Amino Acid Metabolism I - Generasl - Masaryk University
Amino acid metabolism ICatabolism of proteinsGeneral catabolism of amino acidsBiochemistry ILecture 62008 (J.S.)
Digestion of dietary proteins in the GITProtein digestion begins in the stomach, where the acidic environmentfavours protein denaturation. Proteins undergo the primary proteolysiscatalyse by gastric pepsin (the optimal pH 2).Degradation continues in the lumen of the intestine owing to the activityof pancreatic proteolytic enzymes secreted as inactive proenzymesand converted into active enzymes within the intestinal lumen.Digestion is then completed by aminopeptidases located in the plasmamembrane (brush border) of the enterocytes.Single amino acids, as well as di- and tripeptides, are transportedinto the intestinal cells from the lumen and released into2 the blood.
The generation of trypsin leads to the activation of other proenzymes:Enteropeptidase secreted by the mucosa of duodenum initiates the activationof the pancreatic proenzymes by activating trypsin, which then activates otherproenzymes.3
Proteolytic activation of chymotrypsinogenThe active site for binding a part of the substrate is not fully formed in thechymotrypsinogen. Proteolysis enables the formation of the substrate-binding site.4
Proteolytic enzymes of the GITattack peptide bonds of a number of amino acids, but they exhibit thepreference for particular types of peptide bonds:Proteinases (formerly called referentially attacks the bond after:aromatic (Phe, Tyr) and acidic amino acids (Glu, Asp)basic amino acids (Arg, Lys)hydrophobic (Phe, Tyr, Trp, Leu) and acidic AA (Glu, Asp)an amino acid with a small side chain (Gly, Ala, Ser)Peptidases (formerly exopeptidases):Carboxypeptidase ACarboxypeptidase BLeucine aminopeptidaseProlidaseDipeptidasenearly all amino acids (not Arg and Lys)basic amino acids (Arg, Lys)nearly all amino acidsprolinesplits only dipeptides5
The absorption of amino acids in the gutThe absorption of amino acids and peptides is an active proces,obligatorily coupled to the uptake of Na . It is driven by the largedifference in Na concentration across the brush border membrane, thatis maintained by the Na ,K -ATPase.There are at least seven different Na -linked amino acid transporters ofdifferent but overlapping specificities, and also separate transporters fordi- and tripeptides.Na 6
Degradation of intracellular proteins– eliminates abnormal proteins,– permits the regulation of cellular metabolism by elimination of someenzymes and/or regulatory proteins that represent importantmetabolic control points.Normal intracellular proteinsare eliminated at rates that depend ontheir identity.Long-lived proteins, underphysiological conditions, are degradedat nearly constant rates, mostlynonselectively; nutritional deprivationincreases rates of degradation and it isselective.Short-lived proteins degradation isprovided selectively by cytosolicubiquitin systém, or by other systemsnot yet known.7
Systems which select proteins for degradation:– The N-end amino acyl residue – in both prokaryotes and eukaryots the half-lifeof a cytoplasmic protein varies with the identity of its N-terminal amino acyl:– Some sequences of amino acyl residues in the primary structure.Proteins rich in segments Pro-Glu-Ser-Thr (symbol PEST), so-called PEST proteins,are rapidly degraded in the cytosol, t½ about 30 minutes.During a prolonged fasting, proteins with sequences Lys-Phe-Glu-Arg-Gln (symbol KFERQ),KFERQ proteins, in the tissues that atrophy in response to starvation (e.g. liver, kidney)are specifically bound with the protein prp73, delivered to the lysosome, and degraded.Cyclin destruction boxes – amino acid sequences that mark cell-cycle proteins fordestruction.– A highly destabilizing N-terminal residue such as Arg and Leu favours rapidubiquitinization, the ubiquitin-tagged proteins are degraded in the proteasomes.–Other, more complex systems?8
