Amino Acid Metabolism
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Amino Acid Metabolism
- 1. Amino Acid Metabolism PSBCTC302-Intermediary metabolism M.Sc. Biochemistry
- 2. Proteins • Proteins comprise the most abundant organic compound forming major part of body dry weight (10-12 kg). Proteins perform a wide variety of functions including : 1. Static (structural) e.g., collagen – usually stable e.g., crystallin, the lens protein, lasts life time 2. Dynamic (receptors, hormones, enzymes, growth factors) – usually regulatory/short lived • The stability of cellular proteins varies from minutes to months and corresponding degradation in lysosome or proteasome is based on their N-terminus A.A. (N-end rule) or Pro, Glu, Ser, Thr (PEST) sequences. Digestion of dietary proteins into their repeating L-amino acid (A.A.) units, on the other hand, occurs in digestive tract by action of proteases in the digestive juices • About ~300-400 g of body protein (1-2%) gets turned over (synthesized and degraded) everyday into L- amino acid (A.A’s) for energy, elimination of damaged or abnormal proteins and metabolic regulation and homeostasis (synthesis of proteins, N-compounds, glucose and ketone bodies)
- 3. Energy storage Figure. Fuel composition of an average man (70 kg) after overnight fast as % of stored calories • Although protein is not preferred energy source, prolonged starvation or severe chronic illness may lead to breakdown of muscular protein and muscular wasting, a condition called CACHEXIA
- 4. Digestion of proteins • Entry of food stimulates stomach to release hormone gastrin which inturn stimulates release of pepsinogen and HCl. Upto 0.16 N (2.5 l/day) HCl is pumped using H+/K+-ATPase by pariteal/oxyntic cells of stomach. It kills microbes, denatures proteins and activates Pepsinogen (proenzyme/zymogen) to Pepsin (active form). Pepsin, secreted by chief cells, has self-activation activity, and is specific endopeptidase for proteins with tyrosine, tryptophan and phenylalanine • Entry of chyme in duodenum activates release of secretin and cholecystokinin-pancreozymin hormones which inturn stimulate release of pancreatic juice containing bicarbonate, three endopeptidases (trypsin, chymotrypsin and proelastase) and an exopeptidase (carboxypeptidase) • Presence of roughage in diet along with a layer of mucous trapped in mucins (membrane glycoproteins secreted by goblet cells in gut epithelial lining) protects intestine from proteolysis
- 5. Neutral cotransport ATPase Gastric HCl secretion CO2 + H2O H2CO3 Carbonic anhydrase CO2 H+HCO3 - H+ K+ K+ Cl- H+ K+ K+ Cl- HCl Anion channel HCO3 - Cl- CYTOPLASM STOMACH LUMEN (from blood)
- 6. Alkaline proteolysis in intestine Figure: Activation of pancreatic proteolytic enzymes (Zymogen, Active Enzyme) Chymotrypsinogen Chymotrypsin (Aromatic amino acyl bonds) Procaboxypeptidase Carboxypeptidase (C-terminal A.A.) Trypsinogen Trypsin Enteropeptidase Proelastase Elastase (C-terminus to A, G, S) (C-terminus to R, K)
- 7. A.A. absorption • Peptides and ⍺-A.A.’s are absorbed by stereospecific transport in duodenum and jejunum (upper small intestine). A.A.’s enter cell primarily by active Na+ co-transport. A.A.’s are released into the portal blood resulting in a range of A.A. concentrations, e.g., Glu and Gln are present at 24 and 640 µmol / L, respectively, with a total of about 100 gm free A.A. in adult human blood • At least 5 transporters are present in kidneys and intestine to remove amino acids from glomerular filtrate before excretion of urine. HARTNUP DISEASE is due to defective transport of large neutral and aromatic A.A.’s while CYSTIURIA is due to defective transport of basic amino acids
- 8. Inter-organ A.A. exchange A.A.’s Branched chain A.A.’s Glucose Ala Val Urea Citrulline Ser Ala 30% Amm. 70% Amm. Urea Gln G-ase G-ase Urea Cycle
