1. Dr Vivek Joshi, MD

2.  Amino acid pool  Protein Degradation  Amino acid degradation  Disposal of Body Nitrogen Transamination Deamination Transport of ammonia

3.  Amino Acid pool  Amino acids-Not stored in the body.  Maintain a supply of amino acids-Amino acid pool

4.  Affords metabolic flexibility  Protects cells from the accumulation of abnormal proteins  Plays an important role in numerous physiological processes e.g. Eukaryotic cell cycle control/antigen presentation  Measured in half-lives  Structural proteins (Collagen)typically have long half-lives  Regulatory enzymes have half-lives that are typically measured in minutes  Proteins with Ser as N-terminal aa -Half life >20 h  Proteins with shorter half life: Asp as N-terminal aa Rich in proline (P), glutamate (E),serine (S), and threonine (T)

5.  Enzyme systems for degradation of proteins  Energy dependent ubiquitin- proteasome pathway  Cytosolic pathway for degradation of damaged /unneeded proteins  Important in degrading proteins of intracellular origin  Non energy dependent degrading enzymes of lysosmes.  Way by which extracellular and some intracellular proteins are degraded

6.  Present in all eukaryotic cells  Cytosolic proteins destined for degradation are enzymatically tagged with activated ubiquitin  Ubiquitin-tagged proteins are then attacked by cytosolic ATP- dependent proteases Ubiquitin - Proteasomes pathway

7.  Regardless of the source,aa not immediately incorporated into a new protein are rapidly degraded.  AA catabolism involves: Removal of the  amino group as ammonia Conversion of the ammonia into urea Conversion of remaining AA carbon skeleton (  keto acid) into TCA cycle intermediates  Disposal of Body Nitrogen Transamination Deamination Transport of Ammonia Amino acid Degradation

8.  Most common reaction involving free amino acids  Reversible reaction  Transfer of amino group from an amino acid to a keto acid to form a newer keto acid and a newer amino acid respectively.  No release of ammonia  Major process for removing nitrogen from amino acids with EXCEPTION- Lysine,Threonine,proline  Require Pyridoxal phosphate as an essential coenzyme

9. Transamination- Introduction

10. Transamination- General Reaction

11.  Catalyses Transamination reaction  Located in the Cytosol and mitochondria of liver, muscle  Each Aminotransaminases is specific for 1 pair of substrate but non-specific for the other pair  Aminotransaminases are named after the specific amino group donor  Major Aminotransaminases #Aminotransaminases family Conserve amino group of most of the amino acids into Glutamate #Alanine Aminotransaminases(SGPT)- Alanine /Pyruvate conversion #Aspartate Aminotransaminases(SGOT)- Aspartate/Oxaloacetate conversion Amino transferase/ Transaminase

12.  Glutamate All the amino nitrogen from amino acids that undergo transamination can be concentrated in glutamate The acceptor of the amino group is almost always  -keto glutarate Amino Transaminase Family Family

13.  Alanine-The principal amino acid released from muscle during starvation  Pyruvate -Form an important substrate for hepatic Gluconeogenesis. Alanine Amino Transaminase

14.  Aspartate which is a source of nitrogen in the UREA cycle.  Oxaloacetate can form Glucose via Gluconeogenesis Aspartate Aminotransaminases

15. Aminotransaminases- Intracellular enzymes with low levels in plasma Elevated plasma amino transaminase- Cell Damage - Diagnostic purpose Non hepatic diseases Myocardial infarction and muscle disorders Serum AST more specific than ALT Hepatic diseases Serum ALT more specific than serum AST Serum AST more sensitive than serum ALT

17. Amino AcidKeto Acid  ketoglutarate Pyruvate Oxaloacetate Glutamate Alanine Aspartate Transamination-Pair

18. Transamination- Pyridoxal phosphate (PLP)

19.  Removal of amino group as AMMONIA  Types Oxidative Deamination  Oxidase- L. AA oxidase / D-AA oxidase  Dehydrogenase –Glutamate Dehydrogenase Non Oxidative Deamination  Hydroxy and Sulfur containing AA  Catalysed by Dehydratase and Desulfurase  PLP dependent Deamination

20. In mammals: almost entirely confined to the liver mitochondrial matrix / Readily reversible Glutamate –The only amino acid that undergoes rapid oxidative deamination

21.  ADP and GDP Indicative of low cellular energy level Stimulates glutamate degradation  ATP and GTP Indicative of ample energy supply Allosteric activator in the direction of glutamate synthesis  Transamination is followed by Deamination: Transdeamination Oxidative Deamination by Glutamate Dehydrogenase Amino Transaminase Glutamate Dehydrogenase