1 Protein degradation in lysosomesLysosomes contain a large number of proteolytic enzymes, cathepsins, whichare active at pH 5 maintained by the vesicular H pump (and largely inactive atcytosolic pH values).Extracellular proteins that enter the cell via endocytosis are hydrolysed in thephagolysosomes, which are formed by fusing the invaginated vesicles (simpleendosomes or coated vesicles covered with clathrin in receptor-mediatedendocytosis) with lysosomes.Normal long-lived intracellular proteins are also degraded nonselectively inthe lysosomes (autophagy – the digestion of cytoplasmic proteins by the cell sown lysosomes).The protein degradation in lysosomes is ATP-independent. It is probably ofless importance than the turnover of specific proteins in the ubiquitin pathway.)9
2 Cytosolic ubiquitin system (ATP-dependent)Ubiquitin, a small protein (Mr 8500) present in all eukaryotic cells,is the tag that marks proteins for destruction.Ubiquitin is the most highly conserved protein known ineukaryotes. It is identical in humans, toad, trout,Drosophila. Yeast and human ubiquitin differs at only 3of 76 amino acyl residues.The C-terminal glycine residue of ubiquitin (Ub)becomes covalently attached to the ε-aminogroups of several lysine residues (isopeptidebonds) on a protein destined to be degraded.The energy for the formation of these isopeptidebonds comes from ATP hydrolysis.C-endThree enzymes participate in the attachment ofubiquitin to each protein:10
ATPSHPPiE1E1AMPE2SHSHE1E2E1Enzyme 1 – ubiquitin-activatingEnzyme 2 – ubiquitin-conjugatingEnzyme 3 – ubiquitin-protein ligaseE2E3 H3N–ProteinE3ProteinE2E3Ubiquitinated proteinThe ubiquitination reaction is processive – chains of Ub are generated by the linkage of theε-amino group of lysine of one ubiquitin molecule to the terminal carboxylate of another.Chains of four or more ubiquitins are particularly effective in signaling degradation.The ubiquitin-tagged protein heads towards proteasome.11
The proteasomedigests the ubiquitin-tagged proteins. This ATP-driven multisubunitprocess spares ubiquitin, which is then recycled.The 26S proteasome is a complex of a 20S proteasome (it exhibitsat least 5 different proteolytic activities), and two 19S regulatorycomplexes.26S proteasome19S complex20S proteasome20S proteasome19S complex12
The 20S proteasome complex consists of 28 subunits arranged in four ringsof subunits that stack to form a structure resembling a barrel. The activesites of the proteinases are on the interior of the barrel.Substrates are degraded until they are reduced to peptides in lengths fromseven to nine amino acyl residues.The peptide products are further degraded by other cytoplasmic proteases toyield individual amino acids.The 20S proteasome is a sealed barrel. Access to its interior is controlled bytwo 19S regulatory complexes (each made up of 20 subunits), which bindspecifically to polyubiquitin chains and exhibit ATPase activity. ATPhydrolysis may assist the 19S complex to unfold the substrate so that it canbe passed into the center of the proteasome.The 19S complex also cleaves off intact ubiquitin molecules.13
The dynamics of amino acid metabolismand the protein turnoverDIETARY PROTEINSapprox. 80 g dailyPlasma proteins 20 g /dPROTEOSYNTHESISTHE INTESTINE260 g (up to 400 g) dailyVisceral proteins 50 g / dMuscle proteins 50 g / d70 g / dIntestinal secretionProtein breakdownSpecific products140 g / dAbsorptionAMINO ACID POOLAMINO ACIDCATABOLISMFree AA in plasma and cellsapprox. 70gCO2 H2O10 g / d loss in stoolUREAUrinary excretionequivalent to 70 g protein / d14
