1. AMINO ACID METABOLISM Jana Novotná Department of the Medical Chemistry and Biochemistry The 2nd Faculty of Medicine, Charles Univ.

2. Amino acid structure

3. The 20 common amino acids of proteins

4. Metabolic relationship of amino acids BODY PROTEINS Proteosynthesis Degradation AMINO ACIDS DIETARY PROTEINS GLYCOLYSIS KREBS CYCLE Digestion Transamination NONPROTEIN DERIVATIVES Porphyrins Purines Pyrimidines Neurotransmitters Hormones Komplex lipids Aminosugars UREANH 3 Conversion (Carbon skeleton) 250 – 300 g/day ACETYL CoAGLUCOSECO 2 KETONBODIES

5. Endopeptidases – hydrolyse the peptide bond inside a chain: pepsin, trypsin, chymotrypsin Exopeptidases – split the peptide bond at the end of a protein molecule: aminopeptidase, carboxypeptidases Dipeptidases Enzymes cleaving the peptide bond pepsin (pH 1.5 – 2.5) – peptide bond derived from Tyr, Phe, bonds between Leu and Glu trypsin (pH 7.5 – 8.5) – bonds between Lys a Arg chymotrypsin (pH 7.5 – 8.5) – bonds between Phe a Tyr

6. Essential amino acids in humans Arginine* Histidine* Isoleucine Leucine Valine Lysine Methionine Threonine Phenylalanine Tryptophan *Required to some degree in young growing period and/or sometimes during illness.

7. Non-essential and nonessential amino acids in humans Alanine Asparagine Aspartate Glutamate Glutamine Glycine Proline Serine Cysteine (from Met*) Tyrosine (from Phe*) * Essential amino acids Can be formed from  -keto acids by transamination and subsequent reactions.

8. C O R COO - + NH 4 + deamination transamination C O R COO - CH NH 2 R COO - CH NH 2 R COO - oxidative decarboxylation CH 2 NH 3 + RCO 2 + General reactions of amino acid catabolism

9. The fate of the amino group during amino acid catabolism

10. Transamination reaction The first step in the catabolism of most amino acids is removal of a-amino groups by enzymes transaminases or aminotransferases All aminotransferases have the same prostethic group and the same reaction mechanism. The prostethic group is pyridoxal phosphate (PPL), the coenzyme form of pyridoxine (vitamin B 6 )

11. Biosynthesis of amino acid: transamination reactions amino acid 1  keto acid 2 amino acid 2 +  -keto acid 1 Glutamate +  - Ketoglutarate + Pyridoxal phosphate (PLP)- dependent aminotransferase Keto-acid Amino acid

12. Active metabolic form of vitamin B 6

13. Mechanism of transamination reaction: PPL complex with enzyme accept an amino group to form pyridoxamine phosphate, which can donate its amio group to an  -keto acid.

14. All amino acids except threonine, lysine, and proline can be transaminated Transaminases are differ in their specificity for L-amino acids. The enzymes are named for the amino group donor.

15. Clinicaly important transaminases ALT Alanine-  -ketoglutarate transferase ALT (also called glutamate-pyruvate transaminase – GPT) Aspartate-  -ketoglutarate transferase AST (also called glutamate-oxalacetate transferase – GOT) Important in the diagnosis of heart and liver damage caused by heart attack, drug toxicity, or infection.

16. Glucose-alanine cycle Ala is the carrier of ammonia and of the carbon skeleton of pyruvate from muscle to liver. The ammonia is excreted and the pyruvate is used to produce glucose, which is returned to the muscle. Alanine plays a special role in transporting amino groups to liver. According to D. L. Nelson, M. M. Cox :LEHNINGER. PRINCIPLES OF BIOCHEMISTRY Fifth edition

17. Glutamate releases its amino group as ammonia in the liver The amino groups from many of the a-amino acids are collected in the liver in the form of the amino group of L -glutamate molecules.  Glutamate undergoes oxidative deamination catalyzed by L -glutamate dehydrogenase.  Enzyme is present in mitochondrial matrix.  It is the only enzyme that can use either NAD + or NADP + as the acceptor of reducing equivalents.  Combine action of an aminotransferase and glutamate dehydrogenase referred to as transdeamination.

