Amino acid metabolism II Metabolism of individual amino acids

Biochemistry I Lecture 7 2008 (J.S.) The degradation of amino acids usually begins with deamination. However, transamination or oxidative deamination is not the first reaction in catabolism of eight amino acids: Serine and threonine are deaminated by dehydration, and histidine undergoes deamination by desaturation (both reactions were mentioned previously). The five remaining amino acids are deaminated later on, after partial transformation: Arginine – deamination occurs after transfomation to ornithin, lysine – transamination follows the transformation to α-aminoadipate, methionine – deamination of homoserine, proline – deamination after conversion to glutamate, tryptophan – after its transformation to kynurenine, alanine is released.

2 Each carbon skeleton of deaminated amino acids follows a unique metabolic pathway to compounds , which can be completely oxidized by way of the citrate cycle to CO2 and water.

In spite of this common fate, amino acids are classified as glucogenic and ketogenic according to the type of their intermediate metabolites.

The glucogenic amino acids give rise to pyruvate or some of the intermediate of the citrate cycle, which can serve as substrates for gluconeogenesis. The ketogenic amino acids give rise to acetoacetate or acetyl-CoA (from which acetoacetate can be synthesized) that cannot be transformed to glucose.

3 Glucogenic and ketogenic amino acids

4 Irreversible conversions in the metabolism of amino acids show which proteinogenic amino acids are essential:

5 Nonessential amino acids Essential amino acids:

Glycine Threonine Alanine Serine Methionine Cysteine Lysine Aspartate Valine Asparagine Leucine Glutamate Isoleucine Glutamine Histidine Proline Arginine Phenylanine Tyrosine Tryptophan

6 The metabolism of amino acids will be described in the following sequence:

1 The most simple AA that give pyruvate – Ala, Ser, Gly, Thr 2 Amino acids containing sulfur – Met, Cys 3 Sources of one-carbon units and use of those units in syntheses 4 Aspartic acid 5 Glutamic acid and its relation to Arg, Pro, His 6 Branched-chain amino acids – Val, Ile, Leu 7 Lysine 8 Aromatic amino acids – Phe, Tyr, and Trp

7 1 Amino acids that are converted to pyruvate:

Alanine - by transamination. Serine - by deamination catalyzed of dehydratase (hydrolyase). Glycine - by accepting one-carbon group gives serine. Threonine - by splitting gives glycine that may give serine.

Cysteine also gives pyruvate by deamination and desulfuration (see "Amino acids containing sulfur"), as well as tryptophan that after transformation to kynurenin releases alanine (see "Aromatic amino acids").

8 Alanine is nonessential and glucogenic; it undergoes transamination to pyruvate readily: H C–CH–COOH 3 alanine aminotransferase (ALT)

NH2 H3C–CH–COOH H3C–C–COOH

NH2 Glu O pyruvate 2-oxoglutarate Concentrations of alanine in blood plasma are 300 – 400 μmol/l (the second highest next to glutamine). Alanine is released from muscle tissue and serves both as the vehicle for NH3 transport from muscle to liver and a substrate for liver gluconeogenesis. This bidirectional transport is called the alanine cycle (or glucose-alanine cycle). Blood Muscle Liver glucose glucose glycolysis gluconeogenesis pyruvate pyruvate

alanine alanine 9 Serine

CH2–CH–COOH

OH NH2 is nonessential and glucogenic;

– nonessential – synthesis of the carbon skeleton from 3-phosphoglycerate – glucogenic – direct deamination by serine dehydratase to pyruvate

Serine does not take part in transamination, but it is directly deaminated by dehydration:

H O 2 NH CH –CH–COOH CH =C–COOH CH –C–COOH 3 2 2 3 CH3–C–COOH = NH = OH NH2 NH2 O H2O enamine imine serine pyruvate

10 Serine is a substantial source of one-carbon groups:

its -CH2-OH group is readily transferred to tetrahydrofolate

(coenzyme of C1-group transferase), the product is glycine that

is able to yield the second C1-group. The reaction is reversible, but the synthesis of serine from glycine

and a C1-group is not an advantage.

