1. Lecture outline… Protein Metabolism II Urea Cycle 27th Mar 2012
Know the:
1.Urea Cycle: Reaction and Significance.
2.The Importance of the Cytosol and Mitochondria.
3.Enzyme Defect and Subsequent Clinical Diagnosis.
27th Mar 2012

2. Learning Objectives II.Urea Cycle A.Reactions of the urea cycle.
1.Synthesis of carbamoyl phosphate.
2. Production of arginine by urea cycle.
3. Cleavage of arginine to produce urea.
B. Origin of ornithine.
C. Regulation of the urea cycle.
D. Function of the urea cycle during fasting.
III. Disorders of the Urea Cycle. 2

3. What are the Precursors of Urea
What is Urea
Urea is a diamide, chief nitrogenous waste product.
What are the Precursors of Urea
Ammonia +CO2 + Aspartate are the precursors of Urea.
Other nitrogenous waste products which includes uric acid & creatinine. 3

4. Summary of Amino Acid Metabolism
Dietary Proteins
Digestion (Stomach Intestine)
Amino Acids in Blood
Membrane
Other
N-Containing Compounds
Proteins
Amino Acids
Nitrogen
Carbon
g-Aminobutyrate
Dopamine
Norepinephrine
Epinephrine
Serotonin
H2N-C-NH2 Urea O
CO2 + H2O
Energy ATP 4

5. Fate of Amino Acid Nitrogen: Urea Cycle
Amino Acids
Nitrogen
Carbon
Storage
CO2+H2O
NH4+
Energy
ATP
+CO2
COO-
H C- - -
NH2
-C-
Urea O -
NH2
NH2
Urea Cycle
Aspartate
What are the source of amino groups and carbonyl group for the biosynthesis of Urea?. 5

6. Most mammals convert amino-acid nitrogen to urea for excretion
Excretion of Nitogenous Waste Products
The carbon chains are broken down to molecules that feed into the TCA cycle.
Amino acids
Most mammals convert amino-acid nitrogen to urea for excretion
NH4+
Some animals excrete NH4+ or uric acid.
Fish & other aquatic vertebrates
Most terrestrial vertebrates
Birds & reptiles O HN N H
H2N-C-NH2 Urea O
NH4+
Ammonium ion
Uric acid 6

7. An Overview of Urea Cycle
What Are The Sites & The Organ In Which Biosynthesis Of Urea Occurs?
Urea biosynthesis occurs partly in the cytosol and partly in the mitochondria of the liver.
Net reaction:
NH3 + CO2 + 2 ATP
(HCO3)
Carbamoyl-Phosphate +
2 ADP + Pi

8. phosphate synthase I (CPS-I) Transcarbamoylase (OTC)
The Urea Cycle in the Liver
Mitochondrial
Matrix
NH4+ + HCO ATP
Carbamoyl
phosphate synthase I (CPS-I) 1
Carbamoyl phosphate
Ornithine
Transcarbamoylase (OTC) 2
Ornithine
Citrulline
Hepatocyte
Citrulline
Aspartate
Ornithine
Argininosuccinate
Synthetase
ATP 3
AMP+ Pi
Argininosuccinate 5
Argininosuccinate
Lyase 4
Fumerate
Arginase
Arginine
Cytoplasm
Urea

9. What is Normal Value of Urea Is there any Specific functions of Urea
Serum urea concentration is mg/dL.
Accumulation of urea more than normal value in blood is referred as’”Uremia”.
Urea Excretion: 20–30 gm / day.
Is there any Specific functions of Urea
Not yet identified, but probably may involve in ‘’’’’the maintenance of osmotic balance as a ‘’’’’crystalloid. 9

10. Osmolality of Plasma Osmolality is a measure of the solute particles
present in the fluid medium.
Osmolality of Plasma: milliosmoles / Kg.
Plasma Osmolality can be computed from the
concentrations (mmol/l) of Na+, K+, urea, & glucose
as follows.
2(Na+) + 2(K+) + Urea + Glucose
The factor 2 is used for Na+ & K+ ions for the
associated anion concentration.
Plasma Osmolality will be altered in lipidaemias, ESRD,
hyperproteinaemia, ketoacidosis, diabetes insipidus,chronic
alcohol intoxication, diuretics & in chronic diarrhoea.