The minimum protein requirement of adultsis above 0.4 g / kg daily, i.e. 28 g / d for a 70 kg man, supposing that the proteinsgiven will be utilized with maximal efficiency.The recommended daily protein supply for adultsis about 0.8 g / kg, i.e. 56 g / d for a 70 kg man.The protein requirement of children is larger than that of adults – 1.2-1.5 g / kg.The effectiveness of different proteins in the dietAn empirical measure of the efficiency of a protein is the biological value –the mass of body proteins that are synthesized from 100 g of a protein(expressed as percentage).It depends primarily on the sufficient content of the essential amino acidswhich the body cannot make itself, and on the digestibility of a protein.Essential amino inePhenylalanineTryptophanHistidine (for children)Arginine (for children)15
Examples of the biological values of proteins in some foods(%) :Eggs97Milk90Beef77Pork71Casein68Oat flakesWheatBeansLentilsGelatine6253 (low in lysine)46 (low in methionine)45 (low in methionine)25 (very low in tryptophan)Examples of protein content of foods (%) :Cheese25-30Curd30Cottage cheese 20Meat20Eggs13Cow s milk3.3Legumes25-30Yeast11Pasta products8-10Rice7Potatoes2Fruit, vegetables 0.5-216
The amino acid degradationThe first step in degradation of many standard amino acids is theremoval of the α-amino group, i.e. deamination or transamination.The product is mostly the corresponding 2-oxoacid (α-ketoacid) andα-amino group is released as ammonia or ammonium ion.Direct deamination of amino acidsOnly few amino acids are deaminated directly:Glutamate – oxidative deamination catalysed by glutamate dehydrogenaseSerin and threonine – deamination preceded by dehydratationcatalysed by serine dehydratase or threonine dehydratase.Histidine – deamination by elimination of NH3 catalysed by histidine ammonialyase.In peroxisomes, aerobic deamination of various amino acidscatalysed by amino acid oxidase is not very efficient.Direct deamination is therefore unimportant for most L-amino acids; instead,their amino groups are removed indirectly, by transamination.17
Direct oxidative deamination of glutamate by dehydrogenationThe reaction is catalysed by the mitochondrial enzymeglutamate dehydrogenase (GLD). It requires either NAD or NADP as coenzyme,and its activity in mitochondria is 2GlutamateNHNAD(P) 2-IminoglutarateNAD(P)H H H2ONH3The equilibrium favours glutamatesynthesis, but it is pulled in the directionof deamination by the continuousremoval of NH3/NH4 tarate)18
Direct deamination of histidine by elimination (desaturation)Histidase(L-Histidine ammonialyase)NNH–CH2–CH–COOHNH2–CH CH–COOHNHNH3HistidineNUrocanic acid(Urocanate)Direct oxidative (aerobic) deamination of amino acids in peroxisomesis catalysed by L-amino acid oxidase. In spite of this reaction seems to be similar to that catalysedby glutamate dehydrogenase, the amino acid oxidases are flavoproteins which require O2 for reoxidationof FADH2, and they produce hydrogen peroxide.R–CH-COOHNH2Amino acidCatalaseH2O ½O2R–C–COOHNHFADFADH2H2O2O2Imino acidH2ONH3R–C–COOHO2-oxoacid(α-Ketoacid)19
TransaminationThe α-amino group of many amino acids that cannot be deaminateddirectly is transferred to 2-oxoglutarate (α-ketoglutarate) to formglutamate. The product glutamate is oxidatively deaminated byglutamate dehydrogenase to yield ammonium ion NH4 .These transaminations are reversible and can be used to synthesizeamino acids from 2-oxoacids and glutamate.Transaminations are catalysed by aminotransferases, the prostheticgroup is pyridoxal phosphate.The aldehyde group enables binding to α-amino group of anamino acid so that aldimine intermediates (Schiff s bases)are formed.Pyridoxal phosphate (PLP)An aminotransferase reaction is shown on the next picture.Transaminations are reactions with a "ping-pong“ mechanism:the first product leaves the enzyme before the second substrate binds, in the meantimepyridoxal phosphate binds the transferred amino group (as pyridoxamine phosphate).20