- 9. Glutathione Cys-Gly 𝜸-Glu-A.A. • Glutamate uptake by cell Outside cell Cell membrane Inside cell L-Glu 𝜸-GLUTAMYL CYCLE Glutathione Glutathione synthase Cys Gly Dipeptidase 5-Oxoproline Cys-Gly 𝜸-Glu-A.A. 𝜸-Glu-Transpeptidase 𝜸-Glu-Cys
- 10. Protein catabolism Amino acidsProteins Keto acids Acetyl CoA CO2, H2O Fat Ketogenic Pyruvate, ⍺-KG, S-CoA, F, OA Glucose/Glycogen Glucogenic Ammonia Urea • ~75% of liberated A.A.’s are reused while rest serve as precursors for amphibolic intermediates
- 11. Side chains [R-] of L-⍺-A.A.’s Aliphatic R- (G, A, V, L, I) Hydroxyl R- (S, T, Y) Glycine (Gly, G) Serine (Ser, S) Alanine (Ala, A) Threonine (Thr, T) Valine (Val, V) Basic side chain (K, R, H) Leucine (Leu, L) Lysine (Lys, K) Isoleucine (Iso, I) Acidic R- or its amides (D, E, N, Q) Arginine (Arg, R) Aspartic acid (Asp, D) Glutamate (Glu, E) Sulphur containing R- (C, M) Asparagine (Asn, N) Cysteine (Cys, C) Glutamine (Gln, Q) Methionine (Met, M)
- 12. Side chains [R-] of L-⍺-amino acids Aromatic R- (F, Y, W, H, P) Phenylalanine (Phe, F) Tyrosine (Tyr, Y) Tryptophan (Trp, W) Histidine (His, H) Proline (Pro, P)
- 13. Cofactors involved in A.A. metabolism Cofactor Function Structure Chemistry 1 Pyridoxal-5’-Phosphate (PLP) Transamination, decarboxylation, deamination, racemization Vitamin B6 derivative Forms an enzyme bound Schiff’s base (pyridoxamine phosphate) intermediate 2 Tetrahydrofolate (THF) 1-C unit carrier Folic acid derivative Essential vitamin. Multiple oxidation states. 1-C carrier {Methyl (CH3-), methylene (-CH2-), formyl (-CHO), methenyl (-CH=), formimino (-CHNH)} 3 S-adenosylmethionine (SAM) Carboxylation ATP and Methionine Biologically prefered C-donor 4 Biopterin Carboxylation Pteridine ring derivative Involved in oxidation of Phe to Tyr
- 14. Biopterin 5, 6, 7, 8-Tetrahydrobiopterin (BH4/THB/Sapropterin)Dihydrobiopterin (BH2)
- 15. Tetrahydrofolate (THF) Folate Tetrahydrofolate (THF) 2 NADPH + H+ 2 NADP+ Dihydrofolate reductase N10-Formyl-THF HCOO- + ATP ADP + Pi N10-formyl-THF synthetase N5-Methyl-THF 2 NADPH + H+2 NADP+ N5-N10-methenyl-THF cyclohydrolase and reductase {6-methyl pterin with R = p-aminobenzoic acid attached to glutamates (1-6)}
- 16. S-adenosylmethionine (SAM) Methionine Adenosine S-adenosylmethionine Methyl transferase R- R-CH3 S-adenosyl-homocysteine Methionine Methionyl adenosyl transferase THF + ATPN5-Methyltetrahydrofolate + PPi +Pi HomocysteineH2O Adenosine Methyl transferase N5-methyl THF THF Vit. B12
- 17. Pyridoxal phosphate (PLP) Pyridoxal-phosphatePyridoxine PLP-Schiff base Pyridoxamine-phosphate
- 18. Reactions in A.A. metabolism • Transamination followed by deamination is the major route for A.A. catabolism. A.A.’s also undergo direct decarboxylation to amines, which further get deaminated to urea, CO2 and H2O 3 ATP 2 ADP + 2 Pi + AMP +PPi NH3 + HCO3 - + Aspartate Urea + Fumarate • Ammonia is fixed into urea by urea cycle for excretion. Most aquatics are ammonotelic (excrete ammonia). Terrestrial vertebrates and sharks are ureotelic (excrete uric acid) while birds and reptiles are uricotelic (excrete urea) • Transamination by aminotransaminases involves transfer of nitrogen from A.A., such as Ala, to pyridoxal phosphate which becomes pyridoxamine phosphate followed by transference of amine to ⍺-ketoglutarate yielding glutamate. Glutamate dehydrogenase releases ammonia from glutamate by oxidative deamination. Conversion of ⍺-amino nitrogen to ammonia using concerted transamination and deamination is called as transdeamination
- 19. • Objective: Synthesis of non-essential A.A.’s and equilibration of amino groups from excess dietary A.A’s to keto acids or A.A.’s which can be deaminated (e.g., E, A). Undergone by all amino acids except K, T, P, P-OH • Reaction: Reversible conversion of amino acid into corresponding keto acid with concomitant conversion of keto acid into amino acid by ⍺-amino group transfer • Enzyme: Aminotransaminases Transamination • Coenzyme: pyridoxal-5’-phosphate (PLP)
