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3 M1 Renal: Nitrogen Metabolism (and Related Topics) Amino Acid Metabolism (Nitrogen metabolism) Folate Metabolism ( ne-arbon pathways ) Nucleotide Metabolism Dr. Robert Lyons Assistant Professor, Biological hemistry Director, DNA Sequencing ore Web: Fall 008

4 Supplementary study material on the Web:

5 Amino Acid metabolism Amino acids Folate metabolism Glu, Gln, Asp, N Methylene TF Met ycle Urea oxaloacetate P u rin e s DNA RNA P y rim id in e s TA ycle fumarate Uric Acid (energy) Nucleic Acid metabolism

6 Protein Degradation: Endogenous proteins degrade continuously - Damaged - Mis-folded - Un-needed Dietary protein intake - mostly degraded Nitrogen Balance - expresses the patient s current status - are they gaining or losing net Nitrogen?

7 Transaminases ollect Amines General reaction overview: R 1 N amino acid + R R + R 1!-keto acid (typically alpha-ketoglutarate)!-keto acid N amino acid (typically glutamate) Details of reaction mechanism: R P amino acid N + + N pyridoxal phosphate R P N + N R P + N N R P N + N R P!-keto acid + N + N pyridoxamine phosphate

8 Transfer the amine back to an acceptor α-keto acid P N + N pyridoxamine phosphate + R!-keto acid P + N pyridoxal phosphate + R N amino acid

9 In peripheral tissues, transaminases tend to form Glutamate when they catabolize amino acids In other words, alpha-ketoglutarate is the preferred acceptor, and Glutamate is the resulting amino acid: Some amino acid + α-ketoglutarate à some alpha keto acid + Glutamate

10 Glutamate can donate its amines to form other amino acids as needed A specific example - production of Aspartate in liver (described a few slides from now): Glutamate + oxaloacetate à α-ketoglutarate + aspartate

11 Ge tt in g A m in es In to the L iv e r Glutamate Dehydrogenase: NAD(P) N (mito) glutamate NAD(P)!-ketoglutarate + N ammonia Glutamine Synthetase: N glutamate ATP N ADP P i + + N glutamine N

12 . In th e L iv er: Pre cursers for Urea ycle Glutamine is hydrolyzed to glutamate and ammonia: N glutamine N N N glutamate Ammonia can also be formed by the glutamate dehydrogenase reaction and several other reactions as well. Glutamate donates its amino group to form aspartate: Glutamate-aspartate aminotransferase: (- ) N Glutamate + + ( -) ( -) oxaloacetate (- )!-keto glutarate (- ) N aspartate.

13 ATP ADP + P i N arbamoyl phosphate N P N P i N N N rnithine Liver mitochondrion Liver cytoplasm N itrulline N N N N N Urea N 5 rnithine N N N N Arginine ATP AMP + PP i N itrulline N N N N N aspartate Argininosuccinate 4 Fumarate

14 a rb a m o y l p h o s p h a t e s y n th e ta s e I (- ) A T P P N N A T P P N b ic ar b on at e A D P c ar bo ny l ph os p ha te P i ca rb a ma te A D P ca rb a mo yl ph os p ha te

15 rn it h in e T ra n s c a rb a m o y la s e a rb a m o y l p ho s p ha t e N P ( -) P i ( -) (+ ) N (- ) N N N (+ ) N (+ ) rn ith in e it rullin e

16 A rg in in o s u c c in a te s y n t h e t a s e N N itrulline N aspartate N N N N N Argininosuccinate ATP AMP + PP i

17 A r g in in o s u c c in a t e ly a s e N N N N N N Argininosuccinate Fumarate N Arginine N

18 A rg in a s e N N N N Arginine N N Urea N N rnithine

19 ATP ADP + P i N arbamoyl phosphate N P N P i N N N rnithine Liver mitochondrion Liver cytoplasm N itrulline N N N N N Urea N 5 rnithine N N N N Arginine ATP AMP + PP i N itrulline N N N N N aspartate Argininosuccinate 4 Fumarate

20 Urea ycle onnects to TA ycle Urea rnithine Urea ycle itrulline N Aspartate Arginine Argininosuccinate Malate xaloacetate Fumarate TA ycle itrate!-ketoglutarate

21 Ge tt in g A m in es In to the L iv e r Glutamate Dehydrogenase: NAD(P) N (mito) glutamate NAD(P)!-ketoglutarate + N ammonia Glutamine Synthetase: N glutamate ATP N ADP P i + + N glutamine N

