1. 1 Metabolism of amino acids  Department of Biochemistry (J.D.) 2013

2. 2 Glucogenic (13)  pyruvate and/or CAC intermediates Ketogenic (2) = Leu, Lys  acetyl-CoA + acetoacetate Mixed (5) = Thr, Ile, Phe, Tyr, Trp Intermediates of amino acid catabolism

3. 3 Asp Ser, Gly, Thr, Ala, Cys, Trp Intermediates of amino acid catabolism pyruvate glucose fumarate succinyl-CoA 2-oxoglutarate oxaloacetate acetoacetate

4. 4 Transamination of alanine alanine aminotransferase Alanine alanine pyruvate 2-oxoglutarate glutamate

5. 5 Glucose-alanine cycle livermuscle glucose pyruvate alanine glucose pyruvate alanine transamination glycolysis transamination gluconeogenesis transport in blood alanine is non-toxic transport of ammonia from muscles to liver in the liver, alanine is the substrate for gluconeogenesis

6. 6 Alanine - summary readily made from pyruvate (transamination) ALT is clinically important enzyme, mainly in liver, elevated catalytic concentration in blood serum – liver diseases Ala is released to blood mainly from muscles, together with Gln (postresorption phase) semiessential AA (in metabolic stress) – important substrate for gluconeogenesis

7. 7 Hydrolysis of arginine  urea Arginine glutamate no transamination arginine ornithine

8. 8 NO is signal molecule from arginine BH 4 Exogenous NO sources glycerol trinitrate isosorbide dinitrate amyl nitrite isobutyl nitrite sodium nitroprusside citrulline N-hydroxyarginine nitric oxide radical

9. 9 Synthesis of creatine (1. part) glycin arginine ornithine guanidinoacetate from Greek κρέας (meat) glycine

10. 10 Synthesis of creatine (2. part) N 1 -methylation of guanidinoacetate S-adenosylmethionine (SAM) S-adenosylhomocysteine creatine N-methylguanidine-N-acetate guanidinoacetate

11. 11 N 2 -Phosforylation of creatine kreatinkreatinfosfát ATP creatinecreatine phosphate

12. 12 Creatinine is a catabolite of creatine made in non-enzymatic reaction non-enzymatic cyclization dehydratation kreatin creatinine - H 2 O creatine

13. 13 Arginine - summery semiessential AA (childhood) the most basic AA (guanidine, pK B = 0.5) no transamination, Arg releases ornithine + urea Arg + Gly + Met  creatine releases NO (vasodilator) OTC (over-the-counter) preparations in pharmacy

14. 14 Dehydratation + deamination of serine Serine pyruvate imineenamine

15. 15 Conversion of serine to glycine H 2 O + N 5 N 10 -CH 2 -FH 4 methylene FH 4 serine glycine cofactor: tetrahydrofolate (FH 4 )

16. 16 Transamination of serine and glucose formation reverse reaction: synthesis of serine pathway is different - through phosphoserine serinehydroxypyruvateglycerate 3-P-glycerate glucose 2-oxoglutarate

17. 17 Betaine is made by choline oxidation oxidation Decarboxylation of serine gives ethanolamine. Methylation of ethanolamine leads to choline decarboxylationmethylation serineethanolaminecholine betaine choline

18. 18 Serine - summary non-essential glucogenic AA source of C1 fragments (attached to tetrahydrofolate) component of glycerophospholipids decarboxylation gives ethanolamine  choline carbon skeleton used for selenocysteine serine side chain in proteins: the site of phosphorylation the linkage of oligosaccharides (O-glycoside bond) nucleophilic -OH group in active site of enzyme (serine proteases)

19. 19 The complete catabolism of glycine COO H CH 2 NH 2 N 5 N 10 CH 2 FH 4 + + CO 2 NH 3 FH 4 + C1 fragment (methylene) is transferred to tetrahydrofolate Glycine

20. 20 Oxidative deamination of glycine glyoxylate oxalate 60 % catabolism of glycine and ethanolamine 30 % catabolism of vitamin C 10 % food (spinach, rhubarb, mangold, tea, cocoa)