18. Ammonia transport in the form of glutamine Glutamine synthetase Excess ammonia is added to glutamate to form glutamine. Glutamine enters the liver and NH 4 + is liberated in mitochondria by the enzyme glutaminase. Ammonia is remove by urea synthesis.

19. Relationship between glutamate, glutamine and  -ketoglutarate  -ketoglutarateglutamate glutamine NH 3 glutamate + NAD + + H2OH2O  -ketoglutarate NH 3 ++ NADH glutamate NH 3 + glutamine ATP ADP glutamine H2OH2O + glutamate NH 3 + A. Glutamate dehydrogenase B. Glutamine synthetase (liver) C. Glutaminase (kidney) From transamination reactions To urea cycle

20. Oxidative deamination Amino acidsFMNH2OH2O ++  keto acidsFMNH 2 NH 3 L-amino acid oxidase A. Oxidative deamination FMNH2O2H2O2 H2OH2OO2O2 + + + O2O2 catalse B. Nonoxidative deamination serine pyruvate threonine  -ketoglutateNH 3 + + Serin-threonin dehydratase L-amino acid oxidase produces ammonia and  -keto acid directly, using FMN as cofactor. The reduced form of flavin must be regenerated by O 2 molecule. This reaction produces H 2 O 2 molecule which is decompensated by catalase. Is possible only for hydroxy amino acids

21. Amino acid metabolism and central metabolic pathways 20 amino acids are converted to 7 products:  pyruvate  acetyl-CoA  acetoacetate   -ketoglutarate  succynyl-CoA  oxalacetate  fumarate

22. Glucogenic Amino Acids formed:  -ketoglutarate, pyruvate, oxaloacetate, fumarate, or succinyl-CoA Aspartate Asparagine Arginine Phenylalanine Tyrosine Isoleucine Methionine Valine Glutamine Glutamate Proline Histidine Alanine Serine Cysteine Glycine Threonine Tryptophan

23. Ketogenic Amino Acids formed acetyl CoA or acetoacetate Lysine Leucine

24. Both glucogenic and ketogenic amino acids formed:  -ketoglutarate, pyruvate, oxaloacetate, fumarate, or succinyl-CoA in addition to acetyl CoA or acetoacetate Isoleucine Threonine Tryptophan Phenylalanine Tyrosine

25. Alanine Serine Cysteine Threonine The C3 family: alanine, serine, cysteine and threonine are converted to pyruvate Pyruvate

26. The C4 family: aspartate and asparagine are converted into oxalacetate Aspartic acid Asparagine Oxalacetate

27. The C5 family: several amino acids are converted into  -ketoglutarate through glutamate Glutamine Proline Histidine Arginine  ketoglutarate

28. Interconversion of amino acids and intermediates of carbohydrate metabolism and Krebs cycle

29. Metabolism of some selected amino acids

30. Serine biosynthesis from glycolytic intermediate 3-phosphoglycerate Copy from: http://themedicalbiochemistrypage.org/amino-acid-metabolism.html

31. Glycine biosynthesis from serine Reaction involves the transfer of the hydroxymethyl group from serine to the cofactor tetrahydrofolate (THF), producing glycine and N 5,N 10 -methylene-THF. Copy from: http://themedicalbiochemistrypage.org/amino-acid-metabolism.html

32. Glycine oxidation to CO 2 Glycine produced from serine or from the diet can also be oxidized by glycine decarboxylase (also referred to as the glycine cleavage complex, GCC) to yield a second equivalent of N 5,N 10 -methylene-tetrahydrofolate as well as ammonia and CO 2. Copy from: http://themedicalbiochemistrypage.org/amino-acid-metabolism.html