- 20. Examples of transamination 1. Interconversion of aspartate and ⍺-ketoglutarate into oxaloacetate and glutamate by serum glutamate- oxaloacetate-aminotransferase (GOT) also called aspartate aminotransferase (AST) found in cytoplasm as well as mitochondria 2. Interconversion of alanine and ⍺-ketoglutarate into pyruvte and glutamate by serum glutamate-pyruvate aminotransferase (GPT), also called alanine transaminase (ALT) primarily found in liver • Circulating levels of AST and ALT are key liver function test Aspartate Oxaloacetate ⍺-Ketoglutarate Glutamate ⍺-Ketoglutarate Alanine Pyruvate Glutamate
- 21. Oxidative deamination • GDH is a Zn containing mitochondrial enzyme unique in using both NAD+ or NADP+ as cofactor. GDH has six identical subunits of 56 kDa each and is allosterically regulated (inhibited by GTP, ATP, steroid and thyroid hormones; activated by Leu, GDP and ADP) for energy production. GDH is very active in brain to detoxify ammonia • Liberation of ⍺-amino N- of glutamate yields ammonium (NH4 +) ion and ⍺-ketoglutarate in a reversible reaction catalyzed by glutamate dehydrogenase (GDH) L-Glutamate Imino acid (Schiff-base) ⍺-Ketoglutarate
- 22. Minor routes for deamination Hydrolytic Reductive Intramolecular NH4 + Fatty acid Unsaturated fatty acidHydroxy acid OROR
- 23. Decarboxylation Monoamine decarboxylation Diamine decarboxylation H+ CO2 Lysine Cadaverine Ornithine Putrescine H+ CO2 Glutamate GABA H+ CO2 Histidine Histamine H+ CO2 Decarboxylase (PLP)
- 24. Urea Cycle Steps involved in urea cycle 1. A.A. transamination in peripheral cells forms glutamate 2. Deamination of glutamate in liver forms ammonia. (Arginase is found in liver only) 3. Besides, minor pathways of oxidative and non-oxidative deamination produces NH3 in peripheral cells and this ammonia is transported to liver as glutamate, glutamine or alanine 4. Ammonia is detoxified in liver to form urea by five reactions (2 mitochondrial, 3 cytosolic) • Urea cycle involves transference of ammonium ion and amino group of aspartate through a series of intermediates to arginine, which is cleaved to generate urea and regenerate the amino acid ornithine • When urea is not eliminated, ammonium ions concentration increases and crosses BBB. There, it is converted to glutamate by glutamate dehydrogenase, thereby depleting brain ⍺-ketoglutarate, inturn halting TCA cycle. Neural damage and cell death ensues
- 25. Carbamoyl Phosphate HCO- 3 + NH4 + Carbamoyl phosphate synthetase I N’-acetyl-E 2 ATP 2 ADP +Pi H2O Urea UREA CYCLE Arginase Pi Citrulline Ornithine Mitochondria Ornithine trans- carbomylase Arginosuccinate Cytoplasm Aspartate Argininosuccinate synthetase ATP AMP + PPi Arginine Fumarate Argininosuccinase ORNITHINE TRANSLOCASE SYNDROME CITRULLINEMIA I ARGINOSUCCINATE ACID URIA HYPERARGININEMIA HYPERAMMONEMIA
- 26. Fates of C-skeletons of A.A.’s 3. ⍺-Ketoglutarate 2. Oxaloacetate Acetyl CoA Glu Gln Orn Arg ProHis AspAsn Acetoacetate Ketone bodies HMG CoA Ile Lys Trp Alanine 6.Leu 5.Strictly ketogenic 7. FumarateTyrPhe Acetoacetate 4. Succinyl CoA Methylmalonyl CoA Val Ile Met Thr 1. Pyruvate AlaCys Trp Ser Gly
- 27. Serine, Glycine and Cysteine degradation 1. Pyruvate CysSer Gly NH4 + Ser/Thrdehyrdatase Pyruvate N5, N10-Methylene THF THF Serine trans hydroxymethylase Glycine L-Glu ⍺-KG O2, NADP+ NADPH + H+ SO3 - H2O Serine + NH4 + H2O Homocysteine Cysteine Cystathione synthase Cystathione ⍺-Ketobutyrate Cystathionase H2O HOMOCYSTINURIA
- 28. H2O Glutaminase Glutamate Glutamine N5-Formimino-THF TH F Histidine Histidase Uroconase H2O Formimino-L-Glutamate Proline Arginase Ornithine Arginine ⍺-KG L-Glu Glutamate 𝜸-semialdehyde NAD+ NADH + H+ H2O NH4 + ATP ADP +Pi Glutamine synthetase NH4 + Glutamine, Glutamate, Histidine, Arginine and Proline Degradation
