1. Protein Turnover and Amino Acid Catabolism
Chem 454: Biochemistry II
University of Wisconsin-Eau Claire

2. We Are Here Urea Cycle Amino acid metabolism
- Proposed by Hans Krebs and Kurt Henseleit in 1932
- Was the first metabolic cycle to be discovered.
• Before the citric acid cycle
• It is linked to the citric acid cycle.
- One of the N atoms comes directly from ammonium ion, the other from aspartate.
- The C atom comes from HCO3 (CO2).
We Are Here
Amino acid metabolism
Urea Cycle

3. Introduction Proteins are degraded into amino acids.
Protein turnover is tightly regulated.
First step in protein degradation is the removal of the nitrogen
Ammonium ion is converted to urea in most mammals.
Carbon atoms are converted to other major metabolic intermediates.
Inborn errors in metabolism

4. Introduction
Amino acids used for synthesizing proteins are obtained by degrading other proteins
Proteins destined for degradation are labeled with ubiquitin.
Polyubiquinated proteins are degraded by proteosomes.
Amino acids are also a source of nitrogen for other biomolecules.
- What is an example of another type of biomolecule that requires nitrogen?

5. Introduction Excess amino acids cannot be stored.
Surplus amino acids are used for fuel.
Carbon skeleton is converted to
Acetyl–CoA
Acetoacetyl–CoA
Pyruvate
Citric acid cycle intermediate
The amino group nitrogen is converted to urea and excreted.
Glucose, fatty acids and ketone bodies can be formed from amino acids.
- Unlike glucose and fatty acids, which can be stored.

6. 1. Protein Degradation
Dietary proteins are a vital source of amino acids.
Discarded cellular proteins are another source of amino acids.
- Proteins a re hydrolyzed in the stomach and small intestines and the component amino acids absorbed into the bloodstream.

7. 1.1 Dietary Protein Degradation
Dietary proteins are hydrolyzed to amino acids and absorbed into the bloodstream.
- Protein digestion starts in the stomach - acid hydrolysis
- Proteases are synthesized as zymogen and then activated in the stomach and small instestine.
- Aminopeptidase N is located in the plasma membrane of the instestinal cells.

8. 1.2 Cellular Protein Degradation
Cellular proteins are degraded at different rates.
Ornithine decarboxylase has a half-life of 11 minutes.
Hemoglobin lasts as long as a red blood cell.
Υ-Crystallin (eye lens protein) lasts as long as the organism does.
- Ornithine decarboxylase has a half-life of 11 minutes
- Gamma-crystalline lasts the lifetime of the organism

9. 2. Regulation of Protein Turnover
The protein ubiquitin is used to mark cellular proteins for destruction.
- The ubiquitin is attached to the ε-amino groups of lysines by an isopeptide bond to the C-terminus of ubiquitin.
- Ubiquitin is highly conserved throughout evolution: yeast and humans differ by only 3 out of 76 amino acids.

10. 2.1 Ubiquitin
Ubiquitin is activated and attached to proteins using a group of three enzymes
E1 - Ubiquitin activating enzyme
E2 - Ubiquitin-conjugating enyzme
E3 - Ubiquitin-protein ligase
- Reactions are similar to those that activate acyl groups in fatty acid degradation.
- There are only one or a small number of distinct E1 proteins.
- Eukaryotes have many distinct E2 and E3 proteins.
- Only a single family of E2 proteins, but many families of E3 proteins.
• E3 provides most of the substrate specificity. Combination of E2 and E3 allow for fine tuning of specificity.
- The human papilloma virus encodes for an E3 protein which targets the p53 tumor suppressor protein in its host. 90% of the cervical cancers are associtated with this type of activity.
The human papilloma virus encodes for an E3 protein which targets the p53 tumor suppressor protein in its host. 90% of the cervical cancers are associtated with this type of activity.

11. 3. Removal of Nitrogen
The first step in amino acid degradation is the removal of the nitrogen.
The liver is the major site of protein degradation in mammals.
Deamination produces α-keto acids, which are degraded to other metabolic intermediates.

12. 3.1 Conversion to Ammonium Ions
α–Amino groups are converted to ammonium ions by the oxidative deamination of glutamate

13. 3.1 Transamination
Generally these enzyme funnel amino groups to α–ketoglutarate.
Aspartate transaminase
Alanine transaminase
- Aminotransferases (transaminases) catalyze the transfer of an α–amino acid to and α–keto acid.

