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Aa 1
1. Catabolism of amino acids Ammnonia detoxification Biosynthesis of non-essential amino acids Biochemický ústav LF MU (J.D.) 2011
2. Anabolic and catabolic conversions of AA AA pool Endogenous proteins Food proteins FA TAG CO 2 + energy glucose signal molecules (hormone, neurotransmiter) purine / pyrimidine bases porphyrines heme creatine creatinine arginine NO and other ... NH 3 NH 4 + carbon skeleton Synthesis of non-essential AA Urea Glutamine CAC GI tract Intracellular degradation nitrogen compound compound without N
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5. pepsin trypsin pepsinogen activated by HCl carboxypeptidases aminopeptidases AA – resorption into portal blood pH 1-2 chymotrypsin, elastase Digestion of dietary proteins proteases are secreted as inactive proenzymes activated by the cleavage of certain peptide sequence
6. Protein digestion begins in the stomach, acidic environment (pH 1-2) favours denaturation. Proteins undergo proteolysis catalyzed by pepsin. Degradation continues in the lumen of the intestine by pancreatic proteolytic enzymes secreted as inactive proenzymes Digestion is then completed by aminopeptidases located in the plasma membrane (brush border) of the enterocytes. Single amino acids, as well as di- and tripeptides, are transported into the intestinal cells from the lumen and released into the blood.
8. Proteolytic enzymes exhibit the preference for particular types of peptide bonds Proteinases preferentially attacks the bond after: Pepsin aromatic (Phe, Tyr) and acidic AA (Glu, Asp) Trypsin basic AA (Arg, Lys) Chymotrypsin hydrophobic (Phe, Tyr, Trp, Leu) and acidic AA (Glu, Asp) Elastase AA with a small side chain (Gly, Ala, Ser) Peptidases : Carboxy peptidase A nearly all AA ( not Arg and Lys) Carboxy peptidase B basic AA (Arg, Lys) amino peptidase nearly all AA Prolidase proline Dipeptidase only dipeptides
9. Transcellular transport of AA from intestine to portal blood L-amino acids: about seven specific transporters, symport with Na + D-amino acids (trace amounts): nonspecific diffusion, hydrophilic pores in membranes, D-AA cannot be utilized in the body, they are only catabolized to gain energy Also small oligopeptides (symport with H + ) Na +
10. Endogenous proteins have different biological half-lives 12 min 1.3 hour 12 hour 2 days 4 days 19 days 23 days several years whole life (?) Ornithine decarboxylase RNA polymerase I AST Prealbumin Lactate dehydrogenase Albumin IgG Collagen Elastin Half-life Protein
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12. Examples of lysosomal h ydrolas es gly c oside gly c oside gly c oside sulfoester gly c oside peptide peptide peptide phosphodiester ester phosphoester amide Glu c osidas e Gala c tosidas e Hyaluronidas e Arylsulfatas e Lysozym e C athepsin Collagenase Elastase Ribonuclease Lipase Phosphatase Ceramidase Typ e of bond Hydrolas e
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16. Proteasomes degrade regulatory proteins (short half-life) and abnormal or misfolded proteins Ub + short peptides Protein-Ub AA cytosolic peptidases regulation of cell cycle, apoptosis, angiogenesis
17. Spatial model of p roteasom e 28 subunits in 4 rings (4 x 7), yellow chains in beta subunits contain proteolytic active sites on N- termini
18. Bortezomib is inhibitor of proteasom e - boron atom b inds to active site (Thr) ( treatment of m ultiple myelom a ) Synt he tic tripeptid e : pyrazino ic acid -Phe-boro nic acid- Leu boric acid instead of -COOH
