‹#› 1 Catabolism of amino acids Ammonia detoxification Biosynthesis of non-essential amino acids Ó Department of Biochemistry (J.D.) 2013 ‹#› 2 Anabolic and catabolic conversions of AA AA pool endogenous proteins food proteins FA ®TAG CO2 + energy glucose signal molecules (hormone, neurotransmiter) purine / pyrimidine bases porphyrines ® heme creatine ® creatinine arginine ® NO and other ... NH3 D NH4+ synthesis of non-essential AA urea glutamine CAC GI tract intracellular degradation nitrogen compound compound without N ‹#› 3 Amino acid pool •Three sources of AA pool: 1)Proteolysis of dietary proteins (food) 2)Proteolysis of tissue proteins (physiological turnover, more in starvation) 3)Synthesis of non-essential AA (11) 4) •Three utilizations of AA pool: 1)Synthesis of tissue and blood plasma proteins (liver) 2)Synthesis of low-molecular nitrogen compounds (with specific functions) 3)Catabolism: deamination + utilization of carbon skeleton 4) •Three utilizations of AA carbon skeleton 1)Gluconeogenesis (in starvation, most AA are glucogenic) 2)Synthesis of FA and TAG (in AA excess) 3)Metabolic fuel = gain of energy (minor utilization) • ‹#› 4 Degradation of proteins •Exogenous proteins •the lumen of GI tract •stomach – pepsin •intestine – pancreatic proteases (trypsin, chymotrypsin etc.) •Endogenous proteins •intracellular proteases •lysosome •ubiquitin-proteasome •caspases in apoptosis •calpains and others ... • more details in BCH II and physiology ‹#› 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 Enteropeptidase secreted by the mucosa of duodenum initiates the activation of the pancreatic proenzymes ‹#› 7 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: Carboxypeptidase A nearly all AA (not Arg and Lys) Carboxypeptidase B basic AA (Arg, Lys) aminopeptidase nearly all AA Prolidase proline Dipeptidase only dipeptides ‹#› 8 Na+ 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+) Transcellular transport of AA from intestine to portal blood ‹#› 9 Endogenous proteins have different biological half-lives Protein Half-life Ornithine decarboxylase RNA polymerase I AST Prealbumin Lactate dehydrogenase Transferrin Albumin IgG Collagen Elastin 12 min 1 hour 12 hour 2 days 4 days 10 days 19 days 23 days several years whole life (?) ‹#› 10 Degradation of proteins in lysosomes •does not require ATP, non-specific •extracellular and membrane proteins •long-lived intracellular proteins •extracellular glycoproteins – sialic acid at terminal position on oligosaccharide chain is removed = asialoglycoproteins – they are recognized by liver receptors ® degradation in liver lysosomes ‹#› 11 Examples of lysosomal hydrolases Hydrolase Type of bond Glucosidase Galactosidase Hyaluronidase Arylsulfatase Lysozyme Cathepsin Collagenase Elastase Ribonuclease Lipase Phosphatase Ceramidase glycoside glycoside glycoside sulfoester glycoside peptide peptide peptide phosphodiester ester phosphoester amide ‹#› 12 Ubiquitin (Ub) targets proteins for proteasome degradation •small protein, in all cells - ubiquitous •C-terminus binds Lys of proteins to be degraded (kiss of death) •binding Ub to protein has three phases, with three enzymes E1,E2,E3 •binding Ub to E1-SH requires ATP •more Ub molecules are attached - polyubiquitination •Ub-tagged protein is directed to proteasome • ‹#› 13 The targeting of proteins E1 ubiquitin-activating enzyme (ATP) E2 ubiquitin-conjugating enzyme E3 ubiquitin-protein ligase The N-terminal rule Stabilizing AA (long life): •Met, Ser, Ala, Thr, Val, Gly, Cys Destabilizing AA (short life): •Phe, Leu, Asp, Lys, Arg •PEST proteins: segments rich in Pro, Glu, Ser, Thr ‹#› 14 Proteasome •hollow cylindric supramolecule, 28 polypeptides •four cyclic heptamers (4 ´ 7 = 28) •the caps on the ends regulate the entry of proteins into destruction chamber, upon ATP hydrolysis •inside the barrel, differently specific proteases hydrolyze target protein into short (8 AA) peptides •ubiquitin is not degraded, it is released