14. 3.1 Deamination Glutamate dehydrogenase - Uses either NAD or NADP
- Takes place in the mitochondria, where the toxic effects of the ammonium ion can be isolated
- Removal of the ammonium ion pulls this reaction to the right.
- Lowering the energy charge accelerates the oxidation of amino acids
• - GTP, ATP
• + GDP, ADP

15. 3.1 Deamination
In most terrestrial vertebrates the ammonium ion is converted to urea.

16. 3.2 Pyridoxal Phosphate
Pyridoxal phosphate forms a Schiff-base intermediates in aminotransferase reactions.
- We saw pyridoxal phosphate earlier in glycogen phosphorylase, where it played a different role.

17. 3.2 Pyridoxyl Phosphate
Pyridoxyl phosphate can under go acid/base tautomerization.
- The pyridine nitrogen is a weak base, while the phenolic OH is slightly acidic.

18. 3.2 Pyridoxyl Phosphate
The aldehyde forms a Schiff–base with an ε–amino group on the enzyme.
This Schiff-bases can be exchanged for one with the α–amino group of an amino acid
- The Schiff-base becomes protonated and the charge is stabilized by the phenolate ion.
- The Schiff-base is also called an aldimine

19. 3.2 Pyridoxyl Phosphate Transamination mechanism:
The second half of the reaction reverses these steps with a different α–keto acid.

20. 3.2 Pyridoxyl Phosphate
Pyridoxyl phosphate is is a very versatile cofactor
used to make bonds to Cα susceptible to cleavage.

21. 3.4 Serine and Threonine
The β–hydroxy amino acids, serine and threonine, can be directly deaminated

22. 3.5 Transporting Nitrogen to Liver
Urea is produced in the Liver
The alanine cycle is used to transport nitrogen to the liver
- The alanine cycle is reminiscent of the Cori cycle.

23. 4. Ammonium Ion
Ammonium ion is converted into urea in most terrestrial vertebrates

24. 4. The Urea Cycle:reminder
- Proposed by Hans Krebs and Kurt Henseleit in 1932
- Was the first metabolic cycle to be discovered.
• Before the citric acid cycle
• It is linked to the citric acid cycle.
- One of the N atoms comes directly from ammonium ion, the other from aspartate.
- The C atom comes from HCO3 (CO2).
Amino acid metabolism
We Are Here

25. 4. The Urea Cycle - Proposed by Hans Krebs and Kurt Henseleit in 1932
- Was the first metabolic cycle to be discovered.
• Before the citric acid cycle
• It is linked to the citric acid cycle.
- One of the N atoms comes directly from ammonium ion, the other from aspartate.
- The C atom comes from HCO3 (CO2).

26. 4.1 Formation of Carbamoyl Phosphate
Carbamoyl synthetase
Free NH4 reacts with HCO3 to form carbamoyl phosophate.
Reaction is driven by the hydrolysis of two molecules of ATP
- The mammalian enzyme requires the allosteric effector, N-Acetylglutamate for activity.

27. 4.1 Formation of Citrulline
Ornithine transcarbamoylase
Citrulline is formed from transfer of the carbamoyl group to the γ-amino group of ornithine.
- Both ornithine and citrulline are α–amino acids.
- The formation of NH4+ from glutamate dehydrogenase and the subsequent incorporation into the citrulline occurs in the mitochondrial matrix
-The other reactions of the urea cycle take place in the cytosol
-Citrulline is transported to the cytosol.

28. 4.1 Formation of Arginosuccinate
Condensation of citrulline with aspartate to form arginosuccinate
Two equivalent of ATP are required.
- The α–amino group from the aspartate will become the second nitrogen for the urea

29. 4.1 Formation of Arginine and Fumarate
Arginosuccinase
Cleaves arginosuccinate to form arginine and fumarate
- This is an elimination reaction
- Have produced an amino acid and an intermediate of the citric acid cycle.

30. 4.1 Formation of Urea Arginase
The arginine is hydrolyzed to produce the urea and to reform the ornithine.
The ornithine reenters the mitochondrial matrix.

