1. Biochemistry Sixth Edition
Berg • Tymoczko • Stryer
Chapter 23:
Protein Turnover and
Amino Acid Catabolism
Copyright © 2007 by W. H. Freeman and Company

2. Amino Acid Metabolism
Liver is the primary site for amino acid metabolism.
Each amino acid has a pathway for catabolism and separate one for anabolism. Actually the pathways differ in different organisms.
For mammals: Essential amino acids must be obtained from diet. Nonessential amino acids - can be synthesized .

4. Exogenous Protein Digestion
Most dietary protein is hydrolyzed to amino acids and small peptides in the stomach and intestine.
In stomach and intestinal

5. Digestive Proteases Stomach (pH ~ 1-2): Pepsin, Gastricin, Chymosin
Intestine (pH ~ 8): Trypsin, Chymotrypsin, Carboxypeptidase, Elastase Enteropeptidase, Aminopeptidases
Endogenous protein is also catabolized but by a method that differs from that above. Also, endogenous proteins have varying lifetimes.

6. Endogenoous Protein Turnover
Proteins exhibit continuous turnover (synthesis and degradation). Protein half-lives are from minutes to months, most are short. The amino terminal residue is a factor in selection of some protein. ornithine decarboxylase t½ ~11 min liver & plasma protein ~2-10 days muscle protein ~180 days collagen ~1000 days
In eucaryotes, some proteins are targeted for degradation by a covalent attachment through lysine residues of the target protein to the C- terminus of ubiquitin, a small 76 residue peptide.

7. Ubiquitin on Lysine
Attachment of a lysine in the target protein to the C-term glycine of ubiquitin via an isopeptide bond.
The signal for protein death. 

8. Ubiquitin Attachment of ubiquitin to Lys requires three enzymes:
E1. Ubiquitin-activating enzyme (uses ATP)
E2. Ubiquitin-conjugating enzyme (assembles Ubiq., the target protein and E3).
E3. Ubiquitin-protein ligase (forms the Gly-Lys bond).

9. Mechanism of Attachment
an acyl
adenylate
Many isoforms of E3 exist. These select proteins for degradation.

10. Ubiquitin Chains
Sequential attachment of C-term Gly to Lys48 of another ubiquitin forms tetra-ubiquitin. This extended structure serves as an enhanced degradation signal.

11. A Proteasome
A Proteasome is a large ATP dependent complex that hydrolyzes the ubiquitinated proteins.

12. Degradation Events
The core of b subunits contain the active sites and all have an N-term Thr.
The a subunits on the ends serve as regulatory caps that block access to the active sites.

14. Procaryotic vs Eucaryotic
Procaryotes have a proteasome analog of that in eucaryotes but the function is unclear since ubiquitin has not been found. In procaryotes, all α subunits are identical and all β subunits identical whereas in eucaryotes these subunits exhibit a number of isoforms.
Procaryotes do have a ubiquitin-like protein but it is is used in the synthesis of thiamine and not protein degradation.

15. Procaryotic vs Eucaryotic

16. Procaryotic vs Eucaryotic
For thiamine synthesis For protein degradation

17. Amino Acid Catabolism
First step amino acid catabolism is generally removal of the a-amino group.
Carbon chains are then altered for entry into central pathways of carbon metabolism.
The amino acids from either degraded proteins or from a dietary source can be used for the biosynthesis of new proteins.
During starvation proteins are degraded to amino acids to support glucose formation.

18. Methods for Removal of NH3
1. Transamination:
amino acid + a-ketoglutarate  a-ketoacid + Glu
2. Glutamate dehydrogenase:
Glu + NAD+  a-ketoglutarate + NADH + NH4+
3. Direct deamination:
Ser  pyruvate
His  urocanate (resonance driven)
4. Amide hydrolase
Gln or Asn  Glu or Asp + NH4+

19. 1. Transamination
A pyridoxal phosphate (PLP) mediated reaction. a-ketoglutarate, the normal a-keto acid used, forms glutamate.

20. PLP
The aldehyde group forms a schiff base with a Lys on a transaminase enzyme.
PLP enzymes are typically involved in: transamination, decarboxylation, or racemization.

21. Transaminase Mechanism
Lysyl linkage is displaced by an amino acid.

22. Transaminase Mechanism
Loss of a proton and formation of a different Schiff base.

23. Transaminase Mechanism
Reprotonation

24. Transaminase Mechanism
Schiff base hydrolysis removes the amino group and gives an a-ketoacid. Bringing in a-ketoglutarate and reversing these steps gives Glu.

25. Asp Aminotransferase
Amino acid not shown, but Arg binds with –COO-

26. Bond Cleavage

27. 2. Glutamate Dehydrogenase
The requirement for NAD+ or NADP+ in this enzyme varies. Glu (from transamination) -- > a-ketoglutarate

28. 3. Direct Deamination Serine Dehydratase (uses PLP in Ecoli).
In other amino acids direct deamination is driven by extended conjugation.

