1. Amino acid metabolism, concluded Andy Howard Introductory Biochemistry 24 April 2008

2. 24 April 2008 Amino Acid metabolism p. 2 of 44 What we’ll cover Amino acid metabolism Non-essential amino acids Branched-chain Aromatics Histidine Amino acids as metabolites Glucogenic amino acids Ketogenic amino acids Serine biproducts Glycine biproducts

3. 24 April 2008 Amino Acid metabolism p. 3 of 44 Essential and non-essential amino acids An amino acid is defined as essential if it must be obtained within the diet In general the essential amino acids are the ones that have complicated and highly ATP-dependent biosynthetic pathways Of course, it depends on the organism

4. 24 April 2008 Amino Acid metabolism p. 4 of 44 The human list (cf. box 17.3) AAmoles ATPessen- tial? Asp21no  Asn22-24no  Lys50-51yes Met44yes Thr31yes Ala20no  Val39yes Leu47yes Ile55yes Glu30no  Gln31no  AAmoles ATPessen- tial? Arg44no Pro39no Ser18no  Gly12no  Cys19no Phe65yes Tyr62no* Trp78yes His42yes

5. 24 April 2008 Amino Acid metabolism p. 5 of 44 Examples of transaminases Reactants ProductsTransaminase Keto acidamino acidketo acidamino acid Pyruvateglutamate  -k-glutaratealaninepyruvate Pyruvateaspartateoxaloacetatealaninepyruvate Oxaloacetateglutamate  -k-glutarateaspartateaspartate 3-P-OH-pyrglutamate  -k-glutarateP-serphosphoserine 3-OH-phenyl-glutamate  -k-glutaratetyrosinetyrosine pyruvate Glu-g-(glu)(  -k-g)ornithineornithine semialdehyde N-acyl-glutamate  -k-glutarate N-acyl-2.3.1.89 2-amino-2-6,diamino- 6-oxopimelatepimelate

6. 24 April 2008 Amino Acid metabolism p. 6 of 44 Marching through the list of twenty amino acids Amino acids we’ve already covered Acids and amides: glu, gln, asp, asn Simple: ala, ser, gly New but non- essential arg, pro cys Essential but straightforward lys, met, thr val, leu, ile Essential & Ugly phe, tyr, trp his

7. 24 April 2008 Amino Acid metabolism p. 7 of 44 Arginine and proline Two routes: Glutamate to glutamate semialdehyde that cyclizes to  1 -pyrroline 5-carboxylate and thence to proline Glutamate semialdehyde can also be converted to ornithine and thence to arg Alternative: glutamate acetylated to N- acetyl-glutamate-5-semialdehyde and thence to ornithine Glutamate semialdehyde ornithine

8. 24 April 2008 Amino Acid metabolism p. 8 of 44 Glutamate to P5C Single enzyme can interconvert glutamate and  1 -pyrroline carboxylate: 1-pyrroline-5-carboxylate dehydrogenase 3-layer  sandwich protein PDB 2BJA 170 kDa trimer monomer shown Thermus thermophilus

9. 24 April 2008 Amino Acid metabolism p. 9 of 44 Pyrroline-5-carboxylate to proline Pyrroline-5- carboxylate reduced to proline Large, NAD(P)- dependent enzyme Pyrroline-5-carboxylate reductase PDB 2IZZ 354 kDa decamer pentamer shown Human

10. 24 April 2008 Amino Acid metabolism p. 10 of 44 Glutamate to Glu semialdehyde Glu is  -phosphorylated: glu + ATP  glu-5-P + ADP (2.7.2.11) Glu-5-P is reduced and dephosphorylated: glu-5-P + NADPH + H +  glu-5-semialdehyde + NADP +  -glutamyl phosphate reductase PDB 1O20 47 kDa monomer Thermatoga maritima Glu-5-P

11. 24 April 2008 Amino Acid metabolism p. 11 of 44 Glu semialdehyde to ornithine This is just another transamination, catalyzed by ornithine aminotransferase: glu-5-semialdehyde + glu/asp  ornithine +  -keto-glutarate / oxaloacetate Typical PLP- dependent reaction PDB 2OAT 193 kDa tetramer human ornithine

12. 24 April 2008 Amino Acid metabolism p. 12 of 44 Ornithine to citrulline Ornithine condenses with carbamoyl phosphate to form citrulline with the help of ornithine transcarbamoylase Carbamoyl phosphate PDB 1DUV 110 kDa trimer E.coil citrulline

