Chapter 18 Amino Acid Oxidation and The Production of Urea

1. Chapter 18 Amino Acid Oxidation and The Production of Urea

2. Amino Acid Oxidation • Dependency of amino acid as energy source • Carnivores> herbivores> microorganism >> plant • Amino acid degradation in animals • Amino acids for oxidation • Extra amino acid during protein turnover • Protein-rich diet (no storage) • During starvation or in uncontrolled diabetes • Removal of amino group (NH4+) •  a-keto acid (C skeleton of amino acids) •  Oxidation to CO2 & H2O •  Sources of C3 or C4 units for gluconeogenesis or fuels

3. Pathways of amino acid catabolism

4. 18.1 Metabolic Fates of Amino Groups

5. Amino Group Catabolism

6. Amino Group Catabolism • Amino acid metabolism  amino group (= nitrogen metabolism) • Liver is a major site • Recycle for biosynthetic pathways • Excretion; ammnonia, urea, uric acid • Glutamate & glutamine • General collection point for amino group • NH3 from amino acids + a-ketoglutarate  glutamate into mitochondria,  release of NH4+ • Source of ammonia • Dietary protein (major source) • Muscle & other tissues NH4+ + glutamate  glutamine  mitochondria in hepatocytes NH4+ +pyruvate  alanine  hepatocytes

7. Digestion of Dietary Protein • In stomach • Entry of diet  secretion of gastrin from gastric mucosa secretion of HCl (parietal cells), pepsinogen (chief cells) • Acidic gastric juice (pH 1.0 to 2.5) • Antiseptic & denaturing agent (protein unfolding) • Pepsinogen : zymogen • Conversion to active pepsinby autocatalytic cleavage (at low pH) • Digestion of peptide bonds at Phe, Trp, Tyr  mixture of small peptides • In small intestine • Low pH  secretion of secretin  stimulation of bicarbonate secretion from pancreas  neutralization • Arrival in the upper part of intestine (duodenum)  release of cholecystokinin into blood  stimulation of pancreatic zymogens • Trypsinogen : activated by enteropeptidase • Chymotrypsinogen, procarboxypeptidase A and B : activated by trypsin c.f.) Protection of pancreas from proteolytic digestion • Production of zymogens • Pancreatic trypsin inhibitor • Protein digestion by trypsin, chymotrypsin, carboxypeptidase, aminopeptidase • Uptake of amino acids by the epithelial cells

8. Digestion of Dietary Protein Blood capillaries Liver

9. Transamination • 1st step of amino acid catabolism • Transfer of a-amino group to a-ketoglutarate • Generation of L-glutamate & a-ketoacid • Aminotransferase (transaminase) • Amino acid specificity (named after amino acids) • Reversible reaction ; ∆G’° ≈ 0 kJ/mol • Pyridoxal phosphate (PLP) • Bimolecular Ping-Pong reactions

10. Pyridoxal phosphate (PLP) • Coenzyme form of pyridoxine (vitamin B6) • Intermediate carrier of amino group • Electron sink for carbanion (resonance stabilization) • Transamination • Racemization (L- & D-form interconversion) • Decarboxylation

11. PLP-mediated transamination at a-carbon PLP-mediated transamination: Ping-Pong mechanism amino acid a-ketoglutarate pyridoxal phosphate pyridoxamine phosphate pyridoxal phosphate a-keto acid glutamate

12. PLP-mediated amino acid transformations at a-carbon

13. Oxidative Deamination of Glutamate • Oxidative deamination • Mitochondrial matrix of hepatocytes • Glutamate dehydrogenase • Generation of a-ketoglutarate & ammonia • NAD+ or NADP+ as electron acceptor • Allosteric regulation • By ADP (inhibition) • By GTP (activation) • Transdeamination • Transamination of A.a. + oxidative deamination of Glu • A few amino acids undergoes direct oxidative deamination

14. Glutamine as Ammonia Carrier in the Bloodstream • Ammonia generated in extrahepatic tissues • Glutamine synthetase • Incorporation of ammonia into glutamate  glutamine • Transport of gln to the liver via blood • Higher gln concentration than other amino acids in blood • Glutaminase in the liver, intestine, and kidney • Glutamine  Glutamate + NH4+

15. Alanine Transports Ammonia from Skeletal Muscles to the Liver • Glucose-alanine cycle • In muscle • Glycolysis & degradation of amino acids • Alanine aminotransferase • Transfer amino group of glutamate to pyruvate  alanine + a-ketoglutarate • Transport of alanine to the liver • In the liver • Alanine aminotransferase • Transfer amino group of alanine to a-ketoglutarate  glutamate + pyruvate • Gluconeogenesis • Pyruvate , lactate  glucose • Transport of glucose to muscle

16. Ammonia is toxic to animals. Comatose state of brain (high brain’s water content) 1. NH3: alkalization of cellular fluid 2. a-ketoglutarate, NADH, ATP: citric acid cycle & ATP production 3. glutamate and GABA (g-aminobutyrate): neurotransmitter depletion

17. 18.2 Nitrogen Excretion and the Urea Cycle Produced in liver Blood Kidney  urine

19. Urea Cycle in Mitochondria • Formation of carbamoyl phosphate; preparatory step NH4+ + HCO3- + 2 ATP  carbamoyl phosphate + 2 ADP + Pi Carbamoyl phosphate synthetase I - ATP-dependent reaction • 1st step in the urea cycle; Ornitine + carbamoyl phosphate  citrulline + Pi Ornitine transcarbamoylase

