FCH 532 Lecture 24

1. FCH 532 Lecture 24 Chapter 26: Amino acid metabolism Wed. Urea cycle quiz Friday: Ketogenic vs. glucogenic (or both) amino acids-what common metabolites do this amino acids go towards?

2. Figure 26-11 Degradation of amino acids to one of seven common metabolic intermediates. Page 995

3. Met degradation • Met reacts with ATP to form S-adenosylmethionine (SAM). • SAM’s sulfonium ion is a highly reactive methyl group so this compound is involved in methylation reactions. • Methylation reactions catalyzed by SAM yield S-adenosylhomocysteine and a methylated acceptor molecule. • S-adenosylhomocysteine is hydrolyzed to homocysteine. • Homocysteine may be methylated to regenerate Met, in a B12 requiring reaction with N5-methyl-THF as the methyl donor. • Homocysteine can also combine with Ser to form cystathionine in a PLP catalyzed reaction and -ketobutyrate. • -ketobutyrate is oxidized and CO2 is released to yield propionyl-CoA. • Propionyl-CoA proceeds thorugh to succinyl-CoA.

4. Methionine adenosyltransferase Methyltransferase Adenosylhomocysteinase Methionine synthase (B12) Cystathionine -synthase (PLP) Cystathionine -synthase (PLP) -ketoacid dehydrogenase Propionyl-CoA carboxylase (biotin) Methylmalonyl-CoA racemase Methylmalonyl-CoA mutase Glycine cleavage system or serine hydroxymethyltransferase N5,N10-methylene-tetrahydrofolate reductase (coenzyme B12 and FAD) NADH, H+ Page 1002

5. Branched chain amino acid degradation • Degradation of Ile, Leu, and Val use common enzymes for the first three steps • Transamination to the corresponding -keto acid • Oxidative decarboxylation to the corresponding acyl-CoA • Dehydrogenation by FAD to form a double bond. First three enzymes • Branched-chain amino acid aminotransferase • Branched-chain keto acid dehydrogenase (BCKDH) • Acyl-CoA dehydrogeanse

6. Figure 26-21 The degradation of the branched-chain amino acids (A) isoleucine, (B) valine, and (C) leucine. Page 1004

7. Page 1004

8. After the three steps, for Ile, the pathway continues similar to fatty acid oxidation (propionyl-CoA carboxylase, methylmalonyl-CoA racemase, methylmalonyl-CoA mutase). • Enoyl-CoA hydratase - double bond hydration • -hydroxyacyl-CoA dehydrogenase- dehydrognation by NAD+ • Acetyl-CoA acetyltransferase - thiolytic cleavage Page 1004

9. For Valine: • Enoyl-CoA hydratase - double bond hydration • -hydroxy-isobutyryl-CoA hydrolase -hydrolysis of CoA • hydroxyisobutyrate dehydrogenase - second dehydration • Methylmalonate semialdehyde dehydrogenase - oxidative carboxylation Last 3 steps similar to fatty acid oxidation Page 1004

10. For Leucine: • -methylcronyl-CoA carboxylase-carboxylation reaction (biotin) • -methylglutaconyl-CoA hydratase-hydration reaction • HMG-CoA lyase Acetoacetate can be converted to 2 acetyl-CoA Leucine is a ketogenic amino acid! Page 1004

11. Leu and Lys are ketogenic • Leu proceeds through a typical branched amino acid breakdown but the final products are acetyl-CoA and acetoacetate. • Most common Lys degradative pathway in liver goes through the formation of the -ketoglutarate-lysine adduct saccharopine. • 7 of 11 reactions are found in other pathways. • Reaction 4: PLP-dependent transamination • Reaction 5: oxidative decarboxylation of an a-keto acid by a multienzyme complex similar to pyruvate dehydragense and a-ketoglutarate dehydrogenase. • Reactions 6,8,9: fatty acyl-CoA oxidation. • Reactions 10 and 11 are standard ketone body formation reactions.

12. Figure 26-23 The pathway of lysine degradation in mammalian liver. Saccharopine dehydrogenase (NADP+, Lys forming) Saccharopine dehydrogenase (NAD+, Glu forming) Aminoadipate semialdehyde dehydrogenase Aminoadipate aminotransferase (PLP) -keto acid dehydrogenase Glutaryl-CoA dehydrogenase Decarboxylase Enoyl-CoA hydratase -hydroxyacyl-CoA dehydrogenase HMG-CoA synthase HMG-CoA lyase Page 1006

13. Trp is both glucogenic and ketogenic • Trp is broken down into Ala (pyruvate) and acetoacetate. • First 4 reactions lead to Ala and 3-hydroxyanthranilate. • Reactions 5-9 convert 3-hydroxyanthranilate to a-ketoadipate. • Reactions 10-16 are catalyzed by enzymes of reactions 5 - 11 in Lys degradation to yield acetoacetate.