- 29. 4. Succinyl CoA Methylmalonyl CoA Val Ile Met Thr Threonine degradation Ketobutyrate dehydrogenaseThreonine dehydratase (PLP) NAD+ NADH + H+ CoASH CO2 Methylmalonyl CoAPropionyl CoA⍺-KetobutyrateThreonine ATP ADP + Pi CO2 Succinyl CoA Adenosyl cobalamine
- 30. Valine degradation Transamination Keto acid dehydrogenase complex NAD+ NADH + H+ CoASH CO2 FAD FADH2 Dehydrogenase NADH + H+ NAD+ Methylmalonyl semialdehyde Methylmalonyl CoASuccinyl CoA Hydratase H2O H2O CoASH 4. Succinyl CoA Methylmalonyl CoA Val Ile Met Thr Valine ⍺-Ketoisovalerate Isobutyryl CoA Methacrylyl CoA β-hydroxyisobutyryl CoA NADH + H+ NAD+ CoASH Adenosyl cobalamine (B12) Dehydrogenase
- 31. 4. Succinyl CoA Methylmalonyl CoA Val Ile Met Thr Isoleucine degradation + Transamination ⍺-Keto-β-methylvalerate ⍺-Methylbutyryl CoA Tiglyl CoA ⍺-Methyl-β-hydroxybutyryl-CoA⍺-Methyl-acetoacetyl-CoAPropionyl-CoAAcetyl CoA CoASH H2O [2H] Ketomethylvalerate dehydrogenase complex NAD+ NADH + H+ CoASH CO2 FAD FADH2 Dehydrogenase Hydratase Dehydrogenase
- 32. Leucine degradation Leucine ⍺-KG Glu Leucine- ⍺-ketoglutarate transaminase ⍺-Ketoisocaproate NAD+ NADH + H+ Isovaleryl CoA CoASH CO2 ⍺-Ketoisocaproate dehydrogenase, lipoate, TPP, FAD Isovaleryl dehydrogenase FADH2FAD+ 3-Methylcrotonyl CoA3-Hydroxy-3-methyl- glutaryl CoA (HMG CoA) 3-Methylgluconyl CoA hydratase H2O ADP + Pi ATP + H2O 3-Methylcrotonyl CoA carboxylase (biotin) 3-Methylgluconyl CoA CO2 Acetyl CoA Acetoacetate Acetoacetate HMG CoA Leu Acetyl CoA
- 33. Lysine degradation Lysine L-Saccharopine NADPH + H+ NADP+ 5. Acetoacetate Lys Glutamate 2-Aminoadipaldehyde NADH + H+ NAD+ 2-Aminoadipate2-Ketoadipate L-Glu ⍺-KG Crotonyl CoA CO2 CoASH CO2 ⍺-Ketoglutarate
- 34. Tryptophan degradation by kynurenine pathway Tryptophan Tryptophan pyrrolase N-Formyl kynurenine O2 Kynureinine 3-Monoxygenase O2, NADPH + H+ H2O, NADP+ 3-Hydroxykyurenine3-Hydroxyanthranilate Alanine O2 Kyureniase (PLP) Acetoacetate ⍺-Amino-muconate Dioxygenase O2 CO2 NAD+ NADH + H+ NH4 + H2O ⍺-Ketoadipate 1. Pyruvate AlaCys Trp Ser Gly 6. Acetoacetate Trp Alanine L-Kynurenine H2O HCOO- Formamidase
- 35. Phe & Tyr degradation PHENYLKETONURIA Homogentisate p-Hydroxyphenylpyruvate Phenylalanine Fumarylacetoacetate hydrolase Fumarate Acetoacetate + MAA Isomerase 4- Fumarylacetacetate 4-Maleylacetoacetate Homogentisate oxidase TYROSINEMIA I ALKAPTONURIA 7. Fumarate Acetoacetate Tyr Phe TYROSINEMIA II TYROSINEMIA III
- 36. A.A. Biogenesis Glucogenic Ketogenic Gluco- + Ketogenic His Leu Iso Met Lys Phe Thr Try Val Gly Tyr Ser Asp Cys Asn Arg Glu Gln Ala Pro Non-essentialEssential ESSENTIAL AMINO ACID GROUPED AS PER PRECURSOR: 1. Pyruvate precursor - Pyruvate family (Val, Leu) 2. Aspartate precursor - Asp family (Lys, Met, Thr, Iso; this pathway also produces homoserine and homocysteine. Latter can be converted to cysteine using Vitamin B6 and Ser) 3. PEP + E-4-P (+ Ser for Trp) precursor - Aromatic A.A.’s (Tyr, Phe, Trp) 4. 5-Phosphoribosyl-⍺-pyrophosphate – (His) NON ESSENTIAL A.A. SYNTHESIS REQUIRES 3 SETS OF INTERMEDIATES: 1. Transamination of pyruvate, oxaoacetate and ⍺-ketoglutarate forms Ala, Asp, Glu respectively, while amidation of Asp, Glu gives Asn and Gln 2. Glu gives Pro and Arg while Gln gives His 3. 3-Phosphoglycerate forms Ser which in turn forms Cys and Gly. Gly is also produced from CO2, NH4 +, and N5, N10 –methylene-THF
- 37. Acetyl CoA ⍺-Ketoglutarate Oxaloacetate Orn Arg Pro His Glu Gln Pyruvate Phosphoenolpyruvate 3-PhosphoglycerateSerGly O-AcetylserineCys A.A. biogenesis Ala Leu Val 1. Asn Asp-4-semialdehyde Homoserine DAP LysHomocys MetIso Thr Asp2. Erythrose-4-phosphate DAHP Shikimate Chorismate AnthranilateTry Prephenate Tyr Phe 3.