intact ‹#› 15 Proteasomes degrade regulatory proteins (short half-life) and abnormal or misfolded proteins Ub + short peptides Protein-Ub AA cytosolic peptidases important in regulation of cell cycle, growth, differentiation, apoptosis ‹#› 16 20S proteasom (Stryer 2002, 637) Spatial model of proteasome 28 subunits in 4 rings (4 x 7), yellow chains in beta subunits contain proteolytic active sites on N-terminals ‹#› 17 220px-Bortezomib_svg Bortezomib is inhibitor of proteasome - boron atom binds to active site (Thr) (inhibited proteasome does not degrade pro-apoptotic factors – it leads to apoptosis of myeloma cells, treatment of multiple myeloma) Synthetic tripeptide: pyrazinoic acid-Phe-boroLeu boric acid instead of -COOH ‹#› 18 Caspases trigger apoptosis •caspase (cysteinyl aspartate-specific proteinase) •hydrolyse proteins near aspartate •degradation of cellular proteins during apoptosis •formed as inactive precursors (procaspases), activated by the actions of other caspase •initiator caspases start the apoptic pathway •after receiving stimulus they activate effector caspases – cascade of caspases (amplification of the process) •accidentally acivated caspases are neutralized by specific inhibitors • • Calpains – cytosolic proteases activated by Ca2+ ions. They occur in all cells, participate in many cell processes, e.g. the metabolism of cytoskeletal proteins, cell cycle progression etc. ‹#› 19 Proteins in nutrition: Biological value (BV) of proteins refers to how well the body can utilize the proteins we consume Relative amount of nitrogen (%) used to synthesis of endogenous proteins from total protein nitrogen absorbed from food. BV depends on: •total content of essential AA •mutual ratios of essential AA •protein digestibility BVanimal prot > BVplant prot wheat – deficit in Lys, Trp, Thr, Met legumes – deficit in Met, Cys Daily intake of proteins: 0.8 g/kg ‹#› 20 PDCAAS •protein digestibility-corrected amino acid score •a recent method based on essential AA requirement and protein digestibility •reference protein = ideal protein with optimal ratio of all essential AA (often whey or egg white) •true digestibility (%): amount of nitrogen absorbed from food per total food nitrogen amount of limiting AA in test protein -------------------------------------------------------- × true digestibility (%) amount of the same AA in reference protein ‹#› 21 Essential (9) and semiessential (3) amino acids •valine, leucine, isoleucine (BCAA) •threonine (two C*) •lysine, histidine (basic) •phenylalanine, tryptophan (aromatic) •methionine (-S-CH3) • • Semiessential AA • arginine – in childhood • alanine, glutamine – in metabolic stress (Ala-gluconeogenesis, Gln – ammonia detoxification) ----------------------------------------------------------------------------- • about 30 % of methionine need can be substituted by cysteine • about 50 % of phenylalanine need can be substituted by tyrosine ‹#› 22 Quality of some proteins Protein BV (%) PDCAAS (%) Egg white Whey Milk casein Beef Beans Wheat flour Gelatin 100 100 80 80 49 54 25 100 100 100 92 68 40 8 ‹#› 23 Egg white, whey, and gluten •Egg white is a viscous solution of globular proteins (ovalbumin, •ovotransferrin, ovomucoid, ovomucin, ovoglobulins, avidine etc.) Whey is a by-product in cottage chesse (curd) production a yellowish liquid (riboflavin), after precipitation of casein contains high quality proteins (lactoalbumin, lactoglobulins), B-vitamins, and lactose bezlepková dieta Gluten is protein fraction in wheat and other cereals, containing mainly gliadin (high content of Pro and Gln). In genetically predisposed people, it may cause autoimmune celiac disease. GF (gluten free) BL (bezlepkový) ‹#› 24 Quantity of proteins in foodstuffs (%) Parmesan cheese Emmental cheese Curd Beans Meat Eggs Yeast Cereals, rice Milk Potatoes Fruits, vegetables 40 30 25 25 20 13 11 8 4 2 1 ‹#› 25 Alternative protein sources Food Protein content Commentary Šmakoun Robi Seitan Hemp seeds Tofu Tempeh 