1 Metabolism of amino acids Ó Department of Biochemistry (J.D.) 2013 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 Asp Ser, Gly, Thr, Ala, Cys, Trp Intermediates of amino acid catabolism pyruvate glucose fumarate succinyl-CoA 2-oxoglutarate oxaloacetate acetoacetate 4 Transamination of alanine alanine aminotransferase Alanine alanine pyruvate 2-oxoglutarate glutamate 5 Glucose-alanine cycle liver muscle 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 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 Hydrolysis of arginine ® urea Arginine glutamate no transamination arginine ornithine 8 NO is signal molecule from arginine BH4 Exogenous NO sources • glycerol trinitrate • isosorbide dinitrate • amyl nitrite • isobutyl nitrite • sodium nitroprusside citrulline N-hydroxyarginine nitric oxide radical 9 Synthesis of creatine (1. part) glycin arginine ornithine guanidinoacetate from Greek κρέας (meat) glycine 10 Synthesis of creatine (2. part) N1-methylation of guanidinoacetate S-adenosylmethionine (SAM) S-adenosylhomocysteine creatine N-methylguanidine-N-acetate guanidinoacetate 11 N2-Phosforylation of creatine kreatin kreatinfosfát ATP creatine creatine phosphate 12 Creatinine is a catabolite of creatine made in non-enzymatic reaction non-enzymatic cyclization dehydratation kreatin creatinine - H2O creatine 13 Arginine - summery •semiessential AA (childhood) •the most basic AA (guanidine, pKB = 0.5) •no transamination, Arg releases ornithine + urea •Arg + Gly + Met ® creatine •releases NO (vasodilator) •OTC (over-the-counter) preparations in pharmacy 14 Dehydratation + deamination of serine Serine pyruvate imine enamine 15 Conversion of serine to glycine H2O + N5N10-CH2-FH4 methylene FH4 serine glycine cofactor: tetrahydrofolate (FH4) 16 Transamination of serine and glucose formation reverse reaction: synthesis of serine pathway is different - through phosphoserine serine hydroxypyruvate glycerate 3-P-glycerate glucose 2-oxoglutarate 17 Betaine is made by choline oxidation oxidation Decarboxylation of serine gives ethanolamine. Methylation of ethanolamine leads to choline decarboxylation methylation serine ethanolamine choline betaine choline 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 The complete catabolism of glycine C O O H C H 2 N H 2 N 5 N 1 0 C H 2 F H 4 + + C O 2 N H 3 F H 4 + C1 fragment (methylene) is transferred to tetrahydrofolate Glycine 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 Glycine - summary •Catabolism •complete oxidation to CO2 + NH3 •oxidative deamination to oxalate •Anabolic conversions •donor of C1 fragment •serine •porphyrines •purine bases •creatine •glutathione (GSH) •conjugation agent (bile acids, xenobiotics) • 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 pyruvate serine acetaldehyde 23 Methionine is methylation agent (homocysteine side product) B12 ethanolamine noradrenaline guanidinacetate methionine S-adenosylmethionine substrate substrate-CH3 choline adrenaline creatine homocysteine remethylation Methionine no transamination 24 S-Adenosylmethionine (SAM) contains trivalent positively charged sulfur atom cation - sulfonium 25 Cysteine is made from methionine methionine homocysteine cystathionine homoserine cysteine condensation with serine cysteine release pyridoxal-P B12 succinyl-CoA 26 Methionine - summery •essential AA, rather rare in foodstufs •S-adenosylmethionine (SAM) is methylation agent •metabolized to cysteine Þ Cys is non-essential AA •C-skeleton of cysteine comes from serine, sulfur atom from methionine •final catabolite is succinyl-CoA (glucogenic) 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 Cysteine catabolism: oxygenation of -SH group cysteine oxygenation cysteine sulfinate cysteic acid decarboxylation transamination hypotaurine taurine oxygenation sulfinylpyruvate Cysteine oxygenation 29 The formation of sulfite under physiol. pH – dissociation only to HSO3- sulfite sulfinylpyruvate hydrolytic cleavage of sulfite pyruvate 30 Sulfite oxidase catalyzes sulfate formation cysteine HSO3- + H2O ® SO42- + 3H+ + 2e- blood plasma (0.5 mmol/l) acidify body fluids reduce molybdopterine PAPS urine 31 Distinguish Sulfite anion SO32- Sulfide inorganic anion S2- (e.g. ZnS zinc sulfide) Sulfide organic R-S-R dialkylsulfide Sulfate anion SO42- 32 Transamination of cysteine and sulfane production CN- SCN- in smokers SO42- sulfhemoglobin signal molecule ? 