CH2–CH–COOH CH2–COOH + H folate 4 + CH2OH–H4folate OH NH2 NH2

serine glycine hydroxymethyl-H4folate

Decarboxylation of serine results in ethanolamine (a constituent of phospholipids) that gives choline CH by methylation. + 3 HO–CH2–CH2–NH2 HO–CH2–CH2–N– CH3 CH ethanolamine choline 3 11 Demands for serine in the body are great – both one-carbon groups and substrates for the synthesis of complex lipids have to be supplied. Therefore, the synthesis of carbon skeleton from glucose is of great significance:

2-oxoglutarate NAD+ Glu COOH COOH COOH glucose CH–OH – C=O – CH–NH serine O O 2 O– CH2–O–P=O CH2–O–P=O CH2–O–P=O O– O– O– 3-phosphoglycerate 3-P-hydroxypyruvate 3-phosphoserine

12 Utilization of serine: glucose phosphatidyloserines 3-phosphoglycerate

phosphohydroxypyruvate Glu phosphatidylethanolamines phosphoserine ethanolamine glycine serine phosphatidylcholines choline CH -OH–H folate 2 4 acetylcholine

NH3 palmitoyl-CoA pyruvate

sphingosine sphingomyelins

13 Glycine is nonessential and glucogenic; CH2–COOH

NH2 – nonessential – originates from serine or from CO2, NH3, and C1-group

– glycogenic (weakly) – may accept C1-group and give serine Reversible reaction

glycine + CH2OH–H4folate serine + H4folate

(described as an important source of C1-groups) is not a useful

way of glycine catabolism, because it consumpts one C1-group. Transamination of glycine with pyruvate glycine + pyruvate glyoxylate + alanine as well as oxidative deamination of glycine

glycine + FAD glyoxylate + FADH2 are possible, although limited; the enzymes catalyzing those reactions have sufficient activity only in peroxisomes. It is worth mentioning that glyoxylate formed in those minor pathways gives small amounts of unwanted oxalate. High production of oxalate is dangerous. 14 The major pathway of glycine catabolism is oxidative cleavage of glycine in mitochondria:

CH2–COOH 5 10 + H4folate CO2 + NH3 + N ,N -methylene-H4folate NH2 glycine The reaction is reversible and catalyzed by glycine synthase and controlled by respiration and energetic charge of the cell. For the synthesis of glycine, 3 molecules of ATP are lost.

Molecule of glycine is the substrate required for the syntheses of several very important compounds, e.g. purine bases of nucleic acids, porphyrins of haemoproteins, phosphocreatine of skeletal muscles (phosphagen), and tripeptide glutathione (intracellular antioxidant).

15 (oxaluria) Utilization of glycine:

glyoxylate

Ala Synthesis of creatine pyruvate purine serine glycine porphyrin glutathione

hydroxymethyl-H4folate methylene-H4folate glycine conjugates CO NH of bile acids, 2 3 of aromatic acids Gly oxidative splitting / Gly synthesis (mitochondria) (hippuric acids)

16 Threonine is essential and both glucogenic and ketogenic It does not undergo transamination CH3–CH2–CH–COOH OH NH – glucogenic – gives glycine by splitting 2 or succinyl-CoA (by dehydratation and and oxid. decarboxylation to propionyl-CoA) – ketogenic – by splitting to glycine gives acetyl-CoA

Splitting of threonine to glycine

Ser CH OH transferase CH –CH –CH–COOH 2 CH2–COOH 3 2 CH2OH-H4folate NH OH NH2 2 serine glycine Thr dehydrogenase CH3–CH=O acetaldehyde specific lyase oxid. pyruvate 2-amino-3-oxobutyrate oxid.

CH3-CO–S-CoA acetyl-CoA

17 An alternative pathway is the direct deamination of threonine by dehydration:

H2O NH3 CH3–CH2–CH–COOH CH3–CH2–C–COOH [ enamine imine ] = OH NH2 O H2O 2-oxobutyrate

(slow) oxidative decarboxylation

CH3-CH2-CO–S-CoA propionyl-CoA

carboxylation to methylmalonyl-CoA

rearrangement (B12 coenzyme)

HOOC-CH2-CH2-CO–S-CoA sukcinyl-CoA

18 2 Sulfur containing amino acids

CH –CH –CH–COOH is essential and glucogenic Methionine 2 2

CH3–S NH2 – glucogenic (it yields succinyl-CoA)

Methionine is a common methyl donor in the cell:

PPi + Pi CH3 CH2–S ATP Rib-Ade CH2 CH-NH substrates CH2–S–CH3 2 CH COOH 2 methylated substrates CH-NH2 S-adenosylmethionine COOH CH –S methionine 2 Rib-Ade CH2

– CH-NH2 CH2 SH H4folate CH2 COOH

CH-NH2 S-adenosylhomocysteine CH3-H4folate COOH Ado remethylation (B12 coenzyme) homocysteine 19 Activated methionine NH S-adenosylmethionine (S-AM) 2 N N CH3 + N HOOC-CH-CH2-CH2–S N CH 2 O NH2 is the methyl donor. The methyl group may be transferred from a sulfonium ion to various acceptors. OH OH

Examples: synthesis of choline from phosphatidylethanolamine, synthesis of creatine (by methylation of guanidinoacetate), methylation of noradrenaline to adrenaline, inactivation of catecholamines by catechol-O-methyl transferase, methylation of histones, etc.

20 Catabolism of methionine – demethylation to homocysteine methionine – transsulfuration with serine to homoserine and cysteine – conversion to 2-oxobutyrate (homoserine deaminase), S-AM propionyl-CoA, and succinyl-CoA.

–CH3 + Ado

– H2O

serine homocysteine cysteine

cystathionine succinyl-CoA

+ H2O (coenz. B12) methylmalonyl-CoA

CoA-SH CO2

CO (biotin) NAD+ NADH + H+ 2 NH propionyl-CoA homoserine 3 2-oxobutyrate 21 Homocysteine is an important intermediate in metabolism of methionine; it is readily transformed, either remethylated to methionine (the reaction requires tetrahydrofolate and cobalamin) or decomposed to homoserine by transsulfuration with serine,

(vitamin B6 dependent).

If those mechanisms are not sufficient and the concentration of homocysteine in biological fluids increases, injury of endothelial cells by homocysteine (e.g., high production of reactive oxygen species, lipoperoxidation) and decreased vitality of blood platelets may appear.

At present, high concentration of homocysteine in blood plasma is included among other biochemical markers of cardiovascular diseases – as a risk factor for atherosclerosis that is quite independent on the concentration of cholesterol.

22 Cysteine is nonessential and glucogenic – nonessential – synthesis from serine

CH2–CH–COOH (methionine supplies the sulfur atom) – glucogenic – cysteine is converted into pyruvate SH NH2 2– – – (sulfur atom is released as SO3 , HS , or SCN ) The major catabolic pathway is the direct oxidation of SH-group:

CH2–SH O H+ CH-NH2 SO 2– H+O -S=O 4 COOH ll sulfate O oxidation of SH-group sulfite oxidase cysteine (mitochondrial dioxygenase)

+ O + NADPH + H+ O H 2– 2 SO3 H+O -S ll sulfite

O O O

ll ll S-O H+ S-O H+ CH 2-OG Glu 3 CH2 CH 2 C=O CH–NH2 C=O transamination COOH COOH COOH pyruvate cysteine sulfinate 3-sulfinylpyruvate 23 Oxidation of S–II to SIV or SVI (sulfinate, sulfite, sulfate) is a proton-producing process, nonvolatile acids are formed from non-ionized groups. The catabolism of sulfur-containing amino acids slightly acidifies the body.

An alternative catabolic pathway of cysteine is transamination :

CH –SH CH –SH 2 2-oxoglutarate 2 CH3 Glu CH-NH2 C=O desulfuration C=O COOH transamination COOH COOH 3-sulfanylpyruvate cysteine HS– H+ pyruvate

– 2– – Hydrogen sulfide HS ion is mostly oxidized to sulfite SO3 or, if cyanide ion CN is present (e.g. tobacco smokers), hydrogen sulfide gives thiocyanate SCN–.

Sulfite anion is oxidized to sulfate anion, which is either excreted into the urine (approx. 20 – 30 mmol/d) or utilized for sulfations after activation: excretion 2– mitochondrial sulfite oxidase 2– SO3 SO4 (molybdopterin, cyt b ) 3´-phosphoadenosyl sulfite 5 sulfate (ATP) 5´-phosphosulfate, PAPS 24 3‘-Phosphoadenosyl-5‘-phosphosulfate (PAPS)

NH2 is the mixed anhydride of sulfuric and N

phosphoric acid called "active sulfate"; N O

it serves as the sulfate donor in forming O N 5' N of sulfate esters (or N-sulfates). O–S–O–P–O– CH 2 O O O 3' O OH

O–P=O

Examples of sulfations by means of PAPS: O synthesis of proteoglycans (sulfation of glycosaminoglycans), sulfation of saccharidic components in glycolipids and glycoproteins, formation of sulfate esters in inactivation of steroid hormones, catecholamines, and in the phase II of biotransformation of phenols.