13 % 22 % 25 % 30 % 16 % 20 % processed egg white, Czech product rostlinné bílkoviny, cereal + rice proteins isolated wheat proteins good content of essential AA coagulated soy milk proteins fermented soybeans by Rhizopus oligosporus ‹#› 26 Protein supplements •high content of proteins (20 – 90%) •mainly derived from dried whey •and/or free AA (BCAA = Val, Leu, Ile) • •it is a metabolic load for: •digestion (® putrefaction in large intestine, correlates with some types of cancer) •liver (® urea synthesis), kidneys (excretion of urea, NH4+, free AA) •may be adulterated with anabolic steroids !!! ‹#› 27 Catabolic pathway of amino acids Transamination Dehydrogenation + deamination of glutamate Detoxication of ammonia Excretion of nitrogen catabolites ‹#› 28 Proteins NH3 glutamate glutamate + urea (excretion by urine) 2-oxoglutarate + glutamine proteolysis dehydrogenation + deamination detoxication in liver deamidation in kidney amino acids transamination detoxication in other tissues NH4+ (excretion by urine) NH4+ (excretion by urine) deamination in kidney Intake, catabolism, and excretion of nitrogen ‹#› 29 Transamination transfer of -NH2 group from one substrate to other •most AA (not Lys, Thr, Pro, His, Trp, Arg, Met) •amino group is transferred from AA to 2-oxoglutarate •cofactor – pyridoxal phosphate (® Schiff bases) •reversible reaction Þ important for synthesis of AA ‹#› 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 (Lys) ‹#› 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 AA is concentrated in glutamate Glutamate then undergoes dehydrogenation + deamination and releases free ammonia NH3 ! ‹#› 35 Dehydrogenation + deamination of glutamate is reversible reaction NAD(P)+ main source of ammonia in tissues glutamate 2-iminoglutarate 2-oxoglutarate ‹#› 36 Glutamate dehydrogenase (GMD, GD, GDH) •requires pyridine cofactor NAD(P)+ •GMD reaction is reversible: dehydrogenation with NAD+, hydrogenation with NADPH+H+ •two steps: •dehydrogenation of CH-NH2 to imino group C=NH •hydrolysis of imino group to oxo group and ammonia ‹#› 37 1.Deamination of glutamate (GD reaction) in tissues 2.Bacterial putrefaction of proteins in the large intestine produces nitrogen catabolites (e.g. biogenic amines + ammonia), ammonia diffuses freely into portal blood Þ portal blood has high concentration of NH4+ Þ eliminated by liver Two main sources of ammonia in organism ‹#› 38 Other sources of ammonia: deaminations of various substrates •deamination of adenine •oxidative deamination of some AA (® H2O2) •desaturation deamination of histidine ® urocanic acid + NH3 •oxidative deamination of terminal –NH2 in lysine lysyl oxidase(Cu2+): Lys + O2 ® NH3 + allysine + H2O •dehydratation deamination of serine (see next lecture) •oxidative deamination of biogenous amines, MAO monoamine oxidase (® H2O2, see also Med. Chem. II, p. 60) ‹#› 39 Deamination of adenine + NH3 adenosine monophosphate (AMP) inosine monophosphate (IMP) ‹#› 40 Oxidative deamination of some AA uses flavine cofactor and dioxygen • typical for glycine • D-amino acids • side product is H2O2 H2O + ½ O2 2-imino acid catalase ‹#› 41 Oxidative deamination of biogenous amines R-COOH acid biogenous amine monoamine oxidase imine aldehyde ‹#› 42 Desaturation type of deamination in histidine urocanic acid (urocanate) ‹#› 43 Other reactions producing ammonia •non-enzymatic carbamylation of proteins Prot-NH2 + NH2-CO-NH2 ® NH3 + Prot-NH-CO-NH2 •catabolism of pyrimidine bases cytosine/uracil ® NH3 + CO2 + β-alanine thymine ® NH3 + CO2 + β-aminoisobutyrate •synthesis of heme (4 porphobilinogen ® 4 NH3 + uroporphyrinogen) • ‹#› 44 Hydrolysis of amide group in glutamine releases ammonia (deamidation). In kidneys, NH4+ ions are released into urine. glutamate glutamine glutaminase Glutamine is non-toxic transport form of ammonia ‹#› 45 Ammonia production under pathological conditions •bleeding in GIT Þ increased NH3 in portal blood •uroinfections – bacterial urease catalyzes the hydrolysis