22 ATP ADP + P i N arbamoyl phosphate N P N P i N N N rnithine Liver mitochondrion Liver cytoplasm N itrulline N N N N N Urea N 5 rnithine N N N N Arginine ATP AMP + PP i N itrulline N N N N N aspartate Argininosuccinate 4 Fumarate

23 P S I is S t im u la t e d b y N A G ( - ) N ( +) + o A - N -a ce ty l g lu ta m a te s y n th e tas e ( - ) N g lu t a m a t e a c et y l o A N -a c et y l g lu t a m a te (N A G ) ( re p e a t in g t h e fig u re fro m p a g e o f y o u r h an d o u t ) ( -) A T P P N N A T P P N bi ca rb o na te A D P c a rb on y l p ho s ph at e P i c ar ba m at e A D P c ar ba m oy l p ho sp h at e

24 -- Muscle (Amines) Glucose Pyruvate Glutamate Amino acids Alanine!-ketoglutarate blood transport Alanine!-ketoglutarate Urea Glucose Pyruvate Glutamate N Liver

25 omplicating the picture: ther tissues may be involved Muscle: Amino acids: Transamination Deamination purine deamination: Alanine Glutamate N 4 Glutamine Intestine: Glutamine Alanine N 4 itrulline Glutamine Kidney: itrulline N N 4 Arginine Alanine Liver: Glutamine N 4 Arginine Urea Glu Aspartate

26 Why is Ammonia Toxic?

27 Why is Ammonia Toxic? Possible neurotoxic effects on glutamate levels (and also GABA) (due to shifting equilibria of reactions involving these compounds)

28 Why is Ammonia Toxic? Possible neurotoxic effects on glutamate levels (and also GABA) (due to shifting equilibria of reactions involving these compounds) Possible metabolic/energetics effects: - alpha-ketoglutarate levels - glutamate levels - glutamine

29 ATP ADP + P i N arbamoyl phosphate N P N P i N N N rnithine Liver mitochondrion Liver cytoplasm N itrulline N N N N N Urea N 5 rnithine N N N N Arginine ATP AMP + PP i N itrulline N N N N N aspartate Argininosuccinate 4 Fumarate

30 Inherited Defects of Urea ycle Enzymes: Diagnosis Defects are diagnosed based on the metabolites seen in the blood and/or urine. PSD TD ASD ALD AD No elevation except ammonia; diagnosed by elimination. Elevated P causes synthesis of rotate Elevated citrulline Elevated argininosuccinate Elevated arginine

31 ATP ADP + P i N arbamoyl phosphate N P N P i N N N rnithine Liver mitochondrion Liver cytoplasm N itrulline N N N N N Urea N 5 rnithine N N N N Arginine ATP AMP + PP i N itrulline N N N N N aspartate Argininosuccinate 4 Fumarate

32 P S I is S t im u la t e d b y N A G ( - ) N ( +) + o A - N -a ce ty l g lu ta m a te s y n th e tas e ( - ) N g lu t a m a t e a c et y l o A N -a c et y l g lu t a m a te (N A G ) ( re p e a t in g t h e fig u re fro m p a g e o f y o u r h an d o u t ) ( -) A T P P N N A T P P N bi ca rb o na te A D P c a rb on y l p ho s ph at e P i c ar ba m at e A D P c ar ba m oy l p ho sp h at e

33 ATP ADP + P i N arbamoyl phosphate N P N P i N N N rnithine Liver mitochondrion Liver cytoplasm N itrulline N N N N N Urea N 5 rnithine N N N N Arginine ATP AMP + PP i N itrulline N N N N N aspartate Argininosuccinate 4 Fumarate

34 linical Management of Urea ycle Defects Dialysis to remove ammonia Provide the patient with alternative ways to excrete nitrogenous compounds: * Intravenous sodium benzoate or phenylacetate * Supplemental arginine carbamoyl phosphate Excreted by kidney N Urea N rnithine Arginine ATP AMP+PP i itrulline Aspartate X ASD Argininosuccinate Dietary Arginine Levulose - acidifies the gut Low protein diet X ALD Excreted by kidney