33. The sulfur for cysteine synthesis comes from the essential amino acid methionine. SAM serves as a precurosor for numerous methyl transfer reactions (e.g. the conversion of norepinephrine to epinenephrine). Cysteine and methionine are metabolically related Condensation of ATP and methionine yield S-adenosylmethionine (SAM) SAM

34. Cysteine synthesis Copy from: http://themedicalbiochemistrypage.org/amino-acid-metabolism.html 1.Conversion of SAM to homocysteine. 2.Condensation of homocysteine with serine to cystathione. 3.Cystathione is cleavaged to cysteine. Conversion of homocysteine back to Met. N 5 - methyl-THF is donor of methyl group. * *folate + vit B 12

35. Genetic defects for both the synthase and the lyase. Missing or impaired cystathionine synthase leads to homocystinuria. High concentration of homocysteine and methionine in the urine. Homocysteine is highly reactive molecule. Disease is often associated with mental retardation, multisystemic disorder of connective tissue, muscle, CNS, and cardiovascular system. Homocystinuria

36. Biosynthesis of Tyrosine from Phenylalanine Phenylalanine hydroxylase is a mixed-function oxygenase: one atom of oxygen is incorporated into water and the other into the hydroxyl of tyrosine. The reductant is the tetrahydrofolate-related cofactor tetrahydrobiopterin, which is maintained in the reduced state by the NADH-dependent enzyme dihydropteridine reductase

37. Hyperphenylalaninemia - complete deficiency of phenylalanine hydroxylase (plasma level of Phe raises from normal 0.5 to 2 mg/dL to more than 20 mg/dL). The mental retardation is caused by the accumulation of phenylalanine, which becomes a major donor of amino groups in aminotransferase activity and depletes neural tissue of α-ketoglutarate. Absence of α-ketoglutarate in the brain shuts down the TCA cycle and the associated production of aerobic energy, which is essential to normal brain development. Newborns are routinelly tested for blood concentration of Phe. The diet with low-phenylalanine diet. Phenylketonuria

38. valineisoleucineleucine  -ketoglutarateglutamate (transamination)  -ketoisovalerate  -keto-  -methylbutyrate  -ketoisokaproate oxidative decarboxylation Dehydrogenase of  -keto acids* CO 2 NAD + NADH + H + isobutyryl CoA  -methylbutyryl CoA isovaleryl CoA Dehydrogenation etc., similar to fatty acid  -oxidation propionyl CoA acetyl CoA acetoacetate acetyl CoA propionyl CoA ++ Catabolism of branched amino acids

39. Branched-chain aminoaciduria Disease also called Maple Syrup Urine Disease (MSUD) ( because of the characteristic odor of the urine in affected individuals). Deficiency in an enzyme, branched-chain α-keto acid dehydrogenase leads to an accumulation of three branched- chain amino acids and their corresponding branched-chain α-keto acids which are excreted in the urine. There is only one dehydrogenase enzyme for all three amino acids. Mental retardation in these cases is extensive.

40. Histidine Metabolism: Histamine Formation Histidine Histamine Histidine decarboxylase CO 2 Histamine: Synthesized in and released by mast cells Mediator of allergic response: vasodilation, bronchoconstriction

41. Tryptophan catabolism Tryptophan has complex catabolic pathway: 1.the indol ring is ketogenic 2.the side chain forms the glucogenic products Kynurenate and xanthurenate are excrete in the urine.

42. Enzymes which metabolised amino acides containe vitamines as cofactors THIAMINE B 1 (thiamine diphosphate) oxidative decarboxylation of  -ketoacids RIBOFLAVIN B 2 (flavin mononucleotide FMN, flavin adenine dinucleotide FAD) oxidses of  -aminoacids NIACIN B 3 – nicotinic acid (nikotinamide adenine dinucleotide NAD + nikotinamide adenine dinukleotide phosphate NADP + ) dehydrogenases, reductase PYRIDOXIN B 6 (pyridoxalphosphate) transamination reaction and decarboxylation FOLIC ACID (tetrahydropholate) Meny enzymes of amino acid metabolism

43. http://themedicalbiochemistrypage.org/amino-acid-metabolism.html Helpful website