METABOLISM of AMINO ACIDS Dr Shahnaz Khaghani

1. METABOLISM ofAMINO ACIDSDr Shahnaz Khaghani Tehran University of Medical Sciences

2. TRANSAMINATION (Amino nitrogen ) CATABOLISMpyruvateacetyl-CoAacetoacetatesuccinyl-CoA, α-KG GLUTAMATE AMINO ACIDS UREA • Anabolic pathways • Specialized molecules: • creatine, • SAM, cysteine, hormones,NO, • dTMP ABERRANT METABOLISM PKU, MSUD

3. OUTLINE • Incorporation of amino acid nitrogen into urea • A) Transamination • B) Glutamate dehydrogenase generates ammonia • C) Urea synthesis requires ammonia, • bicarbonate, and ATP • 1) hyperammonemia, cause and management • Catabolism (utilization) of amino acid carbon atoms • A) Amino acid carbon atoms pyruvate, thus gluconeogenesis

4. III. Amino acids as substrates in critical anabolic pathways A) Creatine (arginine and glycine) B) Hormones: catecholamines (phenyl- alanine), serotonin (tryptophan), thyroid hormones (phenylalanine) C) “Second messenger,” nitric oxide (NO) (arginine) D) Methyl donor, S-adenosyl-L- methionine or SAM(methionine) E) Cysteine (methionine and serine) F) Methionine resynthesis and DNA synthesis

5. IV. “Inborn errors” of amino acid metabolism A. Phenylketonuria 1) Clinical Manifestations: a) growth failure b) delayed psychomotor development c) seizures d) mental retardation

6. Examples, “inborn errors” of amino acid metabolism • A. PKU • B. MSUD

7. B) Amino acid carbon atoms acetyl-CoA, thus fatty acids and cholesterol C) Amino acid carbon atoms acetoacetate, thus “ketone bodies” D) Amino acid carbon atoms succinyl-CoA, thus TCA cycle E) Amino acid carbon atoms 2-oxoglutarate (α-KG), thus TCA cycle

8. Amino Acid Breakdown: No storage form of Amino Acids, therefore excess need to be converted to other forms to be used as energy or stored as glycogen/fat. R O H2N CH C OH Recycling of the Carbon Skeleton Disposal of Nitrogen Atom (Urea)

9. I. Amino Acid Nitrogen Incorporation into Urea A. TransaminationTransamination requires: 1) An amino acid and an oxo- (keto-) acid 2) Co-factor pyridoxal phosphate 3) An aminotransferase. NH2 O O O | | | | | | | R1CH– COH+ R2 C– C– OH O O NH2 O | | | || | | R1– C – C– O H + R2–CH – C – OH

10. Flow of Nitrogen: In tissues (e.g Muscle), most amino acids transfer their a-amino group to Glutamate Aminotransferase B6 a-ketoglutarate

11. Biosynthesis of Amino Acids: Transaminations Amino Acid1 +a-Keto Acid2 Amino Acid2 +a-Keto Acid1 + Glutamate Pyridoxal phosphate (PLP)- Dependent Aminotransferase + a-Ketoglutarate

12. Transaminations: Role of PLP Tautomerization

13. The N is then transferred from Glutamate to Pyruvate, producing Alanine. a-ketoglutarate Pyruvate Aminotransferase

14. Four of the amino acids, Glycine, Lysine, Threonine and Serine are directly deaminated. Serine Dehydratase

15. NH3 released from Glycine/Lysine/Threonine/Serine is incorporated into Glutamine [ ] Glutamine Synthetase

16. In sum: During amino acid breakdown, the a -amino Nitrogen gets incorporated as the a-amino group in Alanine or the amide group in Glutamine. Alanine and Glutamine are then released to the circulation.

17. Flow of Nitrogen: Alanine and Glutamine released by peripheral tissues are taken up by the Liver. The Nitrogen on Alanine is transferred to a-ketoglutarate to produce Glutamate

18. a-ketoglutarate Glutamate Aminotransferase

19. Glutamate has two fates important for disposal of waste N. Conversion to a-ketoglutarate by Glutamate Dehydrogenase to release NH3 2) As N donor in the transamination of oxaloacetate to Aspartate

20. Conversion to a-ketoglutarate by Glutamate Dehydrogenase to release NH3 [ ] Glutamate dehydrogenase

21. 2) As N donor in the transamination of oxaloacetate to Aspartate Glutamate -ketoglutarate O NH2 O O O O B6 HO-C-CH2-CH-C-OH HO-C-CH2-C-C-OH Aminotransferase Oxaloacetate Aspartate

