Protein & Amino acid Metabolism

1. Protein & Amino acid Metabolism

2. Protein and Amino acid Metabolism Breakdown of tissue proteins and amino acid pool, General Reactions of Amino acids. Disposal of Ammonia: urea cycle, glutamate and glutamine formation. Metabolism of Amino acids,- Glycine, serine

3. Introduction Overview of Amino Acid Metabolism Nitrogen Balance and amino acid pool Protein Turnover Metabolism of Amino Nitrogen Metabolism of Individual Amino Acids – Glycine and serine

4. Proteins are linear hetero polymers of α – L – Amino acids, which are linked by peptide bonds. Nitrogen (N) is characteristic of proteins. Introduction Amino acids are not stored by the body. Hence, they must be obtained from the diet, synthesized de novo, or produced from normal protein degradation. Any amino acids in excess of the biosynthetic needs of the cell are rapidly degraded.

5. Biological importance: 1.Proteins contain nitrogen and they are main source of nitrogen for the body. Dietary Proteins are the sources of essential amino acids for the body. 2.All amino acids are required for the synthesis of proteins and many amino acids serve as precursors for the synthesis of biologically important compounds (Eg: Melanin, serotonin, creatine etc.)

6. Medical importance: 1.Genetic defects in the pathways of amino acid metabolism can cause serious disease. Eg: Albinism, Phenlyketonuria, Alkaptonuria etc.   2. Dietary deficiancy of proteins can result in disease such as P.E.M (protein energy malnutrition)

7. NITROGEN BALANCE Nitrogen balance = Difference between total nitrogen intake and total nitrogen loss from the body. The normal adult is in nitrogen equilibrium, nitrogen intake = nitrogen output.

8. Amino acid catabolism- phases • The first phase of catabolism involves the removal of the α – amino groups (usually by transamination and subsequent deamination) ammonia + corresponding α – Keto acid. Converted to UREA Enters 2nd phase and excreted. (most important route for disposing of nitrogen from the body.)

9. Amino acid catabolism- phases 2. 2nd phase of amino acid catabolism the carbon skeleton of the α – Ketoacids via intermediates of energy producing, metabolic pathways CO2 + H2O, glucose, fatty acids, or ketone bodies • Non essential amino acids are synthesized from the intermediates of metabolism or from essential amino acids

10. Amino acid pool Amino acids released by hydrolysis of dietary or tissue protein or synthesized de novo, and are distributed throughout the body. Collectively, they constitute the amino acid pool.

11. BODY PROTEIN DIATARY PROTEIN Digestion and absorption catabolism synthesis Synthesis of new amino acids AMINO ACID POOL SYNTHESIS OF BIOLOGICALLY IMP. COMPOUNDS CATABOLISM

12. PROTEIN TURNOVER: • the continuous degradation and resynthesis of all cellular proteins • Each day about 1–2% of the total body proteins, principally muscle protein, undergoes turnover. Body proteins Reutilization for new protein synthesis degradation Amino acids Catabolism

13. Metabolism of Amino Nitrogen Overview Transamination Deamination Reactions (Ammonia Formation) • Oxidative deamination • Non-oxidative deamination Ammonia Transport Disposal of Ammonia – Urea cycle.

14. Overview of Metabolism of Amino Nitrogen a Amino acids - Ketoglutarate Transmination Glutamate Keto acids Other Reactions -NH2 Oxidative deamination NH3 CO2 Aspartate -NH2 Urea Urea Cycle H2N-CO-NH2

15. Definition: Transamination is the transfer of the amino group of an amino acid to a keto acid, changing the latter into a new amino acid and the original amino acid into a new keto acid. TRANSAMINATION Transamination reaction is freely reversible and hence involved both in biosynthesis and catabolism of amino acids. Enzyme Involved:“Transaminases” (aminotransferases) – liver, skeletal muscles and heart are particularly rich in transaminases. Cofactor Required:Pyridoxal phosphate (PLP) derived from Vit B6 (pyridoxine).

16. Mechanism: • Pyridoxal phosphate is bound to the transaminase at the catalytic site and during transamination the bound coenzyme serves as a carrier of amino groups. Transamination occurs in 2 stages – 1.Transfer of the amino group of an amino acid to the coenzyme PLP (bound to the enzyme) to form pyridoxamine phosphate and the corresponding -ketoacid. 2.The amino group of pyridoxamine phosphate is then transferred to an -ketoacid to produce a new amino acid and the enzyme with PLP is regenerated.