24.  Nitrogen travels in blood mainly in amino acids, particularly alanine and glutamine.  Alanine and glutamine are synthesized in the peripheral tissues to act as nitrogen carrier  Glutamine Transport of ammonia from peripheral tissue to liver  Alanine Transport of ammonia from the muscle to the liver

25. Glutamine :Transport of ammonia from peripheral tissue to liver  Glutamine Provides a non-toxic transport form of ammonia Transport form of Ammonia from peripheral tissue to the liver Synthesized by Glutamine synthase -Liver, Muscle and Brain Most common free amino acid in human blood plasma. Major mechanism for detoxification of ammonia in the brain. 50% of circulating amino acid molecules are glutamine, an ammonia transporter

26. Most of the tissues NH 3 TRANSAMINATION GLUTAMATE GLUTAMINE Liver,Kidney and Intestine GLUTAMINE GLUTAMATE Glutaminase Glutamine Synthase Glutamine to Glutamate :Liver, Intestine and Kidney NH 3 Glutamine :Transport of ammonia from peripheral tissue to liver

27. Glutamine to Glutamate :Liver

29. Most of the tissues NH 3 TRANSAMINATION GLUTAMATE GLUTAMINE Intestine GLUTAMINE NH 3 GLUTAMATE Glutaminase Glutamine Synthase LIVER Another Source of NH 3 in the intestine :Bacterial flora Glutamine to Glutamate :Intestine Portal Blood

30.  Glutamine is removed from circulation by the kidneys  Glutamine converted into Glutamate by Glutaminase releasing ammonia  Most of the ammonia is excreted in the urine as NH4+  An important mechanism for maintaining the body’s acid-base balance Glutaminase Glutamate Dehydrogenase Glutamine to Glutamate :Kidney

31. Alanine :Transport of ammonia from muscle to liver

32. Glucose –Alanine cycle Alanine :Transport of ammonia from muscle to liver

34.  Major end-product of nitrogen catabolism in humans  Principal Non protein nitrogenous waste products (Uric acid and Creatinine )  Synthesis- liver (Cytosol and mitochondria)  Released in the blood-Cleared by the kidneys  Urea Clearance-Measure of Glomerular filtration rate(GFR)

35.  First metabolic pathway to be discovered  Transdeamination of the AA results in release of NH 4 + in the liver  Ammonia is toxic and is converted into non toxic,water soluble product –UREA.  Mammals are primarily Ureotelic  Birds and reptiles are Uricotelic

36. U R E A C Y C L E

38.  Urea synthesis begins with reaction of ammonia with C0 2 (Bicarbonate) and ATP to give Carbamoyl phosphate  Reaction catalyzed by Carbamoyl phosphate synthetase I  Reaction requires Mg 2+  Carbamoyl phosphate synthetase II-Pyrimidine synthesis Urea Cycle

39. Carbamoyl phosphate synthetase-I  Catalyzes the rate-limiting step in urea cycle  Active only in the presence of the allosteric activator N- acetylglutamate(NAG)  N-Acetyl Glutamate is synthesized from Acetyl CoA and Glutamate by N-Acetyl Glutamate synthase  Binding of NAG to CPS I induces a conformational change that enhances the affinity of the synthase for ATP  Intrahepatic concentration of NAG increases after a protein rich meal –Stimulates UREA synthesis. Know CPS –I …….

41.  Carbamoyl phosphate combines with ornithine to form Citrulline –Ornithine trans Carbomylase(OTC)  Citrulline combines with Aspartate to form Arginosuccinate-Arginosuccinate synthase  Arginosuccinase splits Arginosuccinate into Arginine and Fumarate  Arginase split Arginine into Urea and Ornithine

42. Fate of UREA  Renal failure patients- Urease acts on urea-Important source of NH 3  Oral neomycin administration-Reduces intestinal Bacteria-Decreased NH 3

43.  Overall Reaction: Aspartate+NH 3 +CO 2 +3 ATP Urea+Fumarate +2 ADP +AMP +2Pi +2 PPi +3 H 2 O  4 High energy phosphates –Synthesis of each molecule of urea  Source of one nitrogen of urea-Free ammonia  Source of second nitrogen of urea-Aspartate  In effect, both nitrogen atoms of urea come from glutamate, which in turn gathers nitrogen from other amino acids.

