our lives through doors
We don’t even remember leaving it
open
Dr. Dhiraj J. Trivedi

2. Urea - Introduction
• Urea cycle reactions converts toxic NH3 into non-
toxic and water soluble urea.
• Urea is the principle end product of the protein
or amino acid metabolism in humans.
• 80-90% of urinary nitrogen excreted is in the
form of urea nitrogen.

3. • Krebs and Henseleit were the first to
elucidate the steps. Hence Krebs Henseleit
cycle
• Other name -- Urea cycle,
Ornithine cycle.
• Site of urea synthesis– Liver
• First two reactions occur in the
mitochondria
• and later three reactions occur in cytosol
Urea synthesis

4. Requirements of urea synthesis
• Substrate:
• CO2 --- from HCO3
• NH4 --- Deamination of amino acids
• Aspartic acid --- Transamination of
OAA
• Energy: ATP 3molecules
• Modulators:
• Mg+2, N Acetyl Glutamate (NAG)

5. Steps in the urea cycle are
Cytosol
Step 3: Synthesis of Argininosuccinate
Step 4: Synthesis of Arginine
Step 5: Release of urea and Ornithine
Mitochondria
Step 1: Formation of carbamoyl
phosphate
Step 2: Formation of citrulline

6. CO2 NH3
Carbamoyl phosphate
Carbamoyl phosphate
synthetase - I
N – Acetyl Glutamate
2ATP
2ADP +
2Pi
Mg+2
From HCO3 From deamination
Of amino acids
+
Step 1 : Synthesis of carbamoyl phosphate
Step 1: Takes place in mitochondria

7. • In liver mitochondria
• NAG allosterically activates CPS-I
• Two high energy phosphate bonds are
utilized for the synthesis of carbamoyl
Phosphate

8. Isomers of carbamoyl phosphate synthetase

9. Carbamoyl phosphate
Ornithine trans Carbamoylase
Pi
Ornithine Citrulline
Step 2 : Synthesis of Citrulline
Step 2 : Takes place in mitochondria
Carrier proteins transfer Ornithine from
cytosol to mitochondria matrix

10. Step 3: Takes place in Cytosol
Step 3 : Synthesis of Arginino succinate
Carrier
proteins
Transfer
citrulline
To cytosol
for further
reactions
Arginino succinate
Arginino succinate
synthetase
ATP
AMP
+ PPi
Mg+2
Citrulline
Aspartate
Oxaloacetate
NH4
Malate
TCA cycle

11. Arginino succinate
Arginino succinase
Arginine
Fumarate Malate
Step 4: Takes place in Cytosol
Step 4 : Synthesis of Arginine
Arginine - Essential amino acid synthesized
in adult

12. • Arginino succinate is cleaved to arginine and fumarate
• Enzyme is present in cytoplasm of liver and kidney
tissues
• Link between Urea cycle and TCA cycle
• Fumarate can be converted to malate and then to
oxaloacetate by the intervention of certain TCA cycle
enzyme
• Aspartate may be regenerated by transamination

13. Urea
Arginase
Arginine
Ornithine
Present only in liver
Step 5: Takes place in Cytosol
Step 5 : Release of Ornithine and Urea

14. • Release of urea and ornithine
• The guanido group of arginine
Hydrolytically cleaved by arginase
• The urea diffuses into blood from where
it is cleared by the kidneys
• Ornithine will enter mitochondria and
become substrate for reaction 2

15. Urea
Arginase
Arginine
Ornithine
Arginino succinate
Arginino succinase
Fumarate Malate
Arginino succinate
synthetase
ATP
AMP +PPi
Mg+2
Citrulline
Oxaloacetate
NH4
TCA cycle
Carbamoyl phosphate
Ornithine trans Carbamoylase
Pi
CO2 NH3
Carbamoyl phosphate
synthetase - I
N – Acetyl Glutamate
2ATP
2ADP
+2Pi
Mg+2
Mitochondrial
matrix

16. Regulation of urea cycle
• 1. Substrate availability ( feed forward )
• 2. High protein diet and prolonged starvation
increases urea synthesis
• 3. Allosteric mechanism (by N acetyl glutamate)
• 4. CPS-I, ornithine transcarbamoylase, arginino
succinate synthetase and arginase all undergo
feed back inhibition