14. Page 1007

15. 1. Tryptophan-2,3-dioxygenase, 2. Formamidase, 3. Kynurenine-3-monooxygense, 4. kynureninase (PLP dependent) Page 1007

16. Kynureinase, another PLP mechanism • Reaction 4: cleavage of 3-hydroxykynurenine to alanine and 3-hydroxyanthranilate is catalyzed by the PLP dependent enzyme kynureinase. • This facilitates a C-C bond cleavage. (previous reactions catalyzed the C-H and C-C bond cleavage) • Follows the same steps as transamination but does not hydrolyze the tautomerized Schiff base. • Enzyme amino acid acts as a nucleophile tto attack the carbonyl carbon (Cof the tautomerized 3-hydroxykynurenine-PLP Schiff base.

17. Page 1008

18. 6. Amino carboxymuiconate semialdehyde decarboxylase 7. Aminomuconate semialdehyde dehydrogenase 8. Hydratase, 9. Dehydrogense 10-16. Reactions 5-11 in lysine degradation. Page 1007

19. -keto acid dehydrogenase • Glutaryl-CoA dehydrogenase • Decarboxylase • Enoyl-CoA hydratase • -hydroxyacyl-CoA dehydrogenase • HMG-CoA synthase • HMG-CoA lyase Page 1006

20. Phe and Tyr are degraded to fumarate and acetoacetate • The first step in Phe degradation is conversion to Tyr so both amino acids are degraded by the same pathway. • 6 reactions

21. Phenylanalnine hydroxylase • Aminotransferase • p-hydroxyphenylpyruvate dioxygenase • Homogentisate dioxygenase • Maleylacetoacetate isomerase • Fumarylacetoacetase Page 1009

22. Phenylalanine hydroxylase has biopterin cofactor • 1st reaction is a hydroxylation reaction by phenylalanine hydroxylase (PAH), a non-heme-iron containing homotetrameric enzyme. • Requires O2, FeII, and biopterin a pterin derivative. • Pterins have a pteridine ring (similar to flavins) • Folate derivatives (THF) also contain pterin rings.

23. Figure 26-27 The pteridine ring, the nucleus of biopterin and folate. Page 1009

24. Active BH4 must be regenerated • Active form in PAH is 5,6,7,8-tetrahydrobiopterin (BH4) • Produced from 7,8-dihydrobiopterin via dihydrofolate reductase (NADPH dependent). • 5,6,7,8-tetrahydrobiopterin is hydroxylated to pterin-4a-cabinolamine by phenylalanine hydroxylase. • pterin-4a-cabinolamine is converted to 7,8-dihydrobiopterin (quinoid form) by pterin-4a-carbinoline dehydratase • 7,8-dihydrobiopterin (quinoid form) is reduced by dihydropteridine reductase to regenerate the active cofactor.

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26. NIH shift • A 3H that starts on C4 of Phe’s ring ends up on C3 of Tyr’s ring rather than being lost to solvent. • Mechanism is called the NIH shift • 1st characterized by scientists at NIH

27. 1 and 2: activation of the enzyme’s BH4 and Fe(II) cofactors to yield pterin-4a-carbinolamine and a reactive oxyferryl [Fe(IV)=O2-] 3: Fe(IV)=O2- reacts with Phe to form an epoxide across the 3,4 bond. 4: epoxide opening to form carbocation at C3 5: migration of hydride from C4 to C3 to form more stable carbocation. 6: ring aromatization to form Tyr

28. Phe and Tyr are degraded to fumarate and acetoacetate • The first step in Phe degradation is conversion to Tyr so both amino acids are degraded by the same pathway. • 6 reactions • Reaction 1 = 1st NIH shift • Reaction 3 is also an example of NIH shift (26-31)

29. Phenylanalnine hydroxylase • Aminotransferase • p-hydroxyphenylpyruvate dioxygenase • Homogentisate dioxygenase • Maleylacetoacetate isomerase • Fumarylacetoacetase Page 1009

30. Amino acids as precursors • Amino acids are essential precursors to biomolecules: • Nucleotides • Nucleotide coenzymes • Heme • Hormones • Neurotransmitters • Glutathione