29. 4. Amide Hydrolysis
NH4+ is removed from asparagine and glutamine by the enzymes asparaginase and glutaminase.
asparaginase asparagine > aspartate + NH4+
glutaminase glutamine > glutamate + NH4+

30. Disposition of NH4+
The ammonium ion (which is toxic) formed by action of a transaminase and glutamate dehydrogenase (below) or other reactions, goes to the urea cycle (in the liver).
Transaminase
Glutamate DH

31. Transport of NH4+
NH4+ is transported to the liver by either of two methods.
1. Glucose-Alanine Cycle (next slide)
2. Glutamine
glutamine NH4+ + glutamate + ATP > glutamine + ADP + Pi synthetase
glutaminase glutamine > glutamate + NH4+

32. Glucose-Alanine Cycle

33. NH4+ to Urea
Waste nitrogen must be removed (a high conc. of ammonia is cytotoxic) Fish and many aquatic organisms excrete NH4+, Terrestrial vertebrates synthesize urea, Birds, reptiles synthesize uric acid.
The liver processes NH4+ into urea using carbamoyl-phosphate synthetase I and enzymes of the urea cycle.

34. Incorporation of NH4+ Requires carbamoyl phosphate synthetase.
Carbamoyl phosphate synthetase I catalyzes removal of ammonia using the energy from ATP to form carbamoyl phosphate. This is the normal feeder for the urea cycle which is a liver pathway.
Carbamoyl phosphate synthetase II uses glutamine as a source of ammonia again using the energy from ATP to form carbamoyl phosphate. This provides carbamoyl-P for pyrimidine synthesis.

35. Carbamoylphosphate Synthetase I
A mitochondrial enzyme, requires 2 ATP

36. Urea Cycle Four reactions. One in the mitochondria.
Three in the cytosol.

37. Urea Cycle, Reaction 1
Transfer of the carbamoyl group in the mitochondria.

38. Urea Cycle, Reaction 2
A citrulline:ornithine antiport moves citrulline to the cytosol. The second NH2 for urea comes from Asp. An adenylated citrulline intermediate gives PPi.

39. Urea Cycle, Reaction 3
Cleavage of fumarate, production of arginine

40. Urea Cycle, Reaction 4
Cleavage of urea from arginine gives urea. The ornithine is ready to begin a new cycle.

41. Recycling to Aspartate
Malate dehydrogenase occurs in mitochondria and in the cytosol.

42. N-Acetylglutamate
N-AcGlu is synthesized when NH4+ levels increase during amino acid catabolism.
An activator of CPS I which provides substrate for the urea cycle.
Glu is a product of Gln hydrolysis by CPS II in pyrimidine synth.
An intermediate in ornithine synthesis.

43. Structural similarity
Blue - Ornithine transcarbamoylase from the urea cycle
Red - Aspartate transcarbamoylase from pyrimidine synthesis

44. Functional similarity
Transfer of an amino group by incorporation of aspartate and elimination of fumarate occurs in both the urea cycle and pyrimidine synthesis.

45. AA Carbon Skeletons
In catabolism of their carbon skeletons, amino acids are referred to as being either glucogenic or ketogenic depending upon the structures of the degradation products.
Glucogenic amino acids degrade to pyruvate or citric acid cycle intermediates which can feed gluconeogenesis.
Ala, Cys, Gly, Ser,Asp, Asn, Glu, Gln Thr depends upon the pathway.

46. AA Carbon Skeletons
Ketogenic amino acids degrade to acetylCoA or acetoacetylCoA which can contribute to the synthesis of fatty acids or ketone bodies.
Leu and Lys are the only two purely ketogenic amino acids.
Some amino acids yield both glucogenic and ketogenic parts.
Phe, Tyr, Trp, Ile Thr depends upon the pathway.

47. Pink = glucogenic
Yellow = ketogenic

48. Pyruvate Family

49. a-Ketoglutarate Family
All are converted to Glu first.

50. Histidine

51. Arginine and Proline

52. SuccinylCoA Family
All are converted to propionylCoA first and then follow the odd chain fatty acid pathway.
Thr with a-ketoacid decarboxylase yields propionylCoA (an enzyme similar to PDH).

53. Methionine

54. Ile, Val and Leu
These non-polar, branched chain amino acids follow a similar sequence of three reactions:
1. Transamination giving an a-ketoacid.
2. Oxidative decarboxylation giving an acylCoA.
3. AcylCoA dehydrogenase yields a,b unsaturation.
The residue from Ile follows b-ox to give acetylCoA and propionylCoA.
The residue from Val adds HOH, is then oxidized twice to yield an acid with a b-carbonyl. This decarboxylates to give propionylCoA.

55. Leucine
Transaminase
a-Keto acid decarboxylase
(Like PDH)

56. Leucine, cont.
AcylCoA dehydrogenase
Carboxylase

57. Leucine, cont.
Hydratase
Lyase

58. Conversion of Phe to Tyr
Requires tetrahydrobiopterin

59. Tetrahydrobiopterin
Formation and regeneration

60. Phe and Tyr Degradation

61. Tryptophan Degradation

62. Pool Molecules from Degradation
Amino Acid Pool Molecule
Asp, Asn Oxaloacetate
Ala, Cys, Gly, Ser, Thr, Trp Pyruvate
Arg, His, Pro, Gln, Glu a-ketoglutarate
Met, Ile, Val, Thr SuccinylCoA
Asp, Phe, Tyr, Fumarate
Phe, Tyr, Trp, Ile, Leu, Lys AcetylCoA and/or AcetoacetylCoA

63. Biochemistry Sixth Edition
Berg • Tymoczko • Stryer
End of Chapter 23
Copyright © 2007 by W. H. Freeman and Company