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22. Quality o f some proteins 100 100 80 80 49 54 25 BV (%) 100 100 100 92 68 40 8 Egg white Whey Milk c asein Beef Beans W heat flour Gelatin PDCAAS (%) Protein
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24. Quantity of proteins in foodstuffs (%) D aily intake of protein s : 0.8 g/kg 4 0 30 25 25 20 13 10 11 8 4 2 1 Parmesan cheese Emmental cheese Curd L egumes (beans) M eat Eggs Fish Yeast Cereals , r ice M ilk Potatoes Fruits , vegetables
25. Alternativ e plant protein products Robi Seitan Tofu Tempeh Food ro stlinné bí lkoviny , cereal + rice proteins isolated wheat proteins coagulated s o y milk proteins f erment ed soybeans by Rhizopus oligosporus 22 % 25 % 16 % 20 % Commentary P rotein content
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27. Catabolic pathway of amino acids Transamination Dehydrogenation + deamination of glutamate Detoxication of ammonia Excretion of nitrogen catabolites
28. Proteins NH 3 glutam ate glutam ate + urea (ex c re tion by urine ) 2-oxoglutar ate + glutamin e proteol ysis dehydrogena tion + deamina tion detoxi cation in liver deami d a tion in kidney amino acids transamina tion detoxi cation in other tissues NH 4 + (ex c re tion by urine ) NH 4 + (ex c re tion by urine ) deamina tion in kidney Intake, catabolism, and excretion of nitrogen
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30. General scheme of transamination amino acid 2-oxo acid 2-oxoglutarate glutamate aminotransferase pyridoxal phosphate
31. Pyridoxal phosphate has a reactive aldehyde group aldehyde group covalently linked to enzyme
32. 1. Phase of transamination AA oxoacid pyridoxal-P pyridoxamine-P amino acid oxo acid Schiff base aldimine of pyridoxal Schiff base iminoacid isomeration
33. 2. Phase of transamination 2-oxoglutarate glutamate pyridoxamine-P pyridoxal-P Schiff base aldimine of pyridoxal Schiff base iminoacid 2-oxoglutarate glutamate
34. In transaminations, nitrogen of most A A is c oncent rated in glutam ate Glutamate then undergoes dehydrogen ation + deamina tion and releases free ammonia NH 3 !
35. Dehydrogenation + deamination of glutamate is reversible reaction NAD(P) + main source of ammonia in tissues glutamate 2-iminoglutarate 2-oxoglutarate
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39. Deamina tion of adenin e + NH 3 adenosin e monophosphate (AMP) inosin e monophosphate (IMP)
44. Hydrol ysis of amide group in glutamin e releases am monia (deamidation). In kidneys, NH 4 + ions are released into urine. glutam ate glutamin e glutaminase Glutamine is non-toxic transport form of ammonia
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47. Compare: Ammonium ions in body fluids hydrolysis of Gln, deamination of Glu (tubules) hydrolysis of urea by oral microflora protein putrefaction (GIT), Gln/Glu catabolism (enterocyte) catabolism of AA in tissues 10 – 40 mmol/l 2 – 3 mmol/l 100 – 300 μ mol/l 5 – 30 μ mol/l Urine Saliva Portal blood Venous blood Metabolic origin of NH 4 + Concentration of NH 4 + ions Body fluid
50. T hree ways of ammonia detoxication α -amino acid hydrog. amin. 2-OG GMD 1 NADPH+H + mitochondri a (brain) γ - amid e of Glu Glu + NH 3 Gln-synthetas e 1 ATP cytosol liver, brain , other H 2 CO 3 diamide urea cy cle 5 enzym es 4 ATP mitoch. + cytosol only liver Relevance Compound type Rea ction(s) Enzym e Energ y needs Organelle(s) Org an(s) Glutam ate (Glu) Glutamin e (Gln) Urea Feature
55. 3. The second -NH 2 group comes from aspartate (cytosol) citruline aspartate argininosuccinate