25 Utilization of methionine and cysteine

serine – 2H cystine methionine decarbox. cysteine 2-aminoethanethiol (cysteamine, constituent of coenzyme A) SAM homoserine methylations glutathione (γ-glutamyl-cysteinyl-glycine) succinyl-CoA 3-cysteine mercapturic acids sulfinate (N-acetyl-S-arylcysteines)

decarbox. 2– 2– SO4 SO3 pyruvate PAPS hypotaurine taurine sulfate esters conjugation with of sugars and phenols bile acids 26 Glutathione (GSH, γ-glutamyl-cysteinyl-glycine) is a tripeptide with a free sulfanyl group, γ CO–NH–CH–CO–NH–CH –COOH 2 required to maintain the normal reduced

CH2 CH2–SH state in the cell:

CH2 – 2H α (reduced form) 2 G-SH G-S–S-G CH–NH2 + 2H COOH Functions: 1 Reduced G-SH confronts oxidative stress, it reduces peroxides (lipid hydroperoxides and hydrogen peroxide) in the reaction catalyzed by a selenoprotein glutathione peroxidase, and (non-enzymatically) methaemoglobin (FeIII, hemiglobin) to haemoglobin (FeII) and disulfides to thiols:

L-OOH + 2 G-SH L-OH + G-S–S-G + H2O R-S–S-R + 2 G-SH 2 R-SH + G-S–S-G reduced G-SH can be regenerated by glutathione reductase and NADPH + H+. 2 Conjugation to lipophilic compounds (detoxification of reactive electrophiles). 3 Transport of amino acids into cells with concomitant attachment of γ-glutamyl (group translocation, γ-glutamyl cycle). 27 3 Sources of one-carbon groups and utilization of those groups in syntheses

One-carbon groups are transferred by tetrahydrofolate (H4folate, FH4, tetrahydropteroylglutamate). Mammals can synthesize the pteridine ring, but they are unable to conjugate it to the other two units. They obtain folate from diets or from microorganisms in their intestinal tracts.

Sites for bonding of one-carbon units

COOH OH H N CH2-NH– –CO–NH-CH-CH2-CH2-COOH N 5 10

H N 2 N N H glutamate tetrahydropteroic acid (1 – 5 residues)

28 The one-carbon groups transferred by H4folate exist in three oxidation states:

Example: CH 2 COOH OH

N CH2–N– –CO–NH-CH-CH2-CH2-COOH N 5 10

H N 2 N N H 5 10 N ,N -methylene FH4

(The fully oxidized one-carbon group is CO2, but CO2 is transferred by biotin, not by H folate.) 4 29 methylations

homocysteine S-AM

methionine

5 N -methyl FH4 thymine glycine, serine nucleotides NADH

N5,N10- methylene FH4 purine nucleotides

NADPH NH3 H2O 5 5 10 10 N -formimino FH4 N ,N -methenyl FH4 N -formyl FH4

histidine tryptophan 30 4 Aspartic acid and asparagine Aspartate is nonessential and glucogenic – it gives oxaloacetate by transamination HOOC–CH2–CH–COOH

NH2 Asparagine is the amide of aspartate

H2N-CO–CH2–CH–COOH

NH2 asparagine 2-oxoglutarate glutamate asparaginase

H2O NH3

AST

glutamate glutamine aspartate oxaloacetate + AMP + 2 Pi + ATP Asn synthetase

31 Utilization of aspartate and asparagine

NH3 for the synthesis of urea malate fumarate AMP from IMP purine

AST oxaloacetate aspartate incorporated into the skeleton of Glu pyrimidine bases

NH3 Glu CO2 β-alanine asparagine

NH3 transport in CNS ?