11. Is any Clinical Conditions Are related with Low Urea Level
Impairment of liver functions and congenital deficiencies of urea cycle enzymes will impair the biosynthesis of urea, which ultimately leads to accumulation of ammonia in blood and CSF causes “azotemia”. 11

12. What Is Meant By Azotemia
Accumulation of nitrogenous materials including
ammonia due to impairment in urea biosynthesis due to,
Urea cycle enzyme deficiencies,
Gastrointestinal tract (GI) bleeding,
Valproic acid therapy and
Renal impairments ect.,. 12

13. What Are The Consequences of Uremia
Uremia will leads to ammonia toxicity, hypercapnia & alkalosis which may leads to neurotoxicity depending on the severity.
Impaired renal function leads to erythropoietin deficiency and hence accumulation of CO2. 13

14. What Are The Causes of Uremia
Ureamia may occur as a result of impairment in ‘’’’’the renal functions, which occur as a result of
nephrotic syndrome,
heavy metal toxicities,
ESRD and
nitrogen load due to high protein diet. 14

15. Importance of Urea Cycle
Removal of CO2.
Removal of Ammonia.
Recycling of TCA cycle intermediates.
Recycling of amino acids & ketoacids. 15

16. Involvement of Peripheral Tissues And Liver in the
Ammonia Metabolism
α-KG = α-Ketoglutaratee
GDH = Glutamate Dehydrogenase.

17. Molecualr Interconversions in Handling of Ammonia
α-Ketoglutarate
Aspartate
Alanine
NH4+
NADH+H+
ALT
NADPH+H+
Glutamate
Dehydrogenase
AST
NADPH+
Pyruvate
H2O
NAD
Glutamate
Oxaloacetate
NH4+
ATP
Glutamine
Synthetase
ADP+Pi
Glutamine
The major enzyme responsible for the interconversion of Glutamate into α-KG is
Glutamate Dehydrogenase. ALT= Alanine aminotransferase
AST= Aspartate aminotransferase.

18. Reactions of the Urea Cycle
Synthesis of Carbomoyl Phosphate.
Production of Arginine by the Urea Cycle.
Cleavage of Arginine to Produce Urea.
(3) 18

19. Urea is less toxic than ammonia.
Most terrestrial land animals convert excess nitrogen to urea, prior to excreting it.
Urea is less toxic than ammonia.
The Urea Cycle occurs mainly in liver.
The 2 nitrogen atoms of urea derived from NH3 and the amino nitrogen of aspartate.

20. Carbamoyl Phosphate Synthase is the committed step of the Urea Cycle, and is subject to regulation.
The NH3 and HCO3- that will be part of urea are incorporated first into carbamoyl phosphate (CP).

21. Carbamoyl Phosphate Synthase-I has an absolute requirement for an allosteric activator N-acetylglutamate.
This derivative of glutamate is synthesized from acetyl-CoA & glutamate when cellular [glutamate] is high, signaling an excess of free amino acids due to protein breakdown or dietary intake.

22. Formation & Degradation of N-Acetylglutamate (NAG)
N-Acetyl glucoseamine (NAG) serves as an allosteric activator for the enzyme CPS-I for the biosynthesis of carbamoyl phosphate (CP) with the help of HCO3- and ATP.
NAG, is an allosteric activator of CPS-I.

23. The Reactions of the Urea Cycle
Mitochondria
NAG:N-acetyl glutamate; (in the formation of urea, one amino group is derived from free NH4+ ion, while the other is from aspartate. Carbon is obtained from CO2. (*) mitochondrial enzymes, the rest
of the enzymes are cytosolic).
Cytosol *
NAG * 23

24. Urea Role of Glutamate in Urea Production Glutamate Urea Cycle
Amino acids
α-Ketoglutarate
Transamination
Other reactions
Glutamate
α-Ketoacids
Transamination
Oxalaoacetate
α-Ketoglutarate
NH4+
Aspartate
Urea Cycle
Urea 24

25. Role of Glutamate in Amino Acid Synthesis
α-Ketoglutarate
PLP
GDH
Transamination
Glutamate
α-Ketoacid
PLP
Amino acids
α-Ketoglutarate
Glutamate transfers Nitrogen by means of transamination reactions to α–ketoacids to form AAs. The nitrogen is obtained by glutamate either from transamination of other AAs or from NH4+ by means of the glutamate dehydrogenase (GDH) reaction
PLP= Pyridoxal phosphate 25

26. Oxaloacetate is converted to aspartate via transamination (e. g
Oxaloacetate is converted to aspartate via transamination (e.g., from glutamate).
Aspartate then reenters Urea Cycle, carrying an amino group derived from another amino acid.