- 38. • Ser may be synthesized from 3-phosphoglycerate by oxidation, transamination, and removal of the phosphate by dehydrogenase, transaminase and phosphatase, respectively. Reverse metabolism of Ser to 3-phosphoglycerate occurs by transamination, reduction, and phosphorylation (glycerate kinase) NAD+ NADH + H+ Glu ⍺-Ketoglu H2O Pi 3-phospho- glycerate 3-Phospho hydroxypyruvate 3-Phosphoserine Serine Serine biosynthesis 3-PGSerGly O-AcetylserineCys
- 39. Decarboxylation Transamination Oxidation Oxidation Transamination Ethanolamine Glycolaldehyde Glycolate Glyoxylate GlycineSerine Glycine biosynthesis NADH + H+ N5, N10-methylene THF THF Glycine H2O Serine Serine hydroxymethyl transferase • Glycine aminotransferase can catalyze the synthesis of Gly from glyoxylate and Gln or Ala • Additional routes of Gly synthesis from choline and Ser also exist 3-PGSerGly O-AcetylserineCys
- 40. Asparagine synthesis • Asparagine synthetase catalyzes Asn synthesis from Asp by transamination or direct amination while the reverse reaction is catalyzed by asparaginase Asparagine synthetase Gln, ATP Glu, ADP + Pi Aspartate Asparagine H2O NH4 + Asparaginase Aspartate Asparagine Asparagine synthetase NH4 +, ATP AMP + PPi Asparaginase H2O NH4 + OxaloacetateAsn Asp-4-semialdehyde Homoserine DAP LysHomocys MetIso Thr Asp2.
- 41. 3-Deoxyarabino- heptulosanate 7-phosphate Shikimate Phosphoenolpyruvate Erythrose-4-phosphate ChorismateAnthranilateTryptophan Indole Erythrose-4-phosphate Glu Gln + Pyruvate PPi PRPP + Gly-3-P + CO2 + OH- H2O Ser E-4-P DAHP Shikimate Chorismate AnthranilateTry 3. PEP Tryptophan synthesis ATP ADP + Pi + Pi H2O
- 42. Prephenate Phenylpyruvate p-hydroxyphenylpyruvate Tyrosine Phenylalanine Chorismate Phe and Tyr synthesis Mutase ⍺-KG Glu ⍺-KG Glu NAD+ NADH + CO2 H2O E-4-P DAHP Shikimate Chorismate Prephenate Tyr Phe 3. PEP
- 43. A.A. Precursor Distinguishing features of Pathway Ala Pyruvate By transamination Glu ⍺-Ketoglutarate By reductive amination or transamination Asp Oxaloacetate By transamination Gln Glu ADP + Pi are products Asn Asp and Gln AMP + PPi are products Ser 3-Phosphoglycerate Hydroxypyruvate and phosphoserine intermediates Gly Ser Requires N5, N10 THF Met Homocys Requires N5-methylene THF Cys Ser and Homocys Cystathione intermediate Tyr Phe Requires biopterin Pro Arg Glutamate-𝜸-semialdehyde intermediate Arg Glu Ornithine intermediate Points to remember in A.A. biosynthesis
- 44. Regulation of A.A. metabolism • Glutamine synthetase is the enzyme involved in reversible glutamine biosynthesis from glutamate using ATP. Bacterial enzyme has 12 identical subunits (Hex prism). It undergoes three levels of regulation: a) allosteric feedback inhibition: Increase in concentration of 9 downstream products, 6 of which viz., His, Try, Carbamoyl phosphate, AMP, CTP, glucosamine-6-phosphate, Gly, Val, Ala, Tyr, CDP and ADP are end products of pathways from glutamine, while 3 reflect cellular N-level (Ala, Ser, Gly) b) covalent modification by adenylation of specific Tyr reduces activity c) genetic control at transcriptional level. • Acetoacetate synthase converts pyruvate to acetoacetate. Acetoacetate is used for synthesis of Leu and Val which inhibits acetoacetate synthase • Thr hydratase shows competitive feedback inhibition to product of reaction Iso, which is a structural analogue of substrate Thr
- 45. Regulation of A.A. metabolism • Certain enzymes of A.A. metabolism like Carbamoyl phosphate synthetase I, Tryptophan synthase and Glutamate synthetase exhibit substrate channeling. Substrate channeling increases the rate of a metabolic pathway by generating high local concentrations of intermediates and by preventing their loss or degradation. Reaction intermediates travel some distance from one active site to the next without dissociating from the enzyme
- 46. Metabolism of A.A. precursors Amine derivative Precursor Physiological role 1. Polyamines Ornithine Putrescine, spermine, and spermidine are chromatin associated 2. Creatine Gly + Arg Phosphocreatine in muscle undergoes spontaneous dephosphorylation and is excreted as creatinine 3. Carnitine Lys (-Protein) Shuttles fatty acids across the mitochondrial membrane 4. Neurotransmitters 4.1 𝜸-aminobutyrate (GABA) Glu Inhibits synaptic transmission 4.2 Histamine His Expands capillaries and constricts veins, resulting in increased local blood volume, and edema, with low b.p. during allergies 4.3 Acetylcholine Ser + Met Induces muscular activity • A.A.’s act as precursors to synthesis of physiologically important amines that show potent bioactivity. Examples: Polyamines, creatine, carnitine, neurotransmitters, melanin, betaine, bile salts, haem, purines, choline, glycosamine, purines and pyrimidines, pharmacological amines and hormones