32 5 Glutamic acid, glutamine, and the relationship to proline, arginine, and histidine Glutamate is nonessential and glucogenic – it gives oxaloacetate readily by oxidative

HOOC–CH2–CH2–CH–COOH deamination or transamination NH 2 Glutamine is an amide of glutamate

H2N-CO–CH2–CH2–CH–COOH NH glutamine 2 pyruvate alanine glutaminase

H2O NH3

ALT

ADP + P NH + + ATP i 4 glutamate 2-oxoglutarate Gln synthetase 33 Direct oxidative deamination of glutamate by dehydrogenation

The reaction is catalysed by the mitochondrial enzyme glutamate dehydrogenase (GLD). It requires either NAD+ or NADP+ as coenzyme, and its activity in mitochondria is high.

HOOC-CH-CH2-CH2-COOH HOOC–C–CH2-CH2-COOH

NH2 NH 2-Iminoglutarate NAD(P)+ NAD(P)H + H+

glutamate H2O

NH3

HOOC–C–CH -CH -COOH The equilibrium favours glutamate 2 2 synthesis, but it is pulled in the direction O od deamination by the continuous 2-oxoglutarate + removal of NH3/NH4 . 34 Decarboxylation of glutamate (very active in brain)

– CO 2 γ

γ-aminobutyric acid (GABA) glutamate an inhibitory neurotransmitter in CNS Reversible reduction of glutamate proline

NADH+H+ NAD+ + 2 H H2O ornithine

glutamate glutamate 5-semialdehyde – intermediate in the synthesis and degradation of proline and arginine 35 Utilization of glutamate and glutamine

ornithine proline

glutamate semialdehyde histidine γ-aminobutyrate CO 2 (inhibitory neurotransmitter)

transaminases 2-oxoglutarate glutamate glutamate (excitatory neurotransmitter) AA 2-oxoA pteroate NH3 NH3

cysteine folate glutamine glycine

transport of NH3 to the liver and kidney

donor of NH3 for the syntheses of glutathione carbamoyl phosphate purine amino sugars 36 Glutamate is widely used as a food additive to enhance flavour of dishes, particularly in Chinese cookery in high amounts. Excess in the diet (1 – 5 g of glutamate in one dose, e.g. in the form of "Von-Ton“ soup) can cause unpleasant feelings in sensitive persons – the Chinese restaurant syndrome.

37 Arginine

CH2–CH2–CH2–CH–COOH is nonessential and glucogenic H N NH NH – nonessential in adult man (required in 2 C 2 the diet during the growth) NH – degraded to 2-oxoglutarate

In the liver, arginine is hydrolyzed to ornithine and urea. Ornithine serves as the substrate for ureosynthetic cycle:

arginase + H O 2 +

urea arginine ornithine

CO2 decarboxylation

putrescine (butan-1,4-diamine) for the ureosynthetic cycle synthesis of polyamines 38 After hydrolysis of arginine to ornithine, ornithine is degraded by transamination of the 5-amino group to glutamate 5-semialdehyde that gives glutamate and 2-oxoglutarate.

Nitroxide (nitrogen monoxide, NO) originates from arginine:

O . NADPH O . NADPH 2 2 •N=O + nitroxide (a radical) ω arginine N -hydroxyarginine citrulline

The reaction is a five-electron oxidation catalyzed by nitroxide synthase (NOS),

employing five redox cofactors (NADPH, FAD, FMN, cytochrome, H4biopterin). There are three isoenzymes of NOS: endothelial NOS responsible for vasodilation and inhibition of platelet aggregation, neuronal NOS modulation events on synapses (both are Ca2+-dependent), and NOS in phagocytes (NO – gives bactericidal peroxynitrite ONOO ). 39 Synthesis of creatine Arginine is the donor of amidino group for the synthesis of creatine:

S-AdoHcy (kidney) S-AM (liver)

creatine N1-methylguanidinoacetate

Creatine in skeletal muscles:

ATP ADP

creatine kinase

phosphocreatine (muscle phosphagen) H2O

slow non-enzymatic dehydration creatinine (excreted into the urine) 40 Proline (pyrrolidine-2-carboxylic acid) is nonessential and glucogenic – nonessential – originates from glutamate N COOH – glucogenic – it gives 2-oxoglutarate H

H O NAD+ NADH+H+ 2

1-pyrroline 5-carboxylate glutamate 5-semialdehyde proline (cyclic aldimine) + NAD + H2O NADH+H+ AA 2-oxoA 2-oxoglutarate glutamate