31. 4.2 Linked to Citric Acid Cycle
The urea cycle is linked to the citric acid cycle: Kreb’s Bi-cycle!!

32. 5. Carbon Atoms
The carbon atoms of degraded amino acids emerge as major metabolic intermediates.
Degradation of the 20 amino acids funnel into 7 metabolic intermediates
Acetyl–CoA
Acetoacetyl–CoA
Pyruvate
α-Ketoglutarate
Succinyl–CoA
Fumarate
Oxaoloacetate
Ketogenic
Glucogenic

33. 5. Carbon Atoms Ketogenic Glucogenic Both leucine lysine serine
threonine
aspartic acid
glutamic acid
asparagine
glutamine
glycine
alanine
valine
proline
histidine
arginine
methionine
cysteine
Both
isoleucine
phenylalanine
tryptophan
tyrosine

34. Class problem
Explain the meaning (from a biochemistry perspective) of the saying “fats burn in the flame of carbohydrates.” How would proteins fit into this statement?

35. 5. Carbon Atoms
- Alanine -

36. 5.1 Pyruvate Entry Point - Know • Alanine - transmination
• Serine - deamination

37. 5.2 Oxaloacetate Entry Point
Aspartate
Transamination to oxaloacetate
Asparagine
Hydrolysis to Aspartate + NH4+
Transmination to oxaloacetate

38. 5.3 α–Ketoglutarate Entry Point
Five carbon amino acids

39. 5.3 α–Ketoglutarate Entry Point
Histidine

40. 5.3 α–Ketoglutarate Entry Point
Proline and Arginine

41. 5.4 Succinyl–CoA Entry Point
Methionine, Valine & Isoleucine

42. 5.4 Succinyl–CoA Entry Point
Methionine
Forms S-Adenosylmethionine
S-Adenosylmethionine is

43. 5.6 Branched-chained Amino Acids
- The oxidative decarboxylation occurs using a commplex that is similar to pyruvate and alpha-ketoglutarate dehydrogenases.

44. 5.7 Aromatic Amino Acids
Phenylalanine

45. 5.7 Aromatic Amino Acids
Tetrahydrobiopterin - electron carrier

46. 5.7 Aromatic Amino Acids Phenylalanine & Tyrosine
- p-hydroxyphenylpyruvate reacts with a dioxygenase to produce homogentisate
- the homogentisate reacts with an dioxygenase to cleave it.

47. 5.7 Aromatic Amino Acids
Tryptophan

48. 6. Inborn Errors in Metabolism
Tyrosine related disorders
- Discovered in 1902 by Archibald Gerrod as a mendelian inherited dissease
- Urine turns dark onstanding
- Show student the Online Mendelian Inheritance in Man (OMIM) database.(

49. 6. Inborn Errors in Metabolism
Alcaptonuria
Absence of homogentisate oxidase activity
- Discovered in 1902 by Archibald Gerrod as a mendelian inherited dissease
- Urine turns dark onstanding
- Show student the Online Mendelian Inheritance in Man (OMIM) database.(
urine
sclera

50. 6. Inborn Errors in Metabolism
Tyrosinemia
Absence of activity of fumarylacetoacetase

51. 6. Inborn Errors in Metabolism
Albinism
Absence of melanin pigment
- Phenylalanine is not converted to tyrosine
- Forms phenylpyruvate
- Initially described in 1934
- Observed by reacting phenylpyruvate with FeCl(3) (turns olive green)
- Leads to sever retardation

52. 6. Inborn Errors in Metabolism
Maple syrup urine disease
Lack of branch-chain dehydrogenase activity
Leads to elevation of α–keto banched-chain acids (branched-chain keto aciduria)
- Urine smells like maple syrup
- Mental retardatioin unless put on a diet early in life that is low in valine, isoleucine and leucine
- Detect with 2,4-dinitrophenylhydrazine
An isoleucine-, leucine- and valine-free unflavored powder
detection

53. 6. Inborn Errors in Metabolism
Phenylketonuria
Absence of phenylalanine hydroxylase activity
- Phenylalanine is not converted to tyrosine
- Forms phenylpyruvate
- Initially described in 1934
- Observed by reacting phenylpyruvate with FeCl(3) (turns olive green)
- Leads to severe retardation

54. 6. Inborn Errors in Metabolism