17. Role of CPS – I
in regulation of urea cycle
• CPS-I is the rate limiting enzyme of the urea
cycle
• CPS-I is allosterically activated by N-acetyl
glutamate (NAG), which lowers its Km for ATP
• On binding with NAG, the enzyme changes its
conformation to the active form
• This will exposes specific –SH group and
increases the ATP affinity of the enzyme

18. • This promotes the transfer of phosphate group from
ATP to an enzyme bound carbamate intermediate,
changing the carbamate to carbamoyl phosphate
• Energetic – 3 molecules ATP
• Formation of 1 molecule of urea requires 4 high
energy bonds
Role of CPS – I
in regulation of urea cycle

19. Dependency of Urea cycle on TCA cycle
• For ATP
• CO2 produced in TCA cycle is used for
urea synthesis
• Aspartate supply can be replenished by
converting fumarate produced in urea
cycle to malate and oxaloacetate via
TCA cycle.

20. Urea cycle disorders
• Hyperammonemia Type I
• Hyperammonemia Type II
(Ornithinemia)
• Citrullinemia
• Arginino Succinic Aciduria
• Hyper Argininemia

21. Hyper ammonemia I
Hyper
ammonemia II
Citrullinemia Arginino succinic aciduria
Hyper Argininemia
Carbamoyl phosphate synthetase -
Ornithine trans carbamoylase
Arginino succinate synthetase
Arginino
succinase
Arginase

22. Hyperammonemia Type I
• This is due to the deficiency of carbamoyl
phosphate synthetase-I in mitochondria
• Features are high levels of NH3 in blood and
urine

23. Hyperammonemia Type-II (Ornithinemia)
• Due to the deficiency of ornithine
transcarbamoylase
• Results in elevated levels of NH3 in tissues

24. Citrullinemia
• A rare disorder
• Probably due to a mutational change in the
catalytic site of arginino succinate synthetase
• Blood level and urinary excretion of citrulline
increases

25. Arginino Succinic Aciduria
• Due to the deficiency of Arginino Succinase
• Increased level of Arginino Succinate in blood
and its increased excretion in urine

26. Hyper Argininemia
• Due to the deficiency of Arginase
• Increased level of Arginine in blood and
increased urinary excretion

27. Significance of urea cycle
1. Major route of disposal of toxic Ammonia
2. Synthesis of semi-essential amino acid
ARGININE
3. Urea cycle, Citric acid cycle and
transamination reactions are linked

28. Blood urea
• Normal level- 15 - 45 mg/dl
• Uremia or azotemia is increased blood urea level
due to renal failure
• Normally urea excretion is about 20-30 gm per day
• A high value of blood urea indicates significant
reduction in the GFR and kidney disease
• Causes for reduction in GFR--- Pre renal, Renal and
Post-renal

29. Pre-renal causes- mainly reduction in GFR
(reduction in plasma volume and fall in renal
blood flow)
–Diarrhea
–Vomiting
–Severe dehydration
–Increased protein breakdown observed in
cases of prolonged fevers, diabetic coma,
thyrotoxicosis, after major surgery

30. Renal causes such as-
• Acute glomerular nephritis
• Chronic nephritis
• Nephrosis
• Polycystic kidney
• Hydronephrosis

31. Post renal causes like
• Urinary tract obstruction due to
– Tumors,
–stones in the bladder
–Enlargement of prostate gland
• All these may lead to increased reabsorption of
urea from the renal tubules

32. Hyper ammonemia I
Hyper
ammonemia II
Citrullinemia Arginino succinic aciduria
Hyper Argininemia
Carbamoyl phosphate synthetase -
Ornithine trans carbamoylase
Arginino succinate synthetase
Arginino
succinase
Arginase

33. Common symptoms of urea cycle
disorders includes
• Elevated blood NH3 level
• Aversion to protein intake
• Tendency for vomiting
• Mental retardation
• Coma, convulsions and death
• Lowering the dietary protein intake has been
suggested as a way of treatment

34. 2 -2015