1 / 101

December 7-8, 2011 AMINO ACID METABOLISM I,II,III Lecturer: Eileen M. Lafer

December 7-8, 2011 AMINO ACID METABOLISM I,II,III Lecturer: Eileen M. Lafer Reading: Stryer Edition 6: Chapters 23 and 24 OBJECTIVES: 1. Understand the fates and sources of the amino acids in general terms.

senwe
Download Presentation

December 7-8, 2011 AMINO ACID METABOLISM I,II,III Lecturer: Eileen M. Lafer

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. December 7-8, 2011 AMINO ACID METABOLISM I,II,III Lecturer: Eileen M. Lafer Reading: Stryer Edition 6: Chapters 23 and 24 OBJECTIVES: 1. Understand the fates and sources of the amino acids in general terms. 2. Understand the general features of lysosomal protein degradation, including what types of proteins are degraded in lysosomes. 3. Understand how "controlled proteolysis" is effected in cells. This includes an understanding of the mechanism of selection and attachment of ubiquitin to target proteins, as well as where and how ubiquitinated proteins are degraded. 4. Understand the common pathways for the removal of the a-amino group from an a-amino acid during amino acid catabolism. 5. Understand how the fate of the ammonium ions generated during amino acid degradation differs in the liver versus the peripheral tissues.

  2. 6. Understand the fundamentals of the urea cycle and how it is regulated. Understand how defective urea cycle enzymes can lead to disease, and how those diseases are treated. 7. Understand which a-amino acid carbon skeletons feed into which major metabolic intermediates during amino acid catabolism. 8. Know which amino acids are solely ketogenic, solely glucogenic, and both ketogenic and glucogenic. 9. Know which steps in amino acid degradation lead to the following diseases: methyl-malonic acedemia, homocystinuria, maple syrup disease, phenylketonuria, tyrosinemia I, II and III, and alkaptonuria. For each disease, you should know the name of the defective enzyme, the reaction catalyzed by the enzyme, and the pathway in which the enzyme functions. 10. Know which amino acids are essential and which are non-essential in humans. 11. Know in humans, which major metabolic intermediates are able to serve as carbon skeletons for the biosynthesis of which amino acids. 12. Know which amino acids can act as neurotransmitters. 13. Know which amino acids can be used for the synthesis of which neurotransmitters. 14. Understand in general terms the biosynthesis of spermine, spermidine, creatine and phosphocreatine.

  3. Reminder: to download movies used in my lectures go to my web page: http://www.biochem.uthscsa.edu/~lafer/ (aleternativly go to uthscsa biochem dept., click on faculty, etc. to find above link) Click on links, for a mirror of the ppt files on blackboard, as well as the movie files that are not permitted on blackboard due to size limitations.

  4. SOURCES AND FATES OF AMINO ACIDS IN THE BODY

  5. PROTEIN CATABOLISM Proteolysis of dietary proteins in the stomach and lumen of the small intestine releases free amino acids into the bloodstream. Proteolysis of proteins that move through the endocytic pathway takes place in the lysosomes of all cells. Controlledproteolysis of ubiquitin-tagged intracellular proteins takes place in the proteasomes of all cells. AMINO ACID POOL

  6. LYSOSOMAL DEGRADATION 1. Lysosomes degrade proteins taken up by endocytosis, or proteins that traffic within the endocytic pathway. 2. Lysosomes contain ~50 hydrolytic enzymes (proteases). Their pH optima is acidic. 3. The pH of the lysosome is ~5. 4. In well-nourished cells, lysosomal protein degradation is non-selective. 5. In starving cells, there is a selective pathway that preferentially degrades cytosolic proteins containing the pentapeptide KFERQ (Lys-Phe-Glu-Arg-Gln).

  7. CONTROLLED PROTEOLYSIS 1. Ubiquitin tags proteins for destruction. 2. The proteasome digests the ubiquitin tagged proteins. 3. Protein degradation can be used to regulate biological function.

  8. UBIQUITIN 76 aa polypeptide. C-terminal gly attaches to the e-amino groups of several lys on a protein destined for degradation. Additional ubiquitin molecules can be added to Lys48. The Mark of Death

  9. UBIQUITIN CONJUGATION E1=Ubiquitin-Activating Enzyme E2=Ubiquitin-Conjugating Enzyme E3=Ubiquitin-Protein Ligase

  10. UBIQUITIN IS ATTACHED TO THE e-AMINO GROUP OF LYSINE RESIDUES ON TARGET PROTEINS

  11. CLINICAL CORRELATION: Human papilloma virus (HPV) encodes a protein that activates a specific E3 enzyme. The enzyme ubiquitinates the tumor suppressor p53 and other proteins that control DNA repair, which are then destroyed. The activation of this E3 enzyme is observed in more than 90% of all cervical carcinomas.

  12. A SINGLE UBIQUITIN MOLECULE IS A POORSIGNAL FOR DEGRADATION. CHAINS OF 4 OR MORE UBIQUITIN MOLECULES ARE VERY STRONG SIGNALS FOR DEGRADATION.