21. 21 Glycine - summary Catabolism complete oxidation to CO 2 + NH 3 oxidative deamination to oxalate Anabolic conversions donor of C1 fragment serine porphyrines purine bases creatine glutathione (GSH) conjugation agent (bile acids, xenobiotics)

22. 22 Threonine (4C) is split to glycine (2C) and acetaldehyde (2C) Threonine essential AA two asymmetric C atoms the site of phosphorylation and glycosylation in proteins no transamination glycine pyruvateserine acetaldehyde

23. 23 Methionine is methylation agent (homocysteine side product) B 12 ethanolamine noradrenaline guanidinacetate methionine S-adenosylmethionine substrate substrate-CH 3 choline adrenaline creatine homocysteine remethylation Methionineno transamination

24. 24 S-Adenosylmethionine (SAM) contains trivalent positively charged sulfur atom cation - sulfonium

25. 25 Cysteine is made from methionine methionine homocysteine cystathionine homoserine cysteine condensation with serine cysteine release pyridoxal-P B 12 succinyl-CoA

26. 26 Methionine - summery essential AA, rather rare in foodstufs S-adenosylmethionine (SAM) is methylation agent metabolized to cysteine  Cys is non-essential AA C-skeleton of cysteine comes from serine, sulfur atom from methionine final catabolite is succinyl-CoA (glucogenic)

27. 27 Homocysteine is harmful mechanism of its action is not yet understood direct action on blood vessel epithelium decreases thrombocyte life and fibrinolysis supports formation of oxygen radicals – damage of vessel wall increases LDL lipoperoxidation elevated blood level of homocysteine is risk factor of cardiovascular diseases to eliminate homocysteine - three vitamins are needed: folate, cobalamine, pyridoxin

28. 28 Cysteine catabolism: oxygenation of -SH group cysteine oxygenation cysteine sulfinate cysteic acid decarboxylation transamination hypotaurine taurine oxygenation sulfinylpyruvate Cysteine oxygenation

29. 29 The formation of sulfite under physiol. pH – dissociation only to HSO 3 - sulfite sulfinylpyruvate hydrolytic cleavage of sulfite pyruvate

30. 30 Sulfite oxidase catalyzes sulfate formation cysteine HSO 3 - + H 2 O  SO 4 2- + 3H + + 2e - blood plasma (0.5 mmol/l) acidify body fluids reduce molybdopterine PAPS urine

31. 31 Distinguish Sulfiteanion SO 3 2- Sulfide inorganicanion S 2- (e.g. ZnS zinc sulfide) Sulfide organicR-S-R dialkylsulfide Sulfateanion SO 4 2-

32. 32 Transamination of cysteine and sulfane production CN - SCN - in smokers SO 4 2- sulfhemoglobin signal molecule ? 2-oxoglutarate cysteine glutamate mercaptopyruvate desulfuration pyruvate

33. 33 Cysteine - summary both pathways go to pyruvate (glucogenic) main catabolism: sulfur oxygenation  sulfite  sulfate high protein diet leads to physiologic acidosis cystein provids taurine – conjugation agent (e.g. bile acids) taurine is semiessential AA in metabolic stress taurine is a component of „energy drinks“ cysteine – part of glutathione (GSH) - antioxidant decarboxylation of Cys – cysteamine, in CoA-SH in proteins – disulfide bonds (tertiary structure) cysteine proteases: active site contains –SH group

34. 34 Six amino acids provide pyruvate 1. Serine – dehydratation + deamination 2. Glycine – via serine 3. Threonine – via glycine 4. Alanine – transamination (ALT) 5. Cysteine – both catabolic pathways 6. Tryptophan – via alanine (see later)

35. 35 Transamination of Asp  oxaloacetate (CAC) AST (aspartate aminotransferase) – clinically important enzyme in urea cycle, Asp donates one nitrogen into urea and releases fumarate decarboxylation of Asp  β-alanine (part of coenzyme A) donor of nitrogen in purine synthesis (fumarate released) whole structure given for pyrimidine bases synthesis aspartam (sweetener) condensation with ammonia  asparagine (for cell utilization, not as detoxication of ammonia) Aspartate