56. Less usual hydrolysis of ATP means that two ATP are consumed ATP + H 2 O A M P + P P i PP i + H 2 O 2 P i (diphosphatase, pyrophosphatase) AMP + ATP 2 ADP (adenylate kinase) -------------------------------------------------- summary: 2 ATP + 2 H 2 O 2 ADP + 2 P i
57. 4. The cleavage of argininosuccinate argininosuccinate arginine fumarate
60. Dual function of glutamate in AA catabolism glutamate + NAD + ammonia + 2-OG + NADH+H + GMD UREA oxalacetate AST 2-oxoglutarate aspartate
61. Urea s ynt hesis is proton-produ ctive reaction CO 2 + NH 4 + + aspart ate urea + fumar ate + H 2 O + 2 H + CO(NH 2 ) 2 + - OOC-CH=CH-COO - + H 2 O + 2 H + CO 2 + NH 4 + +
65. Compare liver and other tissues liver only Organe location cytosol mitochondria + cytosol Subcellular location 150 - 400 μ mol/l 2 - 8 mmol/l Serum concentration 0.5 - 1 g/day 20 - 35 g/day Urine excretion 1- 2 % 80 - 90 % Catabolic nitrogen 2,6,8-trihydroxypurin e acidum uricum weak diprotic acid p oor , depeds on pH yes antioxidant yes (two types) a denin e and guanin e carbonic ac. diamid e urea non- ele c trolyt e excelent n o n o a mino acids Chemic al name Latin name In water Solubility in water Redu cing property Salt formation C atabolit e of Uric acid Urea Feature H
66. Glutamine synthesis glutamine synthetase glutamate glutamine 2. way of detoxification
67. In kidneys, ammonia is relased from glutamine and glutamate. Ammonium cation is excreted by mildly acidic urine urine (pH ~ 5) 2-oxo gluta rate glutaminase glutamate dehydrogenase
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69. Glutamate dehydrogenase reaction is reversible ammonia formation 3. way of ammonia detoxication of 2-oxoglutar ate amina tion h ydroge nation of glutam ate deamina tion d ehydrogena tion
70. Subcellular location of AA conversions t ransamination (ALT) glutamate NH 3 glutamate synthesis of urea mitochondria cytosol GMD Glu + NH 3 Gln t ransamination (AST) cytosol
72. Synthesis of glycine 1. The reverse of transamination 2. From serine 2-oxoglutarate glutamate glyoxalate serine glycine
73. Formation of serine from the glycolysis intermediate glu c os e glycolysis 3-P-glycer ate 3-P-hydroxypyruv ate 3-P-serin e transamination
74. Synthesis of alanine from pyruvate and glutamate (ALT = alanine aminotransferase) alanine pyruvate glutamate 2-oxoglutarate
75. Aspartate from oxaloacetate and glutamate (AST = aspartate aminotransferase) AST reaction produces aspartate for urea synthesis aspartate glutamate 2-oxoglutarate oxaloacetate
76. Proline synthesis is the opposite of its catabolism proline glutamate 5-semialdehyde glutamate pyrroline-5-carboxylate oxidation addition of H 2 O ring opening
77. Glutamate is formed by the reductive amination of 2-oxoglutarate (GMD reaction) L-glutamate 2-iminoglutarate 2-oxoglutarate
78. Hydroxyla tion of essential ph enylalanin e gives non-essential tyrosine tetrahydrobiopterin e (BH 4 ) is a donor of 2H to form water from the second oxygen atom phenylalanine tyrosine
79. Glutamine from glutamate and ammonia glutamine synthetase glutamate glutamine Similarly: asparagine from aspartate
80. Cysteine is made from methionine methionine homocysteine cystathionine homoserine cysteine condensation with serine cysteine release
81. Selenocystein e arises c o - transla tionally from serine a nd seleno phosphate Ser yl -tRNA + selenophosphate selenocyste yl -tRNA + phosphate Selenophosphate is made from selenid e ( food ) a nd ATP Se 2- + ATP + H 2 O AMP + P i + few enzymes (redox reactions) contain selenocystein e Glutathione peroxidase (2 GSH + H 2 O 2 2 H 2 O + G-S-S-G) Deiodase of thyronines (thyroxine T4 triiodothyronine T3) Thioredoxin reductase (ribose deoxyribose)