  13. WHAT DETERMINES WHETHER A PROTEIN IS UBIQUITINATED? • The substrate specificity of each E3. • The N-terminal rule: the chemical nature of the amino-terminal amino acid. • For example, a protein with methionine at it s N terminus has a half life of 20 hours, while a protein with an arginine at its N-terminus has a half life of 2 minutes. • 2. Cyclin destructive boxes: specific amino acid sequences that mark cell-cycle proteins for destruction. • 3. PEST sequences: proteins rich in proline, glutamic acid, serine and threonine.

  14. THE 26S PROTEASOME DIGESTS THE UBIQUITIN TAGGED PROTEINS 19S regulatory subunit 20S proteasome (catalytic activity) 19S regulatory subunit The Executioner

  15. THE 20S PROTEASOME 1. 700kD, 28 homologous subunits: 14 of type a and 14 of type b. 2. Subunits are arranged in 4 rings of 7 subunits each to form a sealed barrel. 7 7 7 7

  16. 7 7 7 7 PROTEOLYTIC ACTIVITY RESIDES IN THE N-TERMINALTHREONINE RESIDUES OF THE BETA SUBUNITS

  17. ACCESS TO THE 20S PROTEASOME IS CONTROLLED BY THE 19S CAPS The 19S regulatory subunits bind to polyubiquitin chains.

  18. SOURCES AND FATES OF AMINO ACIDS IN THE BODY

  19. PROTEIN DEGRADATION CAN REGULATE BIOLOGICAL PROCESSES Dynamically alter the stablity of regulatory proteins.

  20. SOURCES AND FATES OF AMINO ACIDS IN THE BODY

  21. AMINO ACID DEGRADATION 1. Any amino acids generated by protein catabolism that are not needed as building blocks for new biomolecular synthetic reactions are degraded to carbon skeletons in the liver. 2. The first step in amino acid degradation is the removal of nitrogen.

  22. a-AMINO GROUPS ARE CONVERTED INTO AMMONIUM IONS BY OXIDATIVE DEAMINATION OF GLUTAMATE The a-amino group of the a-amino acid is transferred to a-ketoglutarate to form glutamate, which is oxidatively deaminated to yield ammonium ion.

  23. 1. THE TRANSAMINATION REACTION: Aminotransferases (also called transaminases) catalyze the transfer of an a-aminogroup from an a-amino acid to an a-keto acid. These enzymes generally utilize a-ketoglutarate as the acceptor. The enzymes are named after their amino acid substrates, i.e. aspartate transaminase catalyzes the transfer of the a-amino group of aspartate to a-ketoglutarate, yielding oxaloacetate plus glutamate.

  24. 2. THE OXIDATIVE DEAMINATION REACTION:The nitrogen atom that is transferred to a-ketoglutarate in the transamination reaction is converted into free ammonium ion by oxidative deamination. This reaction is catalyzed by glutamate dehydrogenase. This reaction takes place in the mitochondria, and is driven by the consumption of ammonia. Dehydrogenation Hydrolysis

  25. aminotransferase dehydrogenase urea cycle excreted The sum of the aminotransferase and glutamate dehydrogenase reactions yield ammonium ion: AMINO ACID 1 ALPHA KETOACID 2 ALPHA KETOACID 1 AMINO ACID 2

  26. ALL AMINOTRANSFERASES CONTAIN THE PROSTHETIC GROUP PYRIDOXAL PHOSPHATE (PLP) PLP is derived from Pyridoxine (Vitamin B6)

  27. The phenolic hydroxyl group is slightly acidic, favoring deprotonation. PLP TAUTOMERS: PHENOLATE The pyridine ring is slightly basic, which favors protonation of the pyrimidine N.

  28. PLP FORMS SCHIFF BASE INTERMEDIATES IN AMINOTRANSFERASES AMINO ACID 1 (schiff-base linkage with the enzyme) (schiff-base linkage with the substrate) The positively charged schiff-base linkages are stabilized by the negatively charged phenolate group.

  29. TRANSAMINATION MECHANISM 1. The schiff base loses a proton from the a-carbon of the amino acid to become a quinonoid intermediate. 2. Reprontonation of the quinonoid at the aldehyde carbon yields a ketimine intermediate. 3. The ketimine is then hydrolyzed to an a-ketoacid and PMP. ALPHA KETOACID 1 1 2 3

  30. ONCE THE AMINO GROUP HAS BEEN TRANSFERRED TO PMP, PMP TRANSFERS THE AMINO GROUP TO ANOTHER ALPHA-KETOACID BY REVERSING THE REACTION SCHEME WE JUST DISCUSSED (FOLLOW THE RED ARROWS): ALPHA KETOACID 2 (ALPHA KETOGLUTARATE) 3 2 1 4 AMINO ACID 2 (GLUTAMATE)

  31. aminotransferase dehydrogenase urea cycle excreted The sum of the aminotransferase and glutamate dehydrogenase reactions yield ammonium ion: AMINO ACID 1 ALPHA KETOACID 2 ALPHA KETOACID 1 AMINO ACID 2

  32. ASPARTATE AMINOTRANSFERASE Active site Arg386 helps orient substrates by binding to their a-carboxylate groups. PLP is bound to active site Lys268 by a Schiff-base linkage.