36. 36 β-Alanine is made by the decarboxylation of aspartate - CO 2 β α in the structure of CoA-SH

37. 37 Glutamate with oxaloacetate afford aspartate (transamination) AST reaction produces aspartate for urea synthesis aspartate aminotransferase Glutamate oxaloacetate aspartate  urea glutamate2-oxoglutarate

38. 38 Dehydrogenative deamination of glutamate is the main producer of ammonia in tissues glutamate dehydrogenase (GD, GDH, GMD) glutamate2-iminoglutarate 2-oxoglutarate (CAC)

39. 39 Decarboxylation of glutamate glutamate

40. 40 Glutamate - summary produced in the transaminations of most AA glutamate dehydrogenase reaction produces most ammonia in body transaminations are reversible, so glutamate can be converted to 2-oxoglutarate (glucogenic) Glu + NH 3  Gln (ammonia detoxification) glutamate is readily made from glutamine, histidine, proline, ornithine pure monosodium glutamate (MSG, E621), flavour enhancer, can cause health problems (chinese restaurant syndrome)

41. 41 See also the previous lecture (AA-1) glutamine synthesis is the way of ammonia detoxification in tissues including liver in kidneys, glutamine releases ammonia (deamidation) metabolic fuel for some tissues (enterocytes, fibroblasts, lymphocytes, macrophages) donor of nitrogen for syntheses (glucosamine, purines) Glutamine

42. 42 Three amino acids donate four N atoms in purine bases synthesis fumarate glutamate aspartate glycine amide group of glutamine

43. 43 proline glutamate 5-semialdehyde glutamate pyrroline-5-carboxylate oxidation addition of H 2 O ring opening Proline is converted to glutamate (and vice versa) Prolineno transamination

44. 44 Hydroxylation of proline with 2-oxoglutarate as reductant - CO 2 proline 2-oxoglutarate 4-hydroxyprolinesuccinate Fe 2+ ascorbate

45. 45 Proline - summary non-essential AA, can be formed from glutamate converted to glutamate (glucogenic) hydroxylation of proline in collagen is post-translation modification, requires ascorbate (vitamin C), Fe 2+, and 2-oxoglutarate (unusual co-reductant) 4-hydroxyproline is catabolized to pyruvate (see Harper)

46. 46 Catabolism of histidine starts with desaturation and deamination urocanic acid (urocanate) Histidineno transamination

47. 47 Urocanate cleavage affords C 1 fragment FIGLU urocanate N-formiminoglutamate (Figlu) glutamate addition of water oxidative ring splitting H2OH2O

48. 48 Decarboxylation of histidine  histamine histidine decarboxylase occurs in mast cells and basophils histamine stimulates HCl production in stomach is released in allergic reactions triggers inflammatory response antihistaminics are drugs blocking the action of histamine - CO 2 histidinehistamine

49. 49 Histidine is responsible for buffering actions of proteins pKB = 8pKB = 8 pK A (His) = 6 pK A (His in proteins) = 6-8

50. 50 Histidine - summary semiessential AA no transamination, catabolism begins with desaturation and deamination the source of 1C groups (formimino) converted to glutamate (glucogenic) histidine is abundant in hemoglobin – buffer system post-translation modification: methylation of His in actine/myosine  3-methylhistidine – its urine excretion is the indicator of muscle proteolysis and nutrition status

51. 51 Leucine (1) - transamination + decarboxylation Leucine, Isoleucine, Valine (BCAA) branched 2-oxoacid oxidative decarboxylation branched acyl-CoA transamination

52. 52 Leucine (2) - dehydrogenation 2,3-dehydrogenation branched unsaturated acyl

53. 53 Leucine (3) – carboxylation at C4 carboxylation acyl of dicarboxylic branched unsaturated acid

54. 54 Leucine (4) – hydratation of double bond

55. 55 Leucine (5) – splitting the C-C bond in HMG-CoA HMG-CoA lyase acetoacetate

56. 56 Compare the final products of BCAA Leucineacetyl-CoA + acetoacetateketogenic Isoleucineacetyl-CoA + succinyl-CoA ketogenic glucogenic Valinesuccinyl-CoAglucogenic B 12