- 47. Metabolism of A.A. precursors A.A. derivative Precursor Physiological effect 4.4 Serotonin Try Induces sleep (W-rich diet) 4.5 Catecholamines (L-dopa, dopamine, epinephrine, and norepinephrine) Tyr Increase cardiac output and cellular metabolism during stress 5 Melanin Tyr Pigmentation 6 Purines Gly + Glu Nucleic acid biosynthesis 7 Glutathione Gly + Glu Reducing environment 8 Nitric oxide Arg Signalling 9 Porphyrins Gly or Glu Nucleus of haem proteins like cytochromes and hemoglobin
- 48. Melatonin Nicotinamide (B3) Serotonin Histidine Histamine Catecholamines Melanin T3, T4 Glutamate 𝜸-Aminobutyric Acid Carnitine Betaine SAM Bile salts Haem Glycine Glutathione Purines Hippuric acid Products from A.A. precursors Creatine Polyamines NO
- 50. Creatine + Glycine Arginine Guanidinoacetate Creatine (methyl guanidinoacetate) S-adenosylhomocysteine (SAdoHcys) S-adenosylmethionine (SAdoMet) Amidinotransferase Ornithine Methyltransferase Creatine kinase ADP ATP PhosphocreatineCreatinine Pi H2O Slow Slow
- 51. Protease Fe2+- TML Hydroxylas e SAdoHcys SAdoMet N6-trimethyllysine (TML) Glycine Protein bound trimethyllysine 3-Hydroxy-N6-trimethyllysine (HTML) HTML Aldolase Carnitine 𝜸-Butyrobetaine (𝜸BB) Carnitine synthesis ⍺-KG Succinate O2 CO2 TMABA Dehydrogenase 4-N-trimethylaminobutyraldehyde (TMABA) NADH NAD+ Fe2+-BBHydroxylase Succinate ⍺-KG CO2 O2 Protein bound lysine
- 52. Tryptophan hydroxylase (H4-Biopterin) 5-HT Decarboxylase (PLP) Tryptophan 5-Hydroxytryptophan Serotonin Serotonin synthesis Serine CO2 Ethanolamine CoASH Choline acetyltransferase Choline SAdoHcys SAdoMet Acetylcholine Acetate H2O Acetylcholine synthesis THB DHB
- 53. Synthesis of Catecholamines and Melanin Tyrosine L-Dopa (3,4-dihydroxyphenylalanine) O2 H2O H4-biopterin H2-biopterin NADPH + H+ NADP+ Norepinepinephrine Epinephrine O2 SAdoMet SAdoCysCu+-Protein Cu2+-Protein Ascorbate Dehydroascorbate Dopamine CO2 ALBINISM Tyrosinase Melanin Dopaquinone Tyrosinase
- 54. Succinyl CoA Glutamate + Porphyrin synthesis 𝛿-Aminolevulinate⍺-Amino-β-ketoadipate CoA-SH CO2 𝛿-aminolevulinate synthase 𝛿-aminolevulinate synthase Glutamyl-tRNAGlu Glutamate 1-semialdehyde ATP AMP + PPi NADPH NADP+tRNAGlu tRNAGlu Glutamyl-tRNA reductase Glutamyl-tRNA reductase Glu-1-semialdehyde aminomutase Protoporphyrin Porphobilinogen H2O Glycine
- 55. Nitric oxide synthesis • Nitric oxide synthase (NOS) is a mixed function oxidase that converts arginine to citrulline and NO. It uses calmodulin and flavin cofactors. NOS is particularly associated with immune responses and nervous and cardiovascular systems • Nitric oxide, with superoxide (O2 -) generates free radicals and has a half life of 5 seconds only. So, the continued activity of NOS is required for the induction of endotoxic shock following a bacterial infection Arginine N-⍵-Hydroxyarginine NADPH + H+ NADP+ NOS H2O Citrulline NADPH + H+ NADP+ NOS O2 H2O + NO Nitric oxide
- 56. Metabolic disorders of A.A. metabolism • MAPLE SYRUP URINE DISEASE is caused due to absence or deficiency of branched chain ⍺-keto acid dehydrogenase resulting in blockage in catabolism of branched chain A.A.’s (L, I, V) and odor of maple syrup in excretory products. Also results in severe neurological symptoms and shortened lifespan. Treatment is dietary restriction of branched chain A.A.’s • ALKAPTONURIA is an autosomal recessive disorder caused due to deficiency of homogentisate oxidase that converts homogestinate to maleylacetoacetate resulting in excretion of homogentistic acid (HA). HA is converted to black color alkapton bodies. Relatively benign condition, though patients might develop arthritis and black pigmentation due to subcutaneous deposition of alkapton bodies in later life • ALBINISM is an inborn error caused due to deficiency of copper-containing tyrosinase in melanocytes causing defective synthesis of melanin from tyrosine. Patient has reduced pigmentation of skin, hair and iris, and eyes may become pink. LEUCODERMA is localized absence of melanin due to degeneration of melanocytes
- 57. Phenylketonuria (PKU) • PKU is a group of genetic disorders of metabolism of phenylalanine. They are characterized by a functional deficiency of phenylalanine hydroxylase. This results in accumulation of phenylpyruvate, phenyllactate, phenylacetate and phenylacetate glutamine from their normal concentration of ~1 mg/dl to 10-60 mg/dl. These metabolites cross BBB causing cellular toxicity • PKU afflicts 1/10-15000 newborn infants. Heterozygotes and less severe mutations have milder symptoms. Untreated infants may lose 50 IQ points a year. Ultimately, 96-98% of untreated classic PKU children have an IQ below 50 • Treatment involves restriction of phenylalanine and supplementation of tyrosine in diet throughout life. 97% of treated PKU infants grow to have normal IQs