27. Regulation of the Urea Cycle
Liver has a vast capacity to convert AA nitrogen to urea
,thereby preventing toxic effects from ammonia.
Urea cycle is regulated by substrate (S) availability; the higher the rate of NH3 production, the higher the rate of urea formation.
Regulation by the S availability is a general characteristics of disposal pathways, such as the urea cycle, which remove toxic compounds from the body.
This is a type of “feed-forward” regulation, in contrast to the “feed-back” regulation characteristic of pathways that produce functional endproducts. 27

28. Regulation of the Urea Cycle…
The other type of regulation control the urea cycle: Allosteric activation of CPS-I by N-acetylglutamate (NAG) and induction/repression of the synthesis of urea cycle enzymes.
NAG is formed specifically to activate CPS-I; it has no other known function in mammals.
The synthesis of NAG from acetyl CoA and glutamate is stimulated by arginine.
Thus, the arginine level increase within the liver, 2 important reactions are stimulated.
The first is the synthesis of NAG, which will increase the rate at which CP is produced.
The 2nd is to produce more ornithine (via arginase rxn), such that the cycle can operate more rapidly. 28

29. D. Functions of Urea Cycle During Fasting
During fasting, the liver maintains blood glucose by gluconeogenesis by utilizing muscle protein AAs as a carbon source.
As AA carbons are converted to glucose, the nitrogens are converted to urea, thus the urinary excretion of urea is high during fasting.
As fasting progresses, the brain begins to use ketone bodies, sparing blood glucose.
Less muscle protein is cleaved to provide AAs for gluconeogenesis,and decreased production of glucose from AAs for gluconeogenesis.
Hence, Decreased production of glucose from AAs is accompanied by decreased production of urea. 29

30. D.Functions of Urea Cycle During Fasting…
The major AA substrate for gluconeogenesis is Alanine, which is synthesized in peripheral tissues to act as a nitrogen carrier.
Glucagon release, which is expected during fasting, stimulates alanine transport into the liver by activating the transcription of transport systems for alanine.
Two molecules of alanine are required to generate one molecule of glucose.
The nitrogen from 2 molecules of alanine is converted to one molecule of urea. 30

31. Hereditary deficiency of any of the Urea Cycle enzymes leads to hyperammonemia - elevated [ammonia] in blood.
Total lack of any Urea Cycle enzyme is lethal.
Elevated ammonia is toxic, especially to the brain.
If not treated immediately after birth, severe mental retardation results.

32. Genetic Deficiency of Urea Synthesis
May be caused by,
Ornithine Transcarbamylase Deficiency (OTC)
Citrullinemia.
Arginase Deficiency
Argininosuccinic Aciduria
Carbamyl Phosphate Synthetase (CPS-I) Deficiency
N-Acetyl Glutamate Synthetase Deficiency (NAGS)

33. Carbamoyl Phosphate Synthetase-I Ornithine Transcarbamylase (OTC)
Genetic Deficiency of Urea Synthesis
Carbamoyl Phosphate Synthetase-I
(CPS-I) Deficiency
Ornithine Transcarbamylase (OTC)
↑[NH4+]; hyperammonemia
↑Blood Glutamine
BUN is decreased
No increase in uracil or orotic acid
↑Uracil & orotic acid in blood & urine
Cerbral edema
Cerebral edema
Lethargy, convulsions, coma, death

34. Metabolic Diseases of the Urea Cycle
Disorders present in infants:
Symptoms: Lethargy, swelling of the brain leads to mental retardation / brain damage.
Diagnosis: Low blood urea nitrogen (BUN) levels -high levels of ammonia in the blood elevated circulating glutamine
-other metabolites that accumulate depend on the specific enzyme defect.
Most common form: Hyperammonemia Type II caused by Ornithine Transcarbamylase (OTC) Deficiency elevated Carbamoyl-Phosphate (CP) levels in this deficiency cause
secondary problems in pyrimidine metabolism.

35. Treatment: Long term, dietary restriction.
Low protein diet. Supplemented with Arginine.
Short term
Dialysis.
Administration of Nitrogen “scavengers”.
e.g. Phenylacetate.

36. Treatment of Hyperammonemia with Phenylacetate:
taking advantage of metabolism
Phenylbutyrate

37. Glycine + Benzoic Acid Hippuric Acid
Excreted in Urine

38. Review Q & As

39. Which biomolecule acts as a positive modulator for the first step of the urea cycle which is catalyzed by carbamoyl phosphate synthetase-I (CPS-I).
A. Gultamate.
B. Glutamine.
C. N-Acetyl Glutamate (NAG).
D. Aspartate.
E. Ammonium ions (NH4+).
Ans: C.

40. 7. Which of the following is a common compound shared by The TCA cycle and the urea cycle?
A. α-ketoglutarate.
B. Succcinyl Coenzyme A (CoA).
C. Oxaloacetate.
D. Fumarate.*
E. Arginine.
Ans: D.

41. 8. Which of the following enzymes requires adenosine triphosphate (ATP) to mediate its reactions?
A. Argininosuccinate lyase.
B. Argininosuccinate synthetase.
C. Arginase.
D. Glutaminase.
E. Ornitine transcarbamoylase.
Ans: B.