57. 57 BCAA - summery all BCAA are essential the first three reactions are the same (transamination, oxid. decarboxylation, dehydrogenation), final products are different leucine – ketogenic, valine – glucogenic, isoleucine – mixed after meal, BCAA make about 70 % of AA in blood, because the liver does not utilize them (lack of aminotransferases) BCAA are most utilized in muscles and brain BCAA infusion are applied in severe catabolic conditions

58. 58 Lysine catabolism (1) Lysineno transamination lysine 2-oxoglutarate ketimine (Schiff base)

59. 59 Lysine catabolism (2) ketimine hydrogenation dehydrogenation saccharopine aldimine

60. 60 Lysine catabolism (3) hydrolysis aldimine allysine glutamate

61. 61 Lysine catabolism (4) allysine dehydrogenation 2-aminoadipate

62. 62 Lysine catabolism (5) 2-aminoadipate 2-oxoglutarate transamination 2-oxoadipate glutamate

63. 63 Lysine is the substrate for carnitine (the transfer of FA from cytosol to mitochondria) carnitine acylcarnitine

64. 64 Cross-links in collagen hydrogenated aldimine lysine (lysyl residue in polypeptide) dehydrated aldol allysine lysine + hydrogenation products of reaction between the amino groups in side chains of lysine with the modified lysine side chains comprising the aldehyde group (the result of oxidation of lysine to allysine) – aldol type or aldimine type of cross-links.

65. 65 Formation of fibrin clot during blood coagulation (cross-linking of fibrin) lysine glutamine cross-linking

66. 66 Lysine - summary essential AA, no transamination ε-amino group is removed as glutamate  -amino group is removed from aminoadipate by transamination final product acetyl-CoA (ketogenic) Other conversions: lysine in many proteins binds ubiquitin (targeting for proteasome) carnitine (transport system for FA to mitochondria) decarboxylation  cadaverine in collagen: cross bridges, hydroxylation  hydroxylysine in fibrin: cross linking during blood coagulation

67. 67 Catabolism (1) Phenylalanine, Tyrosine hydroxylation transamination phenylalaninetyrosine p-hydroxyphenylpyruvate

68. 68 Catabolism (2) p-hydroxyphenylpyruvate homogentisate (2,5-dihydroxyphenylacetate) oxidative decarboxylation rearrangement hydroxylation

69. 69 Catabolism (3) oxidative cleavage of aromatic ring dioxygenase maleylacetoacetate

70. 70 Catabolism (4) maleylacetoacetatefumarylacetoacetate isomeration

71. 71 Catabolism (5) fumarylacetoacetatefumarate acetoacetate

72. 72 Hyperphenylalaninemia and Phenylketonuria deficit of hydroxylase or BH 4 elevated blood Phe and its metabolites excretion of phenylpyruvate by urine phenylalaninetyrosine hydroxylase

73. 73 Metabolites of phenylalanine phenylalaninephenylpyruvate transamination oxid. decarboxylation phenylacetate phenyllactate hydrogenation

74. 74 if not treated properly – mental retardation and other problems treatment – low phenylalanine diet products containing sweetener aspartam must be avoided L-aspartyl-L-phenylalanine methyl ester - phenylalanine is released by hydrolysis: Hyperphenylalaninemia and Phenylketonuria

75. 75 Hydroxylation of phenylalanine gives tyrosine phenylalaninetyrosine co-reductant tetrahydrobiopterine

76. 76 DOPA and dopamine from tyrosine tyrosine dopamine (catecholamine) (3,4-dihydroxyphenylalanine) hydroxylation decarboxylation Tyrosine

77. 77 Linguistic note abbreviation DOPA comes from older English nomenclature oxo group and hydroxyl group were not distinguished properly: DOPA = dioxophenylalanine correct chemical name is: 3-(3,4-dihydroxyphenyl)alanine

78. 78 Two more catecholamines from dopamine Cu 2+ nor- = N-demethyl hydroxylation at C2 N-methylation dopaminenoradrenalineadrenaline O 2, ascorbate