13. 24 April 2008 Amino Acid metabolism p. 13 of 44 Citrulline to arginosuccinate Citrulline condenses with aspartate using ATP hydrolysis to drive it forward to L- arginosuccinate: citrulline + aspartate + ATP  L-arginosuccinate + AMP + PP i Arginosuccinate synthase 200 kDa tetramer monomer shown

14. 24 April 2008 Amino Acid metabolism p. 14 of 44 Arginosuccinate to arginine Fumarate extracted, leaving arginine Arginosuccinate lyase is also  -crystallin, one of the moonlighting proteins: it’s a component of eye lenses PDB 1TJ7 100 kDa dimer (really!) E.coli fumarate

15. 24 April 2008 Amino Acid metabolism p. 15 of 44 Why all that detail? These reactions form 75% of the urea cycle, which is an important path for amino acid and nucleic acid degradation. So we’ll need this later.

16. 24 April 2008 Amino Acid metabolism p. 16 of 44 Cysteine synthesis in plants and bacteria serine + Acetyl CoA  O-acetylserine + HSCoA O-acetylserine + S 2- + H +  cysteine + acetate Ser acetyltransferase is inhibited by cysteine Serine acetyltransferase PDB 1SSQ 176 kDa hexamer dimer shown Haemophilus O-acetylserine

17. 24 April 2008 Amino Acid metabolism p. 17 of 44 Animal pathway to cys Ser + homocysteine (from met) fuse to form cystathionine + H 2 O Cystathionine + H 2 O  NH 4 + + cysteine +  - ketobutyrate Cystathionine  -lyase PDB 1N8P 173 kDa tetramer yeast cystathionine

18. 24 April 2008 Amino Acid metabolism p. 18 of 44 Lys, met, thr asp gets phosphorylated and becomes a source for all of these: Asp + ATP   -aspartyl phosphate + ADP via aspartate kinase  -asp P + NADPH + H + -> Pi + aspartate  -semialdehyde +NADP + This heads to lys or to homoserine Homoserine converts in a few steps to met or thr Aspartate kinase 112 kDa PDB 2CDQ dimer Arabidopsis

19. 24 April 2008 Amino Acid metabolism p. 19 of 44 Asp  -semialdehyde to homoserine  -aldehyde reduced to sec- alcohol, which is homoserine Homo is generally a prefix meaning containing an extra methylene group This is precursor to homocysteine  methionine It also leads to threonine homoserine

20. 24 April 2008 Amino Acid metabolism p. 20 of 44 Homoserine to threonine Homoserine phosphorylated with ATP as phosphate donor Phosphohomoserine dephosporylated with movement of -OH from one carbon to another: threonine results Phospho- homoserine threonine

21. 24 April 2008 Amino Acid metabolism p. 21 of 44 Homoserine to methionine Three reactions convert homoserine to homocysteine 5-methyltetrahydrofolate serves as a methyl donor to convert homocysteine to methionine via methionine synthase This enzyme exists in humans but its activity is low and [homocysteine] is low; So methionine is essential in humans homocysteine

22. 24 April 2008 Amino Acid metabolism p. 22 of 44 Specifics for lysine Aspartyl semialdehyde condenses with pyruvate to form 2-3-dihydropicolinate Reduced again to 2,3,4,5-tetrahydropicolinate Acylated (via AcylCoA) to N-acyl-2-amino-6- oxopimelate Transaminated to N-acyl-2,6-diaminopimelate Deacylated to L,L-N-acyl-2,6-diaminopimelate Epimerase converts that to meso form That’s decarboxylated to lysine 2,3-dihydro- picolinate

23. 24 April 2008 Amino Acid metabolism p. 23 of 44 The human list (cf. box 17.3) AAmoles ATPessen- tial? Asp21no  Asn22-24no  Lys50-51yes  Met44yes  Thr31yes  Ala20no  Val39yes Leu47yes Ile55yes Glu30no  Gln31no  AAmoles ATPessen- tial? Arg44no  Pro39no  Ser18no  Gly12no  Cys19no  Phe65yes Tyr62no* Trp78yes His42yes

24. 24 April 2008 Amino Acid metabolism p. 24 of 44 Branched-chain aliphatics: isoleucine and valine Derived from pyruvate or  - ketobutyrate 2 pyruvate   -ketoisovalerate + CO 2 pyr +  -ketobutyrate   -keto-  -methylvalerate + CO 2 These products are transaminated to ile and val  -ketobutyrate  -keto  -methylvalerate