of urea H2N-CO-NH2 + H2O ® 2 NH3 + CO2 NH3 + H2O D NH4+ + OH- alkaline urine (pH ~ 8) Þ phosphate stones ‹#› 46 Acide-base properties of NH3 •pKB (NH3) = 4.75 (weak base) •NH3 + H2O D NH4+ + OH- •pKA (NH4+) = 14 – 4.75 = 9.25 (very weak acid) • Þ Þ under physiological pH values in ICF and ECF (~ 7.40): 98 % NH4+ 2 % NH3 ‹#› 47 Body fluid Concentration of NH4+ ions Metabolic origin of NH4+ Urine Saliva Portal blood Venous blood 10 – 40 mmol/l 2 – 3 mmol/l 100 – 300 μmol/l 5 – 30 μmol/l 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 Compare: Ammonium ions in body fluids ‹#› 48 1.Low-protein diet (especially important in liver diseases) 2.Alteration of colon microflora by the ingestion of: •Probiotics – live bacteria stimulating saccharolytic (fermentative) processes in large intestine instead of putrefactive ones (Lactobacillus, Bifidobacterium) – yoghurt, kefir milk, sauerkraut etc. •Prebiotics – non-digestible food ingredients (polysaccharides) that stimulate the growth probiotics in the colon (dietary fibre, inulin) How to decrease ammonia production in body? ‹#› 49 Three ways of ammonia detoxification Feature Urea Glutamine (Gln) Glutamate (Glu) Relevance Compound type Reaction(s) Enzyme Energy needs Organelle(s) Organ(s) « « « « « « H2CO3 diamide urea cycle 5 enzymes 4 ATP mitoch. + cytosol only liver « « « « γ-amide of Glu Glu + NH3 Gln-synthetase 1 ATP cytosol liver, brain, other « α-amino acid hydrog. amin. 2-OG GMD 1 NADPH+H+ mitochondria (brain) ‹#› 50 Ureasynthesis in liver five reactions 1. and 2. in mitochondria 3. - 5. in cytosol ‹#› 51 1. Carbamoyl phosphate (matrix) •carbamoyl phosphate synthetase (activated by N-acetylglutamate) •matrix of mitochondria •two moles of ATP •amide bond + mixed anhydride •macroergic compound carbamoyl phosphate synthetase ‹#› 52 Carbamoyl is the acyl of carbamic acid carbamic acid (carbonic acid monoamide) does not exist carbamoyl ‹#› 53 2. Citrulline formation (matrix) citrulline carbamoyl ornithine ornithine carbamoyltransferase ‹#› 54 3. The second -NH2 group comes from aspartate (cytosol) citruline aspartate argininosuccinate argininosuccinate synthetase ‹#› 55 Less usual hydrolysis of ATP means that two ATP are consumed ATP + H2O ® AMP + PPi PPi + H2O ® 2 Pi (diphosphatase, pyrophosphatase) AMP + ATP ® 2 ADP (adenylate kinase) -------------------------------------------------- summary: 2 ATP + 2 H2O ® 2 ADP + 2 Pi ‹#› 56 4. The cleavage of argininosuccinate argininosuccinate arginine fumarate argininosuccinate lyase ‹#› 57 5. Hydrolysis of arginine affords urea arginine ornithine arginase (hydrolase) ‹#› 58 free ammonia aspartate Metabolic origin of nitrogen in urea ‹#› 59 glutamate + NAD+ D ammonia + 2-OG + NADH+H+ GMD UREA oxalacetate AST 2-oxoglutarate aspartate Dual function of glutamate in AA catabolism ‹#› 60 CO2 + NH4+ + aspartate ® urea + fumarate + H2O + 2 H+ CO(NH2)2 + -OOC-CH=CH-COO- + H2O + 2 H+ Urea synthesis is proton-productive reaction CO2 + NH4+ + ‹#› 61 Urea is non-electrolyte •carbonic acid diamide •polar compound (dipole) Þ well soluble in water •diffuses easily through cell membranes (hydrophilic pores) •contributes to blood plasma osmolality • osmolality » 2 [Na+] + [glucose] + [urea] mmol/kg H2O •synthesis only in liver •excretion by urine depends on the amount of food proteins • 330-600 mmol/day (20-35 g/day) ‹#› 62 Urea in blood serum (2-8 mmol/l) •Increased concentration •renal failure •increased protein catabolism (sepsis, burns, polytrauma, fever etc.) •Decreased concentration •lack of proteins in diet •liver failure ‹#› 63 Compare and distinguish urea × uric acid ! ‹#› 64 Compare Feature Urea Uric acid Chemical name Latin name In water Solubility in water Reducing property Salt formation Catabolite of carbonic ac. diamide urea non-electrolyte excelent no no amino acids 2,6,8-trihydroxypurine acidum uricum weak diprotic acid poor, depeds on pH yes Þ antioxidant yes (two types) adenine and guanine Organe location liver only liver and