1st substrateAmino acidR CH COOHNH2HC O–H2OPyridoxal-P2nd substrateAldimine of PLP2-OxoglutarateHOOC–C–CH2-CH2-COOHThe 1st transferO H2OC H2– NH2-H2OKetimine of AAThe 2nd transferPyridoxamine-P1st productKetimine of αKG2nd productOxoacidR C– COOHO H2OGlutamateHOOC-CH-CH2-CH2-COOHNH2Aldimine of PLPHC O21Pyridoxal-P
The product of transamination glutamate is then deaminateddirectly by the action of glutamate dehydrogenase (GLD):Transamination:Oxidative deamination of glutamate2-OxoglutarateAmong other aminotransferases, the two are important (e.g. assays in clinicalbiochemistry, see Practicals):aspartate aminotransferase (AST, L-aspartate:2-oxoglutarate aminotransferase),which catalyses the reactionaspartate 2-oxoglutarateoxaloacetate glutamate , andalanine aminotransferase (ALT, L-alanine:2-oxoglutarate aminotransferase), which inthe liver catalyses the reactionalanine 2-oxoglutaratepyruvate glutamate.22
Indirect deamination of glutamate – so-called "purine nucleotide cycle"In some tissues, in skeletal muscle particularly, glutamate is deaminated not directly butundergoes transamination with oxaloacetate to give aspartate and 2-oxoglutarate.The amino group of aspartate is then transferred to hypoxanthine (the constituent of IMP,inosine-P), and the product (adenosine-P, AMP) adenylate is deaminated by adenylatedeaminase.Aspartate is also the sourceof one of the two nitrogenatoms of urea!OHNNNNRib PNH2H2ONNNNH3NRib P23
In eight proteinogenic amino acids, transamination or oxidative deaminationis not the usual first step in their degradation:Serine and threonine are deaminated by the action of serine dehydratase,histidine undergoes deamination by desaturation –both reactions were already mentioned previously.In five remaining amino acids, only some of their catabolites is deaminated.Arginine – deamination occurs after transformation to ornithin,lysine – after transformation to α-aminoadipate,methionine – deamination of homoserine,proline – deamination after conversion to glutamate,tryptophan – after its transformation to kynurenine, alanine is released.From the quantitative point of view, the most important reactions whichproduce ammonium ion NH4 (and molecules NH3) from the α-aminogroups of amino acids within the cells are:1234Direct deamination of glutamate (glutamate dehydrogenase reaction).Deamination of aspartate within the "purine nucleotide cycle".Aerobic glycine-cleavage system.Direct deamination of histidine by desaturation.24
Transport of "ammonia" in the bloodAmmonia is a weak base, molecules NH3 are mostly protonated to ammonium ion NH4 at physiological pH values. Therefore, we mean both NH4 and NH3 when speaking ofammonia.Ammonia is toxic, particularly to the brain. Regardless of the intensiveamino acid turnover in the body and high normal concetrations of ammonia(about 500 μmol / l) in organs other than brain, the concentration of ammoniain the peripheral blood is quite low, up to 40 μmol / l.The major transport form of ammonia between tissues is non-toxic glutamine(concentration in blood plasma 400 – 700 μmol / l).Synthesis of glutamineCOOHCOOHH2N–CHThe hydrolysis of glutamineto glutamate is catalysed amine synthetaseGlutamateGlutamineAn additional form of amino groups transfer from peripheral tissues, above allskeletal muscles, to the liver is alanine (the glucose-alanine cycle).Alanine concentration in blood plasma is 300 – 400 μmol / l.25
MuscleCentral nervous systemAmino acidsGlucosePyruvateAmino 2-Oxoglutarate CO2PyruvateALANINEIntestinal mucosaLiverOxaloacetateGlutaminePyruvate GlutamateUREAPortal bloodALANINENH4 The transfer of –NH2 from muscleto liver is provided mainly by theglucose-alanine cycle which isanalogous to the glucose-lactate(Cori) cycle.GlutamineGlutamate2-Oxoglutarate CO2OxaloacetatePyruvateBacterial deaminationsUrinary NH4 (depends on ABR)Glutamine is a major energy nutrient forsmall intestine mucosa. The nitrogenatoms of –NH2 are transferred to liveras NH4 (its concentration in portal bloodis very high) and alanine.26
The fate