- 58. Metabolic by-products of phenylalanine that accumulate in PKU Phenylalanine Tyrosine Phenylalanine hydroxylase DHB THB, O2 Phenyl acetatePhenyl pyruvatePhenyl lactate [2H] CO2 Phenylacetyl glutamine Gln H2O NADP+ NADPH + H+ Dihydropteridine reductase O-Tyrosine O-Hydroxy phenylpyruvate O-Hydroxy phenylacetate CO2 O2 [2H] PKU
- 59. Suggested reading • Biochemistry by: Lehninger, Nelson and Cox • Biochemistry by: Voet and Voet • Biochemistry by: Lubert Stryer • Harpers illustrated Biochemistry
Editor's Notes
- Collagen constitutes present in supportive tissue constitutes half of body protein.. Biological value for protein is a measure of its ability to provide essential amino acids. Proteins of animal source like meat, eggs and milk have high BV as compared to those of plant source which might therefore require a combination of different plant based foods in diet. Ubiquitin is a 8.5 kDa protein with extended C-ter that tags proteins to be degraded using its lysine amino groups.
- Only about 10-15% of energy needs are met by protein in the diet.
- Rennin and gastricsin are two other proteolytic enzymes in gastric juice besides pepsin. Renin causes rapid coagulation of ingested caesin. Its pH optima is 5-6, so it only plays important role in children. Digestion of proteins takes place not only in the intestinal cavity but also on the surface of mucosa cells.
- Blood makes a complete circuit of body, in resting state, in 60 seconds. Carbonic anhydrase has a high turnover of the order of 10 to power 6. It increases the rate of reaction over 100 fold. The reaction takes place in erythrocytes and most of bicarbonate produced freely diffuses through RBC membrane into plasma, so that venous plasma has higher bicarbonate as compared to arterial plasma. The transport of Na and K ions is strictly regulated. So, bicarbonate is exchanged for chloride ion to maintain charge neutrality across membrane with bicarbonate diffusion, a process called as chloride shift.
- Enteropeptidase is secreted by intestinal cells. Trypsin also converts prolipase to lipase. Besides their usual activity, pepsin and chymotrypsin also cleave after acidic a.a.’s Glu and Asp. Carboxypeptidase A cleaves nearly all a.a.’s except lys and arg while carboxypeptidase B cleaves only argine and lysine. Amino peptidase cleaves nearly all a.a.’s. Elastase spilts elastin while collaganase spilts collagen. Prolidase cleaves proline. Dipeptidases cleave only dipeptides.
- Na+-K+ pump consumes one third of total energy produced by animal cell.
- Nitrogenous metabolites include purines, pyrimidines, heme, GSH, hormones, neurotransmitters, creatine etc.
- R is essential in children but non-essential in adults. Most arg sythesized is converted to urea via ornithine in urea cycle.
- Pterin ring derivatives. Cofactor for aromatic amino acid hydroxylases required for synthesis of neurotransmitters and catecholamines like dopamine, NO and seotonin. Synthesized denovo from GTP or by salvage pathway.
- Depending on the end from which carboxylate ion is released, there are two types of decarboxylation : ⍺ and ⍵. Amines formed by decarboxylation usually have high physiological activity e.g., histamine from histidine, gamma amino butyric acid from glutamate, and putrescine from ornithine
- PLP functions as an electron sink in the resonance stabilization of a Cα carbanion derived from an amino acid. This carbanion is an intermediate in amino acid transamination, decarboxylation, and the breakage of the Cα—Cβ bond. Transamination is a reversible reaction to transfer α-amino groups of amino acids, ẟ-amino group of ornithine and 𝜀-amino group of lysine.
- ALT elevated in all liver diseases while GOT increased in mycocardial infarction.
- Non-oxidative deamination of Ser, Thr, homoserine, Cys and His uses an alternate route. Dehyratase acts on Ser and Thr to release ammonium with generation of pyruvate and ⍺-ketobutyrate, respectively while a desulfurase acts on Cys and homocys to give pyruvate. Both the enzymes are B6 dependent. Deamination of his by histidase gives urocanate. Besides Glu dehydrogenase, A.A. oxidases also catalyze oxidative deamination. L-amino acid oxidase (FMN cofactor) and catalase catalyze a coupled reaction to release ammonium ion, oxygen and water from A.A.’s. Kidney and liver also have an FAD-containing D-amino acid oxidase
- Microbial enzymes in colon or on dead organisms decarboxylate amino acids with the formation of diamines. Histamine is secreted by mast cells and mediates allergic and inflammatory reactions, gastric acid secretion, and possibly neurotransmission in parts of brain. It’s a powerful vasodilator. Histamine analogues interfere with its action and are used as therapeutics.