20. Urea Cycle in Cytosol • 2nd step; formation of argininosuccinate Incorporation of the second N from aspartate Argininosuccinate synthetase • ATP requirement • Citrullyl-AMP intermediate • 3rd step; formation of arginine & fumarate Argininosuccinase; only reversible step in the cycle • 4th step; Cleavage of arginine to urea & ornithine Arginase

21. Asparatate-argininosuccinate shunt • Metabolic links between citric acid and urea cycles • In cytosol • Fumarate to malate  citric acid cycle in mitochondria • In mitochondria • OAA + Glu  a-ketoglutarate + Asp  urea cycle in cytosol • Energetic cost • Consumption • 3 ATP for urea cycle • Generation • Malate to OAA • 1 NADH = 2.5 ATP

22. Regulation of the Urea Cycle • Long term regulation • Regulation in gene expression • Starving animals & very-high protein diet • Increase in synthesis of enzymes in urea cycle • Short term regulation • Allosteric regulation of a key enzyme • Carbamoyl phosphate synthetase I • Activation by N-acetylglutamate

23. Treatment of genetic defects in the urea cycle • Genetic defect in the urea cycle •  ammonia accumulation; hyperammonemia • Limiting protein-rich diet is not an option • Administration of aromatic acids; benzoate or phenylbutyrate • Administration of carbamoyl glutamate • Supplement of arginine

25. 18.3 Pathways of Amino Acid Degradation

26. Amino Acid Catabolism • Carbon skeleton of 20 amino acids • Conversion to 6 major products - pyruvate - acetyl-CoA - a-ketoglutarate - succinyl-CoA - fumarate - oxaloacetate

27. Glucogenic or Ketogenic Amino Acids • Ketogenic amino acids • Conversion to acetyl-CoA or acetoacetyl-CoA  ketone bodies in liver • Phe, Tyr, Ile, Leu, Trp, Thr, Lys • Leu : common in protein • Contribution to ketosis under starvation conditions • Glucogenic amino acids • Conversion to pyruvate, a-ketoglutarate, succinyl-CoA, fumarate, and OAA  glucose/glycogen synthesis • Both ketogenic and glucogenic • Phe, Tyr, Ile, Trp, Thr

28. Enzyme cofactors in amino acid catabolism • One-carbon transfer reactions ; common reaction type, involvement of one of 3 cofactors • Biotin ;one-carbon tranfer of most oxidized state, CO2 • Tetrahydrofolate (H4 folate) ;One-carbon transfer of intermediate oxidation states or methyl groups • S-adenosylmethionine ; one-carbon transfer of most reduced state, -CH3

29. Tetrahydrofolate • folate (vitamin) to H4 folate • Dihydrofolate reductase • Primary source of one-carbon unit • Carbon removed in the conversion of Ser to Gly • Oxidation states of H4 folate ; One-carbon groups bonded to N-5 or N-10 or both - Methyl group (most reduced) - Methylene group - Methenyl, formyl, formimino group (most oxidized) • Interconvertible & donors of one-carbon units (except N5-methyl-tetrahydrofolate)

30. S-adenosylmethionine (adoMet) • Cofactor for methyl group transfer • Synthesized from Met and ATP • Methionine adenosyl transferase • Unusual displacement of triphosphate from ATP • Potent alkylating agent • Destabilizing sulfonium ion  inducing nucleophilic attack on methyl group

31. Six amino acids are degraded to pyruvate • Ala, Trp, Cys, Ser, Gly, Thr •  pyruvate  acetyl-CoA  citric acid cycle or gluconeogenesis

32. Interplay of PLP and H4folate in Ser/Gly metabolism

33. 3rd pathway of glycine degradation • - D-amino acid oxidase • detoxification of D-amino acid • high level in kidney • - Oxalate  crystals of calcium oxalate (kidney stones)

34. Seven Amino Acids Are Degraded to Acetyl-CoA • Trp, Lys, Phe, Tyr, Leu, Ileu, Thr  acetoacetyl-CoA  acetyl-CoA

35. Intermediates of Trp catabolism be precusors for other biomolecules

36. Catabolic pathways for Phe & Tyr • Phe & Tyr are precusors • dopamine • norephinephrine, epinephrine • melanin

37. Phenylalanine hydroxylase • Mixed function oxidase • ; Substrate hydroxylation + oxygen reduction to H2O • Tetrahydrobiopterin as a cofactor

38. Example of A.a. metabolism defects; Phe catabolism • Phedegradaion fumarate + acetoacetyl-CoA • Defects in Phe catabolism • Phenylketonuria (PKU) • Genetic defect in Phe hydroxylase or dihydrobiopterin reductase • Elevated levels of Phe & phenylpyruvate • Mental retardation • Alkaptonuria • Genetic defect in homogentisate dioxygenase • Oxidation of accumulated homogentisate • Black urine • Arthritis

39. Genetic defects of A.a. metabolism •  defective neural development & metal retardation

40. Five Amino Acids Are Converted to a-ketoglutarate • Pro, Glu, Gln, Arg, His; amino acids with five C skeleton •  converging to Glu

41. Four Amino Acids Are Converted to Succinyl-CoA • Met, Ileu, Thr, Val converging to propionyl-CoA

42. Branched-Chain Amino Acids Are Not Degraded in the Liver • Leu, Ile, Val • Primarily oxidized as fuels in muscle, adipose, kidney, brain • Branched-chain aminotransferases (not in liver) • Branched-chain a-keto acid dehydrogenase complex

43. Asn and Asp are Degraded to Oxaloacetate