44.  Two nitrogen of the Urea comes from # Ammonia # Aspartate Summary-UREA cycle

45.  Constantly produced in the tissues  Toxic to the central nervous system even in trace amounts  Amino acids are quantitatively the most important source of ammonia  Most of the ammonia generated in amino acid degradation is produced by the oxidative deamination of glutamate

46. L –AA Oxidase GLUTAMATE DEHYDROGENASE Non –oxidative Deamination Purines/Pyrimidines Ammonia pool Glutaminase Asparagine Glutamine UREA Bacterial Urease(25%) Amines/Monoamines Urea – formation is quantitatively the most important disposal route for ammonia

47.  Normal Ammonia level -5–50  mol/L  Ammonia is normally detoxified into Urea in the liver  Liver functions compromised- Hyperammonemia (1000  mol/L)  Hyperammonemia-Medical emergency

48.  Ammonia-Directly Neurotoxic- Tremors,slurring of speech and blurring of vision Coma and death at high levels  Brain is particularly vulnerable -Depends on the CAC to maintain its high rate of energy production  Shift in the equilibrium of the glutamate dehydrogenase reaction toward the direction of glutamate formation  Depletes  -ketoglutarate, an essential intermediate in CAC  Results in a decrease in cellular ATP production Hyperammonemia

49. Cirrhosis of the liver Formation of collateral circulation around the liver Portal blood is shunted directly to the systemic circulation (no access to the liver) Severely impaired detoxification of ammonia  Levels of circulating ammonia  Major types are:  Acquired hyperammonemia # Liver diseases # Acute- Viral hepatitis,ischemia, hepatotoxins # Chronic- Cirrhosis of liver Alcoholism,Hepatitis,Biliary obstruction (Porto systemic shunting) Hyperammonemia

50.  Hereditary Hyperammonemia # Inherited deficiency of the enzymes of Urea cycle # Failure to synthesize urea leads to hyperammonemia during the first week following birth # Hyperammonemia Type I- Deficiency of CPS-I # Hyperammonemia Type II -Deficiency of OTC - 1;30,000 live births - Most common of the inherited urea cycle disorders - X-linked (Predominantly affecting males) - All others urea cycle disorders are AR - Presents typically with mental retardation,few weeks after birth #Treatment - Limiting protein in diet - Orally Phenyl butyrate -Converted to phenyl acetate- phenylacetylglutamine -Excreted - Gene Therapy

51. Hyperammonemia IHyperammonemia II CPS –I DeficiencyOTC Deficiency Blood Glutamine increased BUN Decreased No increase in Orotic acid and uracil increased in blood Orotic acid and uracil increased in blood Cerebral edema Lethargy,convulsions, coma,death

52. UCDEnzyme DeficiencySymptoms/Comments Type I Hyperammonemia Carbamoylphosphate synthetase I With 24h - 72h after birth infant becomes lethargic, needs stimulation to feed, vomiting, increasing lethargy, hypothermia and hyperventilation; without measurement of serum ammonia levels and appropriate intervention infant will die: treament with arginine which activates N-acetylglutamate synthetase N-acetylglutamate synthetase Deficiency N-acetylglutamate synthetase severe hyperammonemia, mild hyperammonemia associated with deep coma, acidosis, recurrent diarrhea, ataxia, hypoglycemia, hyperornithinemia: treatment includes administration of carbamoyl glutamate to activate CPS I Type 2 Hyperammonemia Ornithine transcarbamoylase Most commonly occurring UCD, only X-linked UCD, ammonia and amino acids elevated in serum, increased serum orotic acid due to mitochondrial carbamoylphosphate entering cytosol and being incorporated into pyrimidine nucleotides which leads to excess production and consequently excess catabolic products: treat with high carbohydrate, low protein diet, ammonia detoxification with sodium phenylacetate or sodium benzoate Classic Citrullinemia Argininosuccinate synthetase episodic hyperammonemia, vomiting, lethargy, ataxia, seizures, eventual coma: treat with arginine administration to enhance citrulline excretion, also with sodium benzoate for ammonia detoxification Argininosuccinic aciduria Argininosuccinate lyase (Argininosuccinase) episodic symptoms similar to classic citrullinemia, elevated plasma and cerebral spinal fluid argininosuccinate: treat with arginine and sodium benzoate HyperargininemiaArginase rare UCD, progressive spastic quadriplegia and mental retardation, ammonia and arginine high in cerebral spinal fluid and serum, arginine, lysine and ornithine high in urine: treatment includes diet of essential amino acids excluding arginine, low protein diet