4-Hydroxyproline occurs only in collagen, and is formed by posttranslational hydroxylation of prolyl residues in procollagen polypeptide chains. Similarly to proline, 4-hydroxyproline is degraded to 4-hydroxyglutamate, which is cleft to pyruvate and glyoxylate. 41 Histidine is nonessential and glucogenic – nonessential for adults (essential for children) CH –CH–COOH 2 – glucogenic - it gives glutamate and 2-oxoglutarate

N NH2 N Histidine mostly does not undergo transamination, H it is deaminated directly by elimination (desaturation):

H2O NH3-lyase

NH histidine 3 urocanic acid (urocanate) 4-imidazolone-5-propionate

H2O

AA 2-oxoA 2-oxoglutarate glutamate

FH HN=CH–FH4 4 N-formimino-glutamate N5-formimino tetrahydrofolate (FIGLU) 42 Histamine is the product of histidine decarboxylation catalyzed by specific histidine decarboxylase:

CO2 histidine histamine

Histamine is a biogenic amine stored within granules of basophils and mast cells (more than 90 % body stores) and within synaptosomes of certain CNS neurons. When released, histamine induces complex physiological and pathological effects, including immunological reactions (symptoms of allergic conditions of the skin and airways), gastric acid secretion, smooth muscle contractions (e.g. bronchoconstriction), and profound vasodilatation. Histamine exerts its action via at least four distinct histamine receptor subtypes. Released histamine is metabolized by oxidation (to imidazolylacetic acid) or methylation (to tele-N-methylhistamine and tele-N-methylimidazolylacetic acid).

Antihistaminics – drugs which antagonize the effects of histamine. 43 Amino acids metabolized to 2-oxoglutarate – relationships:

proline histidine arginine urea

1-pyrroline-5-carboxylate urocanate ornithine

glutamate semialdehyde N-formiminoglutamate

glutamate 2-oxoglutarate 2-oxoA AA

glutamine 44 6 Branched-chain amino acids Leucine Isoleucine Valine CH CH CH3 3 3 are all essential, their CH CH2 CH CH3 CH3 3 final metabolites are different: CH CH CH2 valine is glucogenic,

CH–NH2 CH–NH2 CH–NH2 leucine is ketogenic, COOH COOH COOH isoleucine both gluco- and ketogenic.

These amino acids are taken up from the blood predominantly by skeletal muscles and their catabolism (transamination) begins there. The three initial catabolic reactions are common to all three branched-chain amino acids: – transamination to corresponding 2-oxoacids, – oxidative decarboxylation catalyzed by 2-oxoacid dehydrogenase producing corresponding acyl-CoA thioesters, and – the second dehydrogenation between carbons α and β catalyzed by flavin dehydrogenase resulting in corresponding 2-alkenoyl-CoA thioesters: 45 The resulting 2-alkenoyl-CoAs after three initial reactions:

valine leucine isoleucine

The following reactions differ (expected addition of water, hydration, occurs as the next reaction only in the case of valine and isoleucine).

Leucine is ketogenic. The alkenoyl-CoA is carboxylated (CO2 donor is carboxy-biotin) and the product hydrated to HMG-CoA that splits to free acetoacetate and acetyl CoA:.

acetoacetate H2O

biotin hydration acetyl-CoA CO2-biotin carboxylation 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) 46 Valine

succinyl-CoA

(B12)

CO2 carboxylation CoA-SH CoA-SH CO2 hydration oxidation decarboxylation propionyl-CoA methylmalonyl-CoA

Isoleucine

succinyl-CoA CoA-SH acetyl-CoA (B12)

carboxylation

hydration oxidation splitting CO2 methylmalonyl-CoA propionyl-CoA 47 Branched-chain amino acids – summary:

isoleucine leucine

valine

3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) acetyl-CoA propionyl-CoA propionyl-CoA acetyl-CoA succinyl-CoA succinyl-CoA acetoacetate

glucogenic ketogenic ketogenic glucogenic

48 7 Lysine is essential and ketogenic – it gives acetoacetyl-CoA CH2–CH2–CH2–CH2–CH–COOH

NH2 NH2

Lysine does not undergo transamination. Primarily, ε-deamination occurs through the formation of saccharopine:

glutamate ε NAD+ + + NADPH+H H2O H O 2 NAD+

reduction oxidation

α 2-oxoglutarate

lysine saccharopine allysine 2-aminoadipate

Transamination of α-amino group in 2-aminoadipate follows: 49 NAD+ 2-oG Glu CoA-SH CO2

CO2 FAD

glutaryl-CoA crotonoyl-CoA 2-aminoadipate 2-oxoadipate H O 2 hydratation NAD+ dehydrogenation

Lysyl side chains in collagen and elastin are oxidatively ε-deaminated to allysyl side chains. The aldehyde groups so formed react non-enzyma- ε acetoacetyl-CoA tically with each other, or with lysyl -NH2, and form covalent crosslinks (pyridinoline type in collagen, isodesmosine in elastin).