other tissues Subcellular location mitochondria + cytosol cytosol Serum concentration 2 - 8 mmol/l 150 - 400 μmol/l Urine excretion 20 - 35 g/day 0.5 - 1 g/day Catabolic nitrogen 80 - 90 % 1- 2 % H ‹#› 65 Glutamine synthesis glutamine synthetase glutamate glutamine 2. way of detoxification ‹#› 66 In kidneys, ammonia is relased from glutamine and glutamate. Ammonium cation is excreted by mildly acidic urine urine (pH ~ 5) 2-oxoglutarate glutaminase glutamate dehydrogenase ‹#› 67 Multiple functions of glutamine •Synthesis of proteins •Metabolic fuel – for some tissues: enterocytes, lymphocytes, macrophages, fibroblasts, kidneys •Source of nitrogen in synthesis – purine, pyrimidines, NAD+, aminosugars •Source of glutamate – glutathione (GSH), GABA, Glu ® ornithine, Glu ® proline •Source of ammonium ions in urine •detoxification of ammonia in tissues and non-toxic transport form of ammonia from tissues to liver ! ‹#› 68 Glutamate dehydrogenase reaction is reversible dehydrogenation deamination of glutamate hydrogenation amination of 2-oxoglutarate ammonia formation ‹#› 69 Subcellular location of AA conversions transamination (ALT) Þ glutamate NH3 glutamate synthesis of urea mitochondria cytosol GMD Glu + NH3 ® Gln transamination (AST) cytosol ‹#› 70 Synthesis of non-essencial amino acids ‹#› 71 Synthesis of glycine 1. The reverse of transamination 2. From serine 2-oxoglutarate glutamate glyoxalate serine glycine ‹#› 72 Formation of serine from the glycolysis intermediate glucose 3-P-glycerate 3-P-hydroxypyruvate 3-P-serine transamination ‹#› 73 Synthesis of alanine from pyruvate and glutamate (ALT = alanine aminotransferase) alanine pyruvate glutamate 2-oxoglutarate ‹#› 74 Aspartate from oxaloacetate and glutamate (AST = aspartate aminotransferase) AST reaction produces aspartate for urea synthesis aspartate glutamate 2-oxoglutarate oxaloacetate ‹#› 75 Proline synthesis is the opposite of its catabolism proline glutamate 5-semialdehyde glutamate pyrroline-5-carboxylate oxidation addition of H2O ring opening ‹#› 76 Glutamate is formed by the reductive amination of 2-oxoglutarate (GMD reaction) L-glutamate 2-iminoglutarate 2-oxoglutarate ‹#› 77 Hydroxylation of essential phenylalanine gives non-essential tyrosine tetrahydrobiopterine (BH4) is a donor of 2H to form water from the second oxygen atom phenylalanine tyrosine ‹#› 78 Glutamine from glutamate and ammonia glutamine synthetase glutamate glutamine Similarly: asparagine from aspartate ‹#› 79 Cysteine is made from methionine methionine homocysteine cystathionine homoserine cysteine condensation with serine cysteine release ‹#› 80 Arginine from glutamate via ornithine glutamate ornithine arginine urea cycle oxidative deamination reductive amination ‹#› 81 Selenocysteine arises co-translationally from serine and selenophosphate Seryl-tRNA + selenophosphate ® selenocysteyl-tRNA + phosphate Selenophosphate is made from selenide (food) and ATP Se2- + ATP + H2O ® AMP + Pi + few enzymes (redox reactions) contain selenocysteine Glutathione peroxidase (2 GSH + H2O2 ® 2 H2O + G-S-S-G) Deiodase of thyronines (thyroxine T4 ® triiodothyronine T3) Thioredoxin reductase (ribose ® deoxyribose) ‹#› 82 Synthesis of non-essential amino acids AA Precursor and reactions Ala pyruvate (transamination, ALT) Glu glutamine (deamidation), 2-OG (reductive amination), proline (catabolism), histidine (catabolism), ornithine (oxidative deamination) Gln glutamate (amidation – synthesis of amide group from ammonia) Asp oxaloacetate (transamination, AST), asparagine (demidation) Asn aspartate (amidation from ammonia) Ser 3-P-glycerate (dehydrogenation, transamination, hydrolysis), glycine (transfer of C1 group) Gly serine (transfer of C1 group), glyoxalate (transamination) Cys methionine (demethylation to homocysteine, condensation with serine, cleavage) Tyr phenylalanine (hydroxylation) Pro glutamate (the reverse of proline catabolism) Arg glutamate (reductive amination to ornithine, urea cycle reactions)