42. 10. Two days after a full-term normal delivery, a neonate begins to hyperventilate, develops hypothermia and cerebral edema, and becomes comatose. Urinalysis reveals high levels of glutamine and orotic acid. The BUN is below normal. Which enzyme is most likely to be deficient in this child?
A. Cytoplasmic glutaminase.
B. Cytoplasmic carbamoyl phosphate synthetase.
C. Cytoplasmic orotidylate decarboxylase.
D. Mitochondrial carbamoyl phosphate synthetase.
E. Mitochondrial ornitihine transcarbamoylase.
Ans: E.

43. 11. Transamination reactions are essential for ammonia assimilation
11. Transamination reactions are essential for ammonia assimilation. What cofactor is required to catalyze transamination reactions?
A. pyridoxal phosphate (PLP).
B. thiamin pyrophosphate.
C. biotin.
D. NAD+.
E. NADPH.
Ans: A.

44. 12. Which of the following enzymes function in the biological
assimilation of ammonia?
A. Glutamate dehydrogenase.
B. Glutamine synthetase.
C. Glutamate synthase.
D. All of the above.***
E. None of the above.
Ans: D.

45. A 55-year-old man suffers from cirrhosis of the liver. Toxins such
as ammonia are not properly metabolized by the liver and can
now damage structures such as the brain. Which of the following
amino acids covalently binds ammonia and transports and stores
it in a nontoxic form?
Aspartate.
Glutamate.
Serine.
Cysteine.
Histidine.
Ans: B.

46. 14. Which amino acid participates as a pathway intermediate in the urea cycle?
Lysine.
Arginine.***
Glutamine.
Histidine.
Tyrosine.
Ans: B

47. Urea Cycle Must Know Key Points
Urea cycle occurs partly in ……….. and partly in the ……….
Ans: Mitochondria, Cytosol / Cytoplasm.
2. The rate-limiting step (committed step) in the urea cycle is
catalyzed by the enzyme …………
Ans: Carbamoyl phosphate synthetase-I (CPS-I).
3. The biomolecule acts as a positive modulator in the first step of
the urea cycle is……………
Ans: N-Acetyl glutamate (NAG).
4. ………….can be converted to arginine by a series of reactions,
some of which requires urea cycle enzymes.
Ans: Glutamate.

48. 5. The enzymes are responsible for producing the direct donors of nitrogen into the pathway producing urea, include ………. And ………..
Ans: Aspartate aminotransferase and carbmoyl phosphate
sythetase.
6. The accumulation of glutamine and orotic acid in the blood and
an ultimate ammonia toxicity in the newborn is due to the
deficiency of the urea cycle enzyme……….
Ans: Mitochondrial ornitihine transcarbamoylase (OTC).
7. The common nitrogen acceptor for all reactions involving
transaminase is ……………
Ans: α-ketoglutarate.
8. The common nitrogen donor for all reactions involving
Ans: Oxaloacetate (OAA).

49. 9. The common compound shared by the TCA cycle and the urea cycle is ………………
Ans: Fumarate.
10. Hyperammonemia caused by a congenital defect of the urea cycle enzyme………..which is characterized by accumulation of excess amount of arginine in the blood.
Ans: Arginase.
11. The urea cycle enzyme which requires adenosine triphosphate (ATP) to mediate its reactions is ?.
Ans: Argininosuccinate synthetase.
12. The reaction catalyzed by an urea cycle enzyme ornithine aminotransferase which requires as a coenzyme, which converts ornithine and α-ketoglutarate into glutamate.
Ans: Pyridoxal phosphate (PLP).

50. Match the Urea Cycle Disorders With the Enzyme DefectNo
Disorder
Answer
Enzyme Defect 1
Hyperammonemia type I
A. Argininosuccinate synthase 2
Hyperammonemia type II
B. Ornithine transcarbomylase (OTC) 3
Citrullinemia
C. Argininosuccinate lyase 4
Argininosuccinic Aciduria
D. Arginase 5
Hyperargininemia
E. Carbamoyl-P synthase-I (CPS)
Answer: 1E; 2-B; 3-A; 4-C; 5-D

51. Genetic Deficiencies in Some of the Urea Cycle Enzymes can be Treated Pharmacologically by eliminating the amino acids such as glycine and glutamine by administering,
A. Aspartic acid.
B. Benzoic acid and phenylacetate.
C. GABA.
D. Pyridoxal phosphate.
E. Methionine.
Ans: B. The amide products of these reactions (hippurate and phenylacetylglutamine) are excreted in the urine. Synthesizing the Gly or Gln removes ammonia.

52. The Reaction Catalyzed by Glutamate Dehydrogenase which
reversibly converts glutamate to α-ketoglutarate require the
cofactor,
ATP
NAD
NAD(P)+ /NAD(P)H.
Biotin
Pyridoxal phosphate.
Ans: C. The cofactors require by the glutamate dehydrogenase
is NAD(P)+ /NAD(P)H.