17. General Reaction: AMINO ACID 1 KETO ACID 1 PLP TRANSAMINASE KETO ACID 2 AMINO ACID2

18. Examples: 1) Alanine Pyruvate ALT PLP a- Ketoglutarate Glutamate 2) Aspartate Oxaloacetate AST PLP a- Keto - glutarate Glutamate

19. Salient features: • All amino acids except lysine, threonine, proline and hydroxyproline undergo transamination. • It is a reversible reaction and can serve in both formation of an amino acid and its catabolism. • For all transaminases, glutamate and -Ketoglutarate are one pair of substrate ( an amino acid and its corresponding keto acid) and differ in the other pair. • The amino acids undergo transamination to finally concentrate nitrogen in glutamate.

20. 1.Diverting excess of amino acids towards catabolism and energy production with simultaneous urea synthesis. Metabolic Functions: 2.Biosynthesis of non-essential amino acids. 3.Producing -keto acids (e.g. oxaloacetate, Pyruvate, -ketoglutarate) for subsequent gluconeogenesis

21. Clinical Aspects: Blood levels of ALT and AST are elevated in liver diseases and AST levels in myocardial infarction. Their estimation is useful in the diagnosis of these conditions. (referEnzymes)

22. Ammonia Formation – Deamination Reactions Ammonia is Produced in the Body by: 1) Cellular Metabolism and 2) In the Intestinal Lumen.

23. 1.Ammonia formation by cellular metabolism • Cells produce ammonia mostly from amino acids by deamination, which may be either • oxidative or • non-oxidative

24. Deamination Reactions(Ammonia formation) • Deamination is removal of amino group from compounds, mostly amino acids, as ammonia (NH3). NH3 +carbon skeleton of amino acid CONVERTED TO UREA (KETOACID)

25. 1.Oxidative deamination a)deamination of glutamate catalyzed by glutamate dehydrogenase. -Most important b)Other Oxidative Deamination Reactions are Mainly Those: -- Catalyzed by Amino Acid Oxidases 2.Non-Oxidative Deamination(less important) Enzymes Involved are: Dehydratases Lyases and Amide Hydrolases Deamination….2types

26. Oxidative Deamination by Glutamate Dehydrogenase (GDH): The removal of the amino group from glutamate to release NH3 and -ketoglutarate coupled with oxidation is known as oxidative deamination • Site: Most active in mitochondria of liver cells, though present in all cells. • Enzyme: Glutamate dehydrogenase (GDH) – a Zn containing mitochondrial enzyme. • Coenzymes: NAD+ or NADP+

27. Oxidative deamination of glutamate…

28. Role of GDH:- • Produces NH3, thus channeling the amino groups of most amino acids for urea synthesis. • Regenerates -ketoglutarate for further collection of amino groups of amino acids by transamination and producing their carbon skeletons. • NADH produced generates ATP in the ETC. • The reverse reaction is required for the biosynthesis of glutamate and in the tissues for fixing ammonia, which is toxic.

29. What Is Transdeamination ?? TISSUES transamination All amino acids Keto acids -KG GLUTAMATE Deamination in liver Carried by blood NH3+-KG Reaches liver UREA

30. Questions: • What are the sources of ammonia in the body? • Explain the biochemical basis: glutamate plays a central role in the catabolism of amino nitrogen of amino acids. • Give 2 examples for each of the following. a)Transaminases b) Reactions forming ammonia • Write the reaction, with cofactors if any, catalyzed by Glutamate dehydrogenase.

31. Oxidative Deamination by Amino Acid Oxidases • Amino Acid Oxidases are: -- Flavoproteins -- Possessing either FMN or FAD Amino Acid FAD/FMN Amino Acid Oxidase FADH2/FMNH2 -Keto Acid + NH3

32. Dehydratase Amino Acid Dehydratases (PLP-dependent) Serine/Threonine Dehydratase PLP NH3 Pyruvate/-Ketobutyrate

33. Amino Acid Lyase Histidine Aspartate Histidase Aspartase NH3 NH3 Urocanate Fumarate

34. 2)NH3 production in intestine Intestinal Lumen -- Another Major Source of Ammonia by the Action of Bacteria on: -- Urea Present in the Intestinal Juice And Dietary Amino Acids. • This Ammonia is Absorbed into Hepatic Vein and Enters Liver Directly.