  33. MECHANISM OF THE AMINOTRANSFERASE REACTION: MOVIE: Movie file 18-01.avi

  34. aminotransferase dehydrogenase urea cycle excreted The sum of the aminotransferase and glutamate dehydrogenase reactions yield ammonium ion: AMINO ACID 1 ALPHA KETOACID 2 ALPHA KETOACID 1 AMINO ACID 2

  35. End of First Lecture

  36. SERINE AND THREONINE CAN BE DIRECTLY DEAMINATED 1. The nitrogen atoms of MOST amino acids are transferred to a-ketoglutarate. 2. The a-amino groups of serine and threonine can be directly converted into ammonium ion by the action of dehydratases. Threonine a-ketobutyrate + NH4+

  37. PERIPHERAL TISSUES TRANSPORT NITROGEN TO THE LIVER BY THE ALANINE CYCLE OR AS GLUTAMINE If amino acids are produced in tissues that lack the urea cycle, they need a mechanism to release nitrogen in a form that can be absorbed by the liver and converted into urea. EXAMPLE: Muscle uses amino acids as fuel during prolonged exercise and fasting.

  38. THE ALANINE CYCLE 1. In peripheral tissues,the a-amino groups of the amino acids are transferred to glutamate by a transamination reaction, as in the liver. 2. However, rather than oxidatively deaminating glutamate to form ammonium ion, the a-amino group is transferred to pyruvate to form alanine. 3. The liver takes up the alanine, and converts it back to pyruvate by another transamination reaction. 4. The pyruvate can be used for gluconeogenesis, and the amino group eventually ends up as urea by the usual pathway. a-ketoglutarate a-ketoglutarate glutamate pyruvate

  39. NITROGEN CAN ALSO BE TRANSPORTED AS GLUTAMINE Glutamine Synthetase: NH4+ + glutamate + ATP glutamine + ADP + Pi Once glutamine is in the liver, it can be metabolized like any other amino acid and the nitrogen can end up in the urea cycle.

  40. SOURCES AND FATES OF AMINO ACIDS IN THE BODY

  41. IN LIVER THE AMMONIUM IONS GENERATED DURING AMINO ACID DEGRADATION FEED INTO THE UREA CYCLE

  42. Urea cycle: importance • NH4+ is a product of the breakdown of amino acids. • NH4+ is required by cells for synthesis of nitrogen-containing compounds. • Excess NH4+ is very toxic. Normal levels in human blood are: [NH4+] < 70 M. • Excess NH4+ is converted to urea via the urea cycle and excreted. The urea cycle accounts of ~80% of the excreted nitrogen.

  43. Urea cycle: location and source of atoms • Urea synthesis takes place mostly in the liver. • One N atom of urea comes from Asp (blue). • One N atom comes from NH4+ (green). • One C atom comes from CO2 (red). • Ornithine acts as a carrier of various atoms in the process of synthesizing urea.

  44. Urea cycle reactions: carbamoyl phosphate synthetase • Catalyzes formation of carbamoyl phosphate from H2O, 2 ATPs, CO2 and NH3. • The positive heterotropic activator, N-acetylglutamate, is required for activity. • Brings one C atom and one N atom into the urea cycle as a carbamoyl group. • Catalyzes the critical step in removing NH4+ from the blood.

  45. Urea cycle reactions: carbamoyl phosphate synthetase • The reaction is made irreversible by cleaving two ATP molecules to two ADP. • One molecule of phosphate is released while the second phosphate ends up as part of carbamoyl phosphate.

  46. Urea cycle reactions: carbamoyl phosphate synthetase • Carbamoyl phosphate synthetase is present at very high concentration in the mitochondrial matrix (~1 mM). • The high enzyme concentration allows the enzyme to work well below the Km ~ 250 M for NH4+. • By operating well below Km, a small increase in NH4+ leads to a large increase in the rate of removal of NH4+ insuring that NH4+ remains low.

  47. Urea cycle reactions: ornithine transcarbamoylase • Catalyzes the formation of citrulline and Pi from ornithine and carbamoyl phosphate. • Transfer of a carbamoyl group to ornithine is facilitated by rupture of a high energy phosphoanhydride bond. • Catalyzes introduction of one C atom and one N atom into the urea cycle from carbamoyl phosphate.

  48. Urea cycle reactions: argininosuccinate synthetase • Catalyzes condensation of citrulline and aspartate to form argininosuccinate. • Catalyzes the introduction of one N atom into the urea cycle from aspartate.

  49. Urea cycle reactions: argininosuccinase • Cleaves argininosuccinate to arginine and fumarate. • Completes the transfer of the amino group from aspartate to make arginine. • Retains the carbon skeleton of aspartate (as a fumarate molecule).

  50. Urea cycle reactions: arginase • Catalyzes hydrolysis of arginine to ornithine and urea. • Ornithine “cycles” back to the first step and picks up another carbamoyl group from carbamoyl phosphate.

More Related