- Increasing concentrations of E stimulate synthesis of N-acetylglutamate, thereby boosting urea cycle. About 25-30 g urea is excreted by an adult human per day. MS of blood measures citrulline and arginine. Disorder in urea cycle lead to hyperammonemia which may lead to anorexia, vomiting, cognitive motor deficits, lethargy, ataxia, asterixis, brain edema, cytotoxic and vasogenic edema, hypothermia, seizures and coma. Disorder correction by limiting protein intake, removal of excess ammonia, and replacement of intermediates like arginine and citrulline.
- Trp-pyrrolase is also called Trp 2,3-dioxygenase. It cleaves the pyrrole ring. Kynureinine 3-monooxygenase hydroxylates remaining benzene ring. Alanine is removed and 3-hyroxyanthranilate is cleaved by another dioxygenase and subsequently processed to acetoacetyl CoA. Nearly all ring cleaves are catalyzed by dioxygenases, a class of enzymes discovered by Osamu Hayaishi. The active site of these enzymes contain iron that is not part of heme or an iron sulphur cluster.
- p-HPPA dioxygenase is a copper containing enzyme that requires ascorbic acid for its activity. It shifts hydroxyl group from para to meta position, incorporates a new hydroxyl group at para position and catalysed oxidative decarboxylation of p-HPPA to generate homogentisate. Homogentisate forms a colored polymer Mutations in PHA gene located on chromosome 12 lead to PKU Tyrosine effects kidney and liver
- Transamination isn’t an option for essential A.A. biosynthesis as enzymes needed to form them are lacking. But they are present in micro-organisms and plants.
- Presence of creatine kinase in plasma is indicative of heart damage, and is used in the diagnosis of myocardial infarction. Phosphocreatine is a high energy compound that provides a small but rapidly mobilized reserve of high energy phosphates that can be reversible transferred to ADP to maintain the intracellular level of ATP during first few minutes of intense muscular contraction. Amount of phosphocreatine is proportional to the muscular mass. Normally, 15 mmol (<100 mg) of creatinine is excreted per day by adult human. When muscle mass decreases, the creatinine content of the urine falls. In contrast, any rise in blood creatinine is a sensitive indicator of kidney malfunction, because creatinine normally is rapidly removed from blood and excreted.
- Only 1% of tryptophan is converted to serotonin (minor Trp degradation pathway). Production of 5-hydroxytryptophan occurs in target tissues only. Most serotonin (5-hydroxytryptamine) production occurs in intestinal mucosa while minor amounts occur in CNS, and blood platlets. It acts as a powerful vasoconstrictor, is necessary for motility of GIT, evokes the release of peptide hormones, is involved in regulation of cerebral activity, pain perception and controls behavioural patters, sleep, appetite, blood pressure and body temperature.
- Outside nervous system, norepinephrine and epinephrine are hormone regulators of carbohydrate and lipid metabolism. They are mainly produced by adrenal medulla and adrenergic nerve endings in response to fright, exercise, cold and low levels of blood glucose. Adrenaline/Epinephrine stimulates lipolysis and glycogenolysis providing energy for increased rate of myocardial contraction resulting in increase in blood pressure for a fight or flight response. It has a half life of 2-5 minutes. Inactivation of catecholamines is catalyzed by monoamine oxidase and catechol-O-methyltransferase which catalyze oxidative deamination and O-methylation, respectively resulting in the formation of vanillylmandelic acid from norepinephrine and epinephrine and homovanillic acid from dopamine, which are excreted. The production of epinephrine is prevented by alpha methyl DOPA which inhibits DOPA decarboxylase, thus acting as an antihypertensive drug. Conversely, monoamine oxidase inhibitors inhibit MAO permitting neurotransmitter molecules to escape degradation, causing activation of norepinephrine and serotonin receptors, leading to antidepressant action of these drugs. In Parkinson disease, the dopamine content in brain is reduced due to idiopathic loss of dopamine producing cells in brain. As dopamine will not enter brain cells, the precursor, L-DOPA (levodopa) is used as a drug in Parkinsonism.
- Porphyrias are caused by inherited or acquired defects in heme synthesis, resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors. Degradation of hemeproteins in reticuloendothelial system, particularly in liver and spleen, leads to the production of green pigment biliverdin, which is subsequently reduced to yellow pigment bilirubin. Jaundice refers to the yellow color of the skin and sclera caused by deposition of bilirubin. Hemolytic, obstructive and hepatocellular are three types of jaundice.
- Maple syrup disease disorder due to mutation in four genes involved in A.A. metabolism: 1. BCKDHA, BCKDHB, DBT and DLD gene Melanin protects underlying cells from the harmful effects of sunlight.