50 8 Aromatic amino acids phenylalanine, tyrosine, and tryptophan

All three amino acids are essential (though tyrosine is also formed by hydroxylation of phenylalanine), and both glucogenic and ketogenic, - phenylalanine and tyrosine give fumarate and acetoacetate, - tryptophan gives alanine and acetoacetyl-CoA.

51 Phenylalanine and tyrosine Hydroxylation of phenylalanine to tyrosine is catalyzed by a monooxygenase – phenylalanine hydroxylase,

for which the reducing coenzyme is tetrahydrobiopterin (BH4):

phenylalanine

O2 + 5,6,7,8-tetrahydrobiopterin NAD

NADH + H+ tyrosine

H2O q-7,8-dihydrobiopterin

Similarly, tyrosine is hydroxylated to DOPA by tyrosine 3-hydroxylase, and tryptophan to 5-hydroxytryptophan by tryptophan 5-hydroxylase. 52 hydroxylation transamination (O , BH ) 4-hydroxy- 2 4 2-oG Glu phenylpyruvate

O dioxygenase phenylalanine tyrosine 2

CO2 homogentisate (2,5-dihydroxy- phenylacetate)

O 2 1,2-dioxygenase

H O fumarate 2 maleinylacetoacetate isomerization acetoacetate fumarylacetoacetate 53 Inborn metabolic disorders of phenylalanine catabolism

(O2, BH4) (O2, BH4) phenylalanine tyrosine DOPA

2-oG ALBINISM PHENYLKETONURIA, Glu hyperphenylalaninaemias 4-hydroxyphenylpyruvate O 2 HYPERTYROSINAEMIA II CO2 homogentisate (2,5-dihydroxyphenylacetate) O 2 ALKAPTONURIA

maleinylacetoacetate

fumarylacetoacetate

H2O HYPERTYROSINAEMIA I fumarate + acetoacetate

54 Hyperphenylalaninaemia type I (classic phenylketonuria, PKU) Phe hydroxylase is a defect in phenylalanine hydroxylase, (O2, BH4) the ability to convert Phe to tyrosine is considerably impaired. (tyrosine) PKU have to be recognized through the compulsory screening of newborn infants phenylalanine and treated by a low-phenylalanine diet till the age of 8 – 10 years. transamination The consequence of untreated PKU is mental retardation (oligophrenia phenylpyruvica). Besides high levels of phenylacetate blood Phe, alternative catabolites are produced and excreted in high amounts (a "mousy" odour of the urine) : phenylpyruvate

o-hydroxyphenylacetate Malignant hyperphenylalaninaemias type IV and V

BH4 (tetrahydrobiopterin) is lacking due to the defective dihydrobiopterin biosynthesis from guanylate, or an ineffective reduction of BH2 to BH4. 55 Hypertyrosinaemias occur in several forms. They may be caused by a deficit of enzymes which catalyze either the transamination of tyrosine, or oxidation of p-hydroxyphenylpyruvate and hydrolysis of fumarylacetoacetate. A low-tyrosine diet may be very useful. Plasma levels of tyrosine are elevated, and large amounts of tyrosine, p-hydroxyphenylpyruvate, –lactate, and –acetate are excreted into the urine (tyrosyluria).