35. Transport of Ammonia Ammonia is toxic to tissues, especially to brain (see Ammonia Toxicity). Ammonia that is constantly produced in the tissues is transported to liver for detoxification by urea synthesis. Ammonia is transported in blood as 1) free NH3, as 2) glutamate or as 3) glutamine.

36. Transport of Ammonia… • NH3is transported in 3 forms. 1) As free NH3Ammonia, whose blood level is 10 to 80  gm/dl, is rapidly removed from the circulation by the liver and converted to urea. 2) asglutamateInside the cells of almost all tissues ammonia combines with -Ketoglutarate to form glutamate by GDH and is transported to the liver.

37. Transport of Ammonia… 3) as glutamine.Ammonia is also trapped by glutamate in the tissues, especially in the brain, to form glutamine, which is catalyzed by glutamine synthetase NH3 Glutamine synthetase Glutamate glutamine .Mg2+ ATP ADP+Pi Transported to liver via blood

38. This reaction may be considered as the first line of detoxification of NH3 in the brain. Glutamine is then transported through circulation (highest blood level among all amino acids) to liver In liver, this reaction is reversed to release NH3.

39. In the liver.. Glutaminase Glutamine glutamate NH3 H2O UREA

40. UREA CYCLE (Detoxification of Ammonia) Contents: • Synonyms • Site • Sources of Atoms of Urea • Reactions • Functions • Ammonia Toxicity – Hyperammonemia

41. UREA CYCLE .(Detoxification of Ammonia) • Ammonia is Toxic to the Body. • Hence it is Necessary that the NH3 Produced During Metabolism of Amino Acids be Removed Immediately. • This is Done by Conversion of Toxic NH3 into Harmless Water-soluble Urea in the Liver by Urea Cycle.

42. UREA CYCLE (Detoxification of Ammonia) • Synonyms: Urea Cycle Ornithine Cycle Krebs-Henseleit Cycle • Site: Urea Synthesized in Liver Carried by Blood And Excreted by Kidneys

43. Sources of Atoms of Urea O || NH2 C  NH2 NH3 CO2 Aspartate

44. UREA CYCLE (Detoxification of Ammonia) • Urea Synthesis: -- A 5-step Cyclic Process • Enzymes of the First 2 Steps: -- Present in Mitochondria • While the Rest: -- Located in the Cytosol

45. Reactions of Urea Cycle CO2 + NH3 + 2 ATP Carbamoyl Phosphate Synthetase–I(CPS-I) Carbamoyl Phosphate + 2 ADP + Pi Urea Ornithine Arginase Arginine Ornithine Transcarbamoylase Fumarate Arginosuccinase Citruline Arginosuccinate Aspartate Arginosuccinate Synthetase ATP AMP + PPi TCA cycle

46. Functions of Urea Cycle 1.Detoxification of NH3 2.Biosynthesis of Arginine.

47. Ammonia Toxicity – Hyperammonemia • Ammonia Concentration Rises in the Blood (Hyperammonemia) and in other Tissues in: -- Liver Failure and -- Inborn Errors of Urea Synthesis (that is, due to Genetic Defect) • This Produces Ammonia Toxicity in Many Ways.

48. Causes Of Hyperammonemia • Causes may be 1.Acquired or 2.Inherited 1.Acquired Causes • Liver Diseases (e.g. Cirrhosis and Severe Hepatitis) -- Liver is Unable to Convert Ammonia into Urea • -- Blood Ammonia Level Rises.

49. 2.InheritedCauses -- Defects Associated with each of the Enzymes of Urea Cycle Exist. -- The Levels Substrate of the Defective Enzyme Rises in the Cells. -- This Causes Product Inhibition of the Enzyme Catalyzing the Earlier Step. -- Leading to Accumulation Ultimately of the StartingSubstrate, Namely, NH3

50. Inherited Causes of Hyperammonemia Disease Enzyme involved Hyperammonemia Type-ICPS-I Hyperammonemia Type –IIOrnithine Transcarbamoylase CitrullinemiaArgininosuccinate Synthetase Argininosuccinic AciduriaArgininosuccinase HyperarginemiaArginase