22. Glutamine is hydrolyzed by Glutaminase to release NH3 Glutaminase NH3

23. Nitrogen flow in Liver Alanine Glutamate Aspartate NH3 Glutamine NH3

24. The oxoacid can be either pyruvate, which produces alanine, oxaloacetate, which produces aspartate 2-oxoglutarate, which producesglutamate

25. Transamination with 2-oxoglutarate yields glutamate. Glutamate yields ammonia. Ammonia enters the urea cycle. NH2 O | | | R– CH– C– O H + 2-OG O NH2 O | | | | | HO– C – CH2 – CH2 –CH – C– OH NH3

26. B. Glutamate dehydrogenase Glutamatetoammonia catalyzed by glutamate dehydrogenase (uses NAD+) O NH2 O | | | | | HO–C– CH2– CH2– CH - C – OH 2-oxoglutarate + NH3

28. C. Urea cycle Rate-determining step of the urea cycle requires ammonia, catalyzed by carbamoyl phosphate synthetase. NH3 + HCO3- + 2 ATP CARBAMOYL PHOSPHATE+ Pi + 2ADP

29. There are two different enzymes with this name. The urea cycle enzyme is carbamoyl phosphate synthetase I or CPS I

30. Carbamoyl phosphate contains nitrogen from ammonia (amino acids), carbon from bicarbonate, and phosphate from ATP. O O │ │ │ │ 2 H N - C - O - P - OH │ OH

31. Next, carbamoyl phosphate reacts with ornithine in a reaction catalyzed by ornithine carbamoyl transferase. Carbamoyl phosphate + ornithine citrulline

33. Citrulline reacts with aspartate to yield argininosuccinate. Second nitrogen of urea from aspartate. citrulline + aspartate argininosuccinate synthetase argininosuccinate + AMP + PPi

35. Argininosuccinate is cleaved by arginino-succinate lyase to yield arginine and fumarate. argininosuccinate arginine + fumarate

37. Final reaction, arginine is cleaved by arginase to yield urea and regenerate ornithine (it’s a cycle!) arginine urea+ ornithine

39. CARBAMOYL PHOSPHATE + UREA Ornithine Citrulline Arginine + UREA CYCLE Aspartate Fumarate Argininosuccinate

40. REGULATION OF THE UREA CYCLE Acute: N-acetylglutamate, allosteric effector, up regulates CPS I

41. N-Acetylglutamate is synthesized from glutamate and acetyl-CoA by a mitochondrial NAG synthase.

42. Metabolic Diseases of the Urea Cycle Arginase Deficiency Argininosuccinic acidemia Type II Hyperammonemia: Type I Citrullinuria

43. 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 deficiency Elevated Carb-P levels in this deficiency cause secondary problems in pyrimidine metabolism

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

45. Excessive ammonia is toxic to the central nervous system. Alternative pathway therapy: Sodium benzoate to produce hippuric acid Sodium phenylacetate or phenyl-butyrate to produce phenylacetylglutamine

46. NH3 + CO2 + 5,10-methylenetetra- hydrofolate NH2O|| |Glycine CH2 – C – OH Sodium Benzoate Bz-N-H O | | | CH2 – C - OH Benzoyl glycine or hippuric acid excreted

47. NH3 + glutamate + ATP → glutamine + ADP + Pi sodium phenylacetate + CoA → phenylacetyl-CoA Then glutamine + phenylacetyl-CoA → phenylacetylglutamine (excreted)

48. sodium phenylbutyrate + CoA → phenylbutyryl-CoA phenylbutyryl-CoA undergoes β-oxidation → phenylacetyl-CoA As above, phenylacetyl-CoA + glutamine→ phenylacetylglutamine (excreted)

49. II. Catabolic Fates of Amino-acid Carbon Atoms Post-transamination carbon atoms become those of pyruvateacetoacetate acetyl-CoA succinyl-CoA 2-oxoglutarate

50. Phenylalanine Methionine (partial) Leucine Tyrosine (50% of carbons) Cysteine Glycine Alanine Serine Aspartate Asparagine Tyrosine (50% of carbons) Phenylalanine Isoleucine (partial) Valine (partial) Methionine(partial) Glutamate Glutamine Proline Acetyl CoA GLUCOSE Pyruvate Oxaloacetate Citrate Malate Isocitrate Fumarate Succinate -Ketoglutarate Succinyl CoA Amino acids discussed in previous lectures Amino acidsto be discussed in this lecture Carbon end products from the degradation (catabolism) of amino acids