Alkaptonuria homogentisate 1,2-dioxygenase is an inborn deficit of homogentisate (homogentisate oxidase) oxidase characterized by the excretion of homogentisate in the urine. Except for the darkening of the urine on the air, there are no clinical manifestations in (maleinylacetoacetate) homogentisate youth until the second or third decade, when deposits of pigments in the connective tissue begins to appear oxidation to benzoquinoneacetate (ochronosis – bluish colouring of the by polyphenol oxidase scleras, the ear and nasal cartilages, or by the O2 in the air etc.) which are the cause of deforming and polymerization to black- arthritis. brown pigments 56 Biosynthesis of catecholamines

3-hydroxylation decarboxylation

(O , BH ) 2 4 - CO2

tyrosine DOPA (3,4-dihydroxyphenylalanine)

β-hydroxylation N-methylation

(O2, L-ascorbate) (S-AM)

dopamine noradrenaline adrenaline (norepinephrine) (epinephrine)

Inactivation of catecholamines occurs by means both oxidative deamination (monoamine oxidase, MAO) to acidic metabolites and 3-O-methylation (catechol-O-methyl transferase, COMT) to metanephrines. 57 Intermediates in the melanin biosynthesis Pigments melanins occurs in the eye, skin, and hair. The initial steps are a hydroxylation of tyrosine to DOPA and oxidation of DOPA to dopaquinone – both reaction in the pigment-forming cells are catalyzed by the copper-containing enzyme tyrosinase. The products of oxidation readily and spontaneously undergo polymerization resulting in insoluble dark pigments.

tyrosine DOPA dopaquinone

dopachrome

indole-5,6-quinone

black eumelanins ochre pheomelanins 58 Biosynthesis of the thyroid hormones Within the thyroid cell, at the cell-colloid interface, iodide anions are oxidized (to I+, IO–, or •I ?) by thyroperoxidase (TPO) and incorporated into tyrosyl residues of thyroglobulin:

tyrosyl residues 3,5-diiodotyrosyl residues TPO I–, H O 2 2 dehydroalanyl residue

TPO, H2O2

proteolysis

The coupling of iodotyrosyl residues iodinated thyronyl residue in thyroglobulin is also catalyzed by thyroperoxidase. Proteolysis of thyroxine (T4) thyroglobulin follows in lysosomes 3,5,3´,5´-tetraiodothyronine and thyroxine (or 3,3´,5´-T3) is secreted. 59 Tryptophan is essential and both glucogenic and ketogenic – after opening of the indole pyrrole ring, it releases alanine, the carbon atoms of aromatic ring give acetoacetate.

Tryptophan mostly does not undergo transamination. Catabolism of tryptophan is usually initiated by cleavage of the pyrrole ring of indole by tryptophan dioxygenase (tryptophan pyrrolase):

H2O Trp dioxygenase

+ O2, NADPH + H HCOO– N-formylkynurenine formate kynurenine

10 N -formyl TH4 pyruvate alanine

hydroxylation H2O + (O2, NADPH + H )

(B6)

3-hydroxyanthranilate 3-hydroxykynurenin60 (tryptophan)

3-hydroxyanthranilate

O2

CO 2 NAD+

CoA-SH CO2 hydrogenation

FAD NH3 CO2 2-aminomuconate crotonoyl-CoA 6-semialdehyde 2-oxoadipate glutaryl-CoA

H2O hydratation NAD+ dehydrogenation

acetoacetyl-CoA 61 Utilization of tryptophan

Nicotinate ring synthesis for NAD(P)+: Humans can provide nearly all of their nicotinamide requirement from tryptophan, if there is a sufficient amount of tryptophan in the diet. Normally, about two-thirds comes from this source:

3-hydroxyanthranilate

O2

PRPP

H O 2 PP i ⊕ CO 2-amino-3-carboxy- 2 quinolinate ribosyl 5´-phosphate muconate 6-semialdehyde nicotinate mononucleotide

2 ATP Gln AMP 2 PPi Glu 62 NAD+ 5-hydroxylation

O2 H2O

BH4 BH2 CO tryptophan 5-hydroxytryptophan 2 serotonin (5-hydroxytryptamine, 5-HT)

large intestine Serotonin is a neurotransmitter in CNS and a local hormone decarboxylation of argentaffin cells of the intestinal mucosa. It is degraded to 5-hydroxyindoleacetic acid (5-HIAA).

pineal gland (N--acetylation 5-O-methylation) tryptamine

melatonin (N-acetyl-5-methoxytryptamine) Secretion of melatonin from the pineal gland is increased in indole skatole darkness. Its physiologic roles remains to be elucidated, but they involve chronobiologic rhythms. (In frogs, melatonin is an antagonist of the melanocyte- stimulating hormone, MSH.) 63 The fate of the carbon skeleton of amino acids – summary:

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