1. Amino Acid Oxidation and The Production of Urea
Chapter 18
Amino Acid Oxidation and The Production of Urea

2. Amino Acid Oxidation Dependency of amino acid as energy source
Carnivores> herbivores> microorganism >> plant
Amino acid degradation in animals
Amino acids for oxidation
Extra amino acid during protein turnover
Protein-rich diet (no storage)
During starvation or in uncontrolled diabetes
Removal of amino group (NH4+)
 a-keto acid (C skeleton of amino acids)
 Oxidation to CO2 & H2O
 Sources of C3 or C4 units for gluconeogenesis or fuels

3. Pathways of amino acid catabolism

4. 18.1 Metabolic Fates of Amino Groups

5. Amino Group Catabolism

6. Amino Group Catabolism
Amino acid metabolism
 amino group (= nitrogen metabolism)
Liver is a major site
Recycle for biosynthetic pathways
Excretion; ammnonia, urea, uric acid
Glutamate & glutamine
General collection point for amino group
NH3 from amino acids + a-ketoglutarate
 glutamate
into mitochondria,  release of NH4+
Source of ammonia
Dietary protein (major source)
Muscle & other tissues
NH4+ + glutamate  glutamine
 mitochondria in hepatocytes
NH4+ + pyruvate  alanine  hepatocytes

7. Digestion of Dietary Protein
In stomach
Entry of diet  secretion of gastrin from gastric mucosa
 secretion of HCl (parietal cells), pepsinogen (chief cells)
Acidic gastric juice (pH 1.0 to 2.5)
Antiseptic & denaturing agent (protein unfolding)
Pepsinogen : zymogen
Conversion to active pepsin by autocatalytic cleavage (at low pH)
Digestion of peptide bonds at Phe, Trp, Tyr  mixture of small peptides
In small intestine
Low pH  secretion of secretin
 stimulation of bicarbonate secretion from pancreas  neutralization
Arrival in the upper part of intestine (duodenum)
 release of cholecystokinin into blood
 stimulation of pancreatic zymogens
Trypsinogen : activated by enteropeptidase
Chymotrypsinogen, procarboxypeptidase A and B : activated by trypsin
c.f.) Protection of pancreas from proteolytic digestion
Production of zymogens
Pancreatic trypsin inhibitor
Protein digestion by trypsin, chymotrypsin, carboxypeptidase, aminopeptidase
Uptake of amino acids by the epithelial cells

8. Digestion of Dietary Protein
Blood capillaries
Liver

9. Transamination 1st step of amino acid catabolism
Transfer of a-amino group to a-ketoglutarate
Generation of L-glutamate & a-ketoacid
Aminotransferase (transaminase)
Amino acid specificity (named after amino acids)
Reversible reaction
; ∆G’° ≈ 0 kJ/mol
Pyridoxal phosphate (PLP)
Bimolecular Ping-Pong reactions

10. Pyridoxal phosphate (PLP)
Coenzyme form of pyridoxine (vitamin B6)
Intermediate carrier of amino group
Electron sink for carbanion (resonance stabilization)
Transamination
Racemization (L- & D-form interconversion)
Decarboxylation

11. PLP-mediated transamination at a-carbon
PLP-mediated transamination: Ping-Pong mechanism
amino acid a-ketoglutarate
pyridoxal phosphate pyridoxamine phosphate pyridoxal phosphate
a-keto acid glutamate

12. PLP-mediated amino acid transformations at a-carbon

13. Oxidative Deamination of Glutamate
Mitochondrial matrix of hepatocytes
Glutamate dehydrogenase
Generation of a-ketoglutarate & ammonia
NAD+ or NADP+ as electron acceptor
Allosteric regulation
By ADP (inhibition)
By GTP (activation)
Transdeamination
Transamination of A.a. + oxidative deamination of Glu
A few amino acids undergoes direct oxidative deamination

14. Glutamine as Ammonia Carrier in the Bloodstream
Ammonia generated in extrahepatic tissues
Glutamine synthetase
Incorporation of ammonia into glutamate  glutamine
Transport of gln to the liver via blood
Higher gln concentration than other amino acids in blood
Glutaminase in the liver, intestine, and kidney
Glutamine  Glutamate + NH4+

15. Alanine Transports Ammonia from Skeletal Muscles to the Liver
Glucose-alanine cycle
In muscle
Glycolysis & degradation of amino acids
Alanine aminotransferase
Transfer amino group of glutamate to pyruvate  alanine + a-ketoglutarate
Transport of alanine to the liver
In the liver
Transfer amino group of alanine to a-ketoglutarate  glutamate + pyruvate
Gluconeogenesis
Pyruvate , lactate  glucose
Transport of glucose to muscle

16. Ammonia is toxic to animals.
Comatose state of brain (high brain’s water content)
1. NH3: alkalization of cellular fluid
2. a-ketoglutarate, NADH, ATP:
citric acid cycle & ATP production
3. glutamate and GABA (g-aminobutyrate):
neurotransmitter depletion

17. 18.2 Nitrogen Excretion and the Urea Cycle
Produced in liver
Blood
Kidney  urine

19. Urea Cycle in Mitochondria
Formation of carbamoyl phosphate; preparatory step
NH4+ + HCO ATP  carbamoyl phosphate + 2 ADP + Pi
Carbamoyl phosphate synthetase I
- ATP-dependent reaction
1st step in the urea cycle;
Ornitine + carbamoyl phosphate  citrulline + Pi
Ornitine transcarbamoylase