25. 24 April 2008 Amino Acid metabolism p. 25 of 44 Leucine Also derived from  -ketoisovalerate; An extra methylene is inserted between the polar end and the isopropyl group Final reaction is another transamination

26. 24 April 2008 Amino Acid metabolism p. 26 of 44 Aromatics: phe and tyr Common pathways for phe,tyr,trp via shikimate and chorismate For phe, tyr: chorismate converted to prephenate Prephenate can be aromatized with or without a 4-OH group to lead to phe,tyr chorismate shikimate

27. 24 April 2008 Amino Acid metabolism p. 27 of 44 Reaction specifics Prephenate is oxidized and dehydroxylated in two steps to phenylpyruvate Or it is oxidized to 4-OH- phenylpyruvate Transaminations of those  -ketoacids yield the final amino acids prephenate 4-hydroxy- phenyl- pyruvate

28. 24 April 2008 Amino Acid metabolism p. 28 of 44 Chorismate mutase Isomerase, converts chorismate to prephenate In E.coli: 2 versions depending on which path the product is heading to Active sites are similar in all organisms but architecture is very different Catalytic triad similar to serine proteases PDB 1DBF 42 kDa trimer B.subtilis

29. 24 April 2008 Amino Acid metabolism p. 29 of 44 Path to tryptophan: anthranilate synthase Chorismate reacts with glutamine and is aromatized to anthranilate: chorismate + gln  anthranilate + pyruvate + glutamate anthranilate PDB 1I1Q 157 kDa heterotetramer heterodimer shown Salmonella

30. 24 April 2008 Amino Acid metabolism p. 30 of 44 Anthranilate to indole Four-step pathway: phosphoribosyl pyrophosphate (PRPP) contributes a phosphoribosyl group Sugar ring opens and rearranges Result is decarboxylated and forms a second ring to form indole 3-glycerinphosphate Glyceraldehyde-3-P is released to leave indole

31. 24 April 2008 Amino Acid metabolism p. 31 of 44 Tryptophan synthase Indole + ser  tryptophan + H 2 O PLP-dependent enzyme, but different in how it uses PLP from the transaminases PDB 2CLF 146 kDa heterotetramer; heterodimer shown Salmonella

32. 24 April 2008 Amino Acid metabolism p. 32 of 44 Genetic control of aromatic aa synthesis In E.coli and other bacteria, a single operon controls several chorismate- related genes

33. 24 April 2008 Amino Acid metabolism p. 33 of 44 Histidine (fig. 17.22) Start with PRPP and ATP: form phosphoribosyl ATP 3 reactions involving glutamine as nitrogen donor for ring lead to imidazole glycerol phosphate That gets modified and transaminated t make histidine

34. 24 April 2008 Amino Acid metabolism p. 34 of 44 What do we do with amino acids? Obviously a lot of them serve as building- blocks for protein and peptide synthesis via ribosomal mechanisms Also serve as metabolites, getting converted to other compounds or getting oxidized as fuel

35. 24 April 2008 Amino Acid metabolism p. 35 of 44 Gluogenic and ketogenic amino acids Degradation of many amino acids lead to TCA cycle intermediates or pyruvate therefore these can be built back up to glucose; these are called glucogenic Degradation of others leads to acetyl CoA and related compounds these cannot be built back up to glucose except via the glyoxalate shuttle these are called ketogenic

36. 24 April 2008 Amino Acid metabolism p. 36 of 44 Serine-based metabolites Serine is a building block for sphinganine and therefore for sphingolipids Serine also leads to phosphatidylserine, which is important by itself and can be metabolized to phosphatidylethanolamine and phosphatidylcholine

37. 24 April 2008 Amino Acid metabolism p. 37 of 44 Glycine-based metabolites Glycine is a source for purines, glyoxylate, creatine phosphate, and (with the help of succinyl CoA) porphobilinogen, whence we get porphyrins, and from those we get chlorophyll, heme, and cobalamin porphobilinogen

38. 24 April 2008 Amino Acid metabolism p. 38 of 44 We’ll continue amino acids next time… But first, a sneak preview of our coverage of nucleic acid chemistry, which we’ll do on Tuesday!

39. 24 April 2008 Amino Acid metabolism p. 39 of 44 Pyrimidines Single-ring nucleic acid bases 6-atom ring; always two nitrogens in the ring, meta to one another Based on pyrimidine, although pyrimidine itself is not a biologically important molecule Variations depend on oxygens and nitrogens attached to ring carbons Tautomerization possible Note line of symmetry in pyrimidine structure 1 2 3 4 5 6