2-oxoglutarate cysteine glutamate mercaptopyruvate desulfuration pyruvate 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 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 •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 β-Alanine is made by the decarboxylation of aspartate - CO2 β α in the structure of CoA-SH 37 Glutamate with oxaloacetate afford aspartate (transamination) AST reaction produces aspartate for urea synthesis aspartate aminotransferase Glutamate oxaloacetate aspartate ® urea glutamate 2-oxoglutarate 38 Dehydrogenative deamination of glutamate is the main producer of ammonia in tissues glutamate dehydrogenase (GD, GDH, GMD) glutamate 2-iminoglutarate 2-oxoglutarate (CAC) 39 Decarboxylation of glutamate glutamate 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 + NH3 D 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 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 Three amino acids donate four N atoms in purine bases synthesis fumarate glutamate aspartate glycine amide group of glutamine amide group of glutamine 43 proline glutamate 5-semialdehyde glutamate pyrroline-5-carboxylate oxidation addition of H2O ring opening Proline is converted to glutamate (and vice versa) Proline no transamination 44 Hydroxylation of proline with 2-oxoglutarate as reductant - CO2 proline 2-oxoglutarate 4-hydroxyproline succinate Fe2+ ascorbate 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), Fe2+, and 2-oxoglutarate (unusual co-reductant) •4-hydroxyproline is catabolized to pyruvate (see Harper) 46 Catabolism of histidine starts with desaturation and deamination urocanic acid (urocanate) Histidine no transamination 47 Urocanate cleavage affords C1 fragment FIGLU urocanate N-formiminoglutamate (Figlu) glutamate addition of water oxidative ring splitting H2O 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 - CO2 histidine histamine 49 Histidine is responsible for buffering actions of proteins pKB = 8 pKA (His) = 6 pKA (His in proteins) = 6-8 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 Leucine (1) - transamination + decarboxylation Leucine, Isoleucine, Valine (BCAA) branched 2-oxoacid oxidative decarboxylation branched acyl-CoA transamination 52 Leucine (2) - dehydrogenation 2,3-dehydrogenation branched unsaturated acyl 53 Leucine (3) – carboxylation at C4 carboxylation acyl of dicarboxylic branched unsaturated acid 54 Leucine (4) – hydratation of double bond 55 Leucine (5) – splitting the C-C bond in HMG-CoA HMG-CoA lyase acetoacetate 56 Compare the final products of BCAA Leucine acetyl-CoA + acetoacetate ketogenic Isoleucine acetyl-CoA + succinyl-CoA ketogenic glucogenic Valine succinyl-CoA glucogenic B12 B12 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 Lysine catabolism (1) Lysine no transamination lysine 2-oxoglutarate ketimine (Schiff base) 59 Lysine catabolism (2) ketimine hydrogenation dehydrogenation saccharopine aldimine 60 Lysine catabolism (3) hydrolysis aldimine allysine glutamate 61 Lysine catabolism (4) allysine dehydrogenation 2-aminoadipate 62 Lysine catabolism (5) 2-aminoadipate 2-oxoglutarate transamination 2-oxoadipate glutamate 63 Lysine is the substrate for carnitine (the transfer of FA from cytosol to mitochondria) carnitine acylcarnitine 64 Cross-links in collagen hydrogenated aldimine lysine (lysyl residue in polypeptide) dehydrated aldol allysine 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 Formation of fibrin clot during blood coagulation (cross-linking of fibrin) lysine glutamine cross-linking 66 Lysine - summary •essential AA, no transamination •ε-amino group is removed as glutamate •a-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 Catabolism (1) Phenylalanine, Tyrosine hydroxylation transamination phenylalanine tyrosine p-hydroxyphenylpyruvate 68 Catabolism (2) p-hydroxyphenylpyruvate homogentisate (2,5-dihydroxyphenylacetate) oxidative decarboxylation rearrangement hydroxylation 69 Catabolism (3) oxidative cleavage of aromatic ring dioxygenase maleylacetoacetate 70 Catabolism (4) maleylacetoacetate fumarylacetoacetate isomeration 71 Catabolism (5) fumarylacetoacetate fumarate acetoacetate 72 Hyperphenylalaninemia and Phenylketonuria •deficit of hydroxylase or BH4 •elevated blood Phe and its metabolites •excretion of phenylpyruvate by urine phenylalanine tyrosine hydroxylase 73 Metabolites of phenylalanine phenylalanine phenylpyruvate transamination oxid. decarboxylation phenylacetate phenyllactate hydrogenation 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 Hydroxylation of phenylalanine gives tyrosine phenylalanine tyrosine co-reductant tetrahydrobiopterine 76 DOPA and dopamine from tyrosine tyrosine dopamine (catecholamine) (3,4-dihydroxyphenylalanine) hydroxylation decarboxylation Tyrosine 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 Two more catecholamines from dopamine Cu2+ nor- = N-demethyl hydroxylation at C2 N-methylation dopamine noradrenaline adrenaline O2, ascorbate 79 ® ® ® melanin condenzation DOPA dopaquinone Conversion of tyrosine to melanin, a dark pigment of skin, hair, fur - 2H 80 Conversion of tyrosine to thyroxine bound to thyreoglobulin tyrosine 3’,5’-diiododtyrosine thyroxine 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 Catabolism (1) O2 Tryptophan no transamination tryptophan oxidative cleavage of aromatic ring N-formylkynurenine 83 Catabolism (2) hydrolysis of amide group formamide hydroxylation formate 84 Catabolism (3) hydrolytic cleavage of alanine 3-hydroxyanthranilate 85 Catabolism (4) 3-hydroxyanthranilate nicotinamide 2-oxoadipate 86 Decarboxylation of tryptophan tryptophan tryptamine 87 Conversion of tryptophan to melatonin sleep-wake cycle the hormone of darkness Trp hydroxylation 5-hydroxytryptophan decarboxylation N-acetylation O-methylation 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 Five vitamins are formed in the body, only four are utilized Vitamin Where and how produced Niacin Biotin Phylloquinone Calciol Cobalamine in tissues, from tryptophan large intestine (bacteria) large intestine (bacteria) skin, from cholesterol (UV radiation) large intestine (bacteria) – not absorbed! 90 Seven amino acids do not undergo transamination Amino acid a-NH2 group is removed as Arginine Lysine Methionine Threonine Tryptophan Proline Histidine ornithine 2-aminoadipate homoserine glycine alanine glutamate NH3 (desaturation deamination) 91 AA Biochemically 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, SO42-, PAPS, cysteamine (CoA) donor of -NH2 (urea, pyrimidines), oxaloacetate, fumarate, β-alanine (CoA) NH4+, 2-oxoglutarate, GABA, ornithine NH4+, donor of -NH2 (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 Overview: decarboxylation of amino acids AA Product Comments Ser ethanolamine part of phospholipids, precursor of choline Cys cysteamine part of coenzyme A (CoA-SH) Phe phenethylamine structural part of stimulants (amphetamine, ephedrine etc.) Tyr tyramine occurs in some foods, may cause migraine Asp b-alanine part of pantothenic acid, CoA-SH, carnosine Glu GABA gama-aminobutyric acid, inhibition neurotransmiter Lys cadaverine product of putrefaction (decay of proteins) Arg agmatine signal molecule in CNS His histamine triggers allergic reactions Trp tryptamine precursor of serotonine and melatonine DOPA dopamine catecholamine, precursor of noradrenaline/adrenaline Ornithine putrescine putrefaction product; precursor of spermidine/spermine