20. Urea Cycle in Cytosol 2nd step; formation of argininosuccinate
Incorporation of the second N from aspartate
Argininosuccinate synthetase
ATP requirement
Citrullyl-AMP intermediate
3rd step; formation of arginine & fumarate
Argininosuccinase; only reversible step in the cycle
4th step; Cleavage of arginine to urea & ornithine
Arginase

21. Asparatate-argininosuccinate shunt
Metabolic links between citric acid and urea cycles
In cytosol
Fumarate to malate  citric acid cycle in mitochondria
In mitochondria
OAA + Glu  a-ketoglutarate + Asp  urea cycle in cytosol
Energetic cost
Consumption
3 ATP for urea cycle
Generation
Malate to OAA
1 NADH = 2.5 ATP

22. Regulation of the Urea Cycle
Long term regulation
Regulation in gene expression
Starving animals & very-high protein diet
Increase in synthesis of enzymes in urea cycle
Short term regulation
Allosteric regulation of a key enzyme
Carbamoyl phosphate synthetase I
Activation by N-acetylglutamate

23. Treatment of genetic defects in the urea cycle
Genetic defect in the urea cycle
 ammonia accumulation; hyperammonemia
Limiting protein-rich diet is not an option
Administration of aromatic acids; benzoate or phenylbutyrate
Administration of carbamoyl glutamate
Supplement of arginine

25. 18.3 Pathways of Amino Acid Degradation

26. Amino Acid Catabolism Carbon skeleton of 20 amino acids
Conversion to 6 major products
- pyruvate
- acetyl-CoA
- a-ketoglutarate
- succinyl-CoA
- fumarate
- oxaloacetate

27. Glucogenic or Ketogenic Amino Acids
Conversion to acetyl-CoA or acetoacetyl-CoA
 ketone bodies in liver
Phe, Tyr, Ile, Leu, Trp, Thr, Lys
Leu : common in protein
Contribution to ketosis under starvation conditions
Glucogenic amino acids
Conversion to pyruvate, a-ketoglutarate, succinyl-CoA, fumarate, and OAA
 glucose/glycogen synthesis
Both ketogenic and glucogenic
Phe, Tyr, Ile, Trp, Thr

28. Enzyme cofactors in amino acid catabolism
One-carbon transfer reactions
; common reaction type, involvement of one of 3 cofactors
Biotin
; one-carbon tranfer of most oxidized state, CO2
Tetrahydrofolate (H4 folate)
; One-carbon transfer of intermediate oxidation states or methyl groups
S-adenosylmethionine
; one-carbon transfer of most reduced state, -CH3

29. Tetrahydrofolate folate (vitamin) to H4 folate Dihydrofolate reductase
Primary source of one-carbon unit
Carbon removed in the conversion of Ser to Gly
Oxidation states of H4 folate
; One-carbon groups bonded to N-5 or N-10 or both
- Methyl group (most reduced)
- Methylene group
- Methenyl, formyl, formimino group (most oxidized)
Interconvertible & donors of one-carbon units (except N5-methyl-tetrahydrofolate)

30. S-adenosylmethionine (adoMet)
Cofactor for methyl group transfer
Synthesized from Met and ATP
Methionine adenosyl transferase
Unusual displacement of triphosphate from ATP
Potent alkylating agent
Destabilizing sulfonium ion  inducing nucleophilic attack on methyl group

31. Six amino acids are degraded to pyruvate
Ala, Trp, Cys, Ser, Gly, Thr
 pyruvate  acetyl-CoA  citric acid cycle or gluconeogenesis

32. Interplay of PLP and H4folate in Ser/Gly metabolism

33. 3rd pathway of glycine degradation - D-amino acid oxidase
detoxification of D-amino acid
high level in kidney
- Oxalate  crystals of calcium oxalate (kidney stones)

34. Seven Amino Acids Are Degraded to Acetyl-CoA
Trp, Lys, Phe, Tyr, Leu, Ileu, Thr  acetoacetyl-CoA  acetyl-CoA

35. Intermediates of Trp catabolism be precusors for other biomolecules

36. Catabolic pathways for Phe & Tyr
Phe & Tyr are precusors
dopamine
norephinephrine, epinephrine
melanin

37. Phenylalanine hydroxylase
Mixed function oxidase
; Substrate hydroxylation + oxygen reduction to H2O
Tetrahydrobiopterin as a cofactor

38. Example of A.a. metabolism defects ; Phe catabolism
Phe degradaion fumarate + acetoacetyl-CoA
Defects in Phe catabolism
Phenylketonuria (PKU)
Genetic defect in Phe hydroxylase or dihydrobiopterin reductase
Elevated levels of Phe & phenylpyruvate
Mental retardation
Alkaptonuria
Genetic defect in homogentisate dioxygenase
Oxidation of accumulated homogentisate
Black urine
Arthritis

39. Genetic defects of A.a. metabolism
 defective neural development & metal retardation

40. Five Amino Acids Are Converted to a-ketoglutarate
Pro, Glu, Gln, Arg, His; amino acids with five C skeleton
 converging to Glu

41. Four Amino Acids Are Converted to Succinyl-CoA
Met, Ileu, Thr, Val converging to propionyl-CoA

42. Branched-Chain Amino Acids Are Not Degraded in the Liver
Leu, Ile, Val
Primarily oxidized as fuels in muscle, adipose, kidney, brain
Branched-chain aminotransferases (not in liver)
Branched-chain a-keto acid dehydrogenase complex

43. Asn and Asp are Degraded to Oxaloacetate