79. 79    melanin condenzation DOPAdopaquinone Conversion of tyrosine to melanin, a dark pigment of skin, hair, fur - 2H

80. 80 Conversion of tyrosine to thyroxine bound to thyreoglobulin tyrosine 3’,5’-diiododtyrosine thyroxine

81. 81 Phenylalanine, tyrosine - summary Phe is essential amino acid, Tyr not Tyr is made by Phe hydroxylation (tetrahydrobiopterine cofactor) catabolism is the same for both AA (mixed AA) provide fumarate for CAC (glucogenic) acetoacetate (ketone body) tyrosine is converted to hormones (catecholamines, thyronines) and dark skin pigment melanin

82. 82 Catabolism (1) O2O2 Tryptophanno transamination tryptophan oxidative cleavage of aromatic ring N-formylkynurenine

83. 83 Catabolism (2) hydrolysis of amide group formamide hydroxylation formate

84. 84 Catabolism (3) hydrolytic cleavage of alanine 3-hydroxyanthranilate

85. 85 Catabolism (4) 3-hydroxyanthranilate nicotinamide 2-oxoadipate

86. 86 Decarboxylation of tryptophan tryptophan tryptamine

87. 87 Conversion of tryptophan to melatonin sleep-wake cycle the hormone of darkness Trp hydroxylation 5-hydroxytryptophan decarboxylation N-acetylation O-methylation

88. 88 Tryptophan - summary essential AA complicated catabolism donor of 1C fragment (formic acid - formate) no transamination, amino group leaves as alanine (glucogenic) final product acetyl-CoA (ketogenic) source of nicotinamide and NAD + bacterial decomposition in large intestine  indole and skatole (3-methylindole) – exhibit strong fecal odor

89. 89 Five vitamins are formed in the body, only four are utilized VitaminWhere and how produced Niacin Biotin Phylloquinone Calciol Cobalamine in tissues, from tryptophan large intestine (bacteria) skin, from cholesterol (UV radiation) large intestine (bacteria) – not absorbed!

90. 90 Seven amino acids do not undergo transamination Amino acid  -NH 2 group is removed as Arginine Lysine Methionine Threonine Tryptophan Proline Histidine ornithine 2-aminoadipate homoserine glycine alanine glutamate NH 3 (desaturation deamination)

91. 91 ABiochemically relevant product Ala Arg Ser Gly Met Cys Asp Glu Gln Pro His Lys Tyr Trp pyruvate  glucose urea, NO, creatine ethanolamine  choline  betaine; donor of 1C fragment, selenocysteine heme, creatine, GSH, conjugation reagent (e.g. glycocholate) donor of methyl, creatine, homocysteine, cysteine glutathione (GSH), taurine, SO 4 2-, PAPS, cysteamine (CoA) donor of -NH 2 (urea, pyrimidines), oxaloacetate, fumarate, β-alanine (CoA) NH 4 +, 2-oxoglutarate, GABA, ornithine NH 4 +, donor of -NH 2 (synthesis of glucosamine, purines) glutamate, hydroxyproline glutamate, histamine, donor of 1C fragment glutamate, allysine (collagen), carnitine, cadaverine fumarate, catecholamines, thyroxine, melanins nicotinamide, serotonin, melatonin, donor of 1C fragment, indole, skatole

92. 92 Overview: decarboxylation of amino acids AAProductComments Serethanolaminepart of phospholipids, precursor of choline Cyscysteaminepart of coenzyme A (CoA-SH) Phephenethylaminestructural part of stimulants (amphetamine, ephedrine etc.) Tyrtyramineoccurs in some foods, may cause migraine Asp  -alanine part of pantothenic acid, CoA-SH, carnosine GluGABAgama-aminobutyric acid, inhibition neurotransmiter Lyscadaverineproduct of putrefaction (decay of proteins) Argagmatinesignal molecule in CNS Hishistaminetriggers allergic reactions Trptryptamineprecursor of serotonine and melatonine DOPAdopaminecatecholamine, precursor of noradrenaline/adrenaline Ornithine putrescineputrefaction product; precursor of spermidine/spermine