35 Degrading the Amino Acid arbon Backbone

36 Easily-degraded products after transamination: N aspartate transamination oxaloacetate N glutamate N alanine transamination transamination!-ketoglutarate pyruvate We also already know how to degrade Glutamine: glutaminase Glutamine > glutamate + ammonia and by analogy, how to degrade Asparagine: asparaginase Asparagine > aspartate + ammonia

37 Amino Acids are categorized as Glucogenic or ketogenic or both. Many amino acids are purely glucogenic: Glutamate, aspartate, alanine, glutamine, asparagine, Some amino acids are both gluco- and ketogenic: Threonine, isoleucine, phenylalanine, tyrosine, tryptophan The only PURELY ketogenic Amino Acids: leucine, lysine

38 N N N Arginine N Amino acids with 5-carbon backbones tend to form α ketoglutarate Urea (via the urea cycle) N N N rnithine!-ketoglutarate glutamate glutamate semialdehyde NAD(P) N Proline NAD(P) N glutamate N N glutamine N! - ketoglutarate

39 Degradation and Biosynthesis of Serine and Glycine NAD NAD Glycine Synthase: N + N 4 Glycine TF 5 10 N -N - methylene TF Serine ydroxymethyltransferase: N N Serine TF 5 10 N -N - methylene TF Glycine N 4 Serine Dehydratase: N Serine N N

40 S N Methionine ATP + PPi + Pi S Adenine N S-Adenosyl Methionine Methionine ycle And Biological Methyl Groups tetrahydrofolate N5 methyl tetrahydrofolate S N omocysteine Bios ynthetic Methylation reaction S Adenine Methyl acceptor * see examples Methylated acceptor N S-Adenosyl omocysteine N Serine S N ysteine (remainder of homocysteine degraded for energy)

41 Phenylalanine and Tyrosine (Normal path shown in black, pathological reaction shown in red) N Phenylalanine Phenylketonuria (no phenylalanine hydroxylase) Tetrahydrobiopterin + Dihydrobiopterin + Enzyme: Phenylalanine hydroxylase N Tyrosine omogentisate Deficiency: Alkaptonuria chronosis Enzyme: homogentisate dioxygenase Phenylpyruvate (you don t need to know the rest)

42 Branched hain Amino Acids Isoleucine Leucine Valine N N N (+ )!"KG!"KG!"KG Transamination Glu Glu Glu + NAD, oas + + NAD, oas NAD, oas --- Branched-chain!-keto acid dehydrogenase --- NAD + S-oA NAD + NAD + S-oA S-oA (continues on to degradation path similar to #-oxidation of fatty acids)

43 Synthesis of Bioactive Amines N Tyrosine Tyrosine hydroxylase N Dihydroxyphenylalanine (L-DPA) N Dopamine N Norepinephrine N Epinephrine

44 Synthesis of Bioactive Amines N Tryptophan N Tryptophan hydroxylase N 5-hydroxytryptophan N PLP-dependent decvarboxylation NAD+ N N Serotonin

45 Synthesis of Bioactive Amines Glutamate N Glutamate decarboxylase (PLP-dependent ) N!-aminobutyric acid (GABA) N N istidine N (- ) istidine decarboxylase (PLP-dependent ) N N N istamine

46 NN-Essential Amino Acids: Glutamate, aspartate, alanine, glutamine, asparagine, (proline), glycine, serine (cysteine, tyrosine) Essential Amino Acids: Arginine (!), phenylalanine, methionine, histidine, Isoleucine, leucine, valine, threonine, tryptophan, lysine

47 Additional Source Information for more information see: Slide 4: Robert Lyons Slide 5: Robert Lyons Slide 7: Robert Lyons Slide 8: Robert Lyons Slide 11: Robert Lyons Slide 1: Robert Lyons Slide 1: Robert Lyons Slide 14: Robert Lyons Slide 15: Robert Lyons Slide 16: Robert Lyons Slide 17: Robert Lyons Slide 18: Robert Lyons Slide 19: Robert Lyons Slide 0: Robert Lyons Slide 1: Robert Lyons Slide : Robert Lyons Slide : Robert Lyons Slide 4: Robert Lyons Slide 5: Robert Lyons Slide 9: Robert Lyons Slide 1: Robert Lyons Slide : Robert Lyons Slide : Robert Lyons Slide 4: Robert Lyons Slide 6: Robert Lyons Slide 8: Robert Lyons Slide 9: Robert Lyons Slide 40: Robert Lyons Slide 41: Robert Lyons Slide 44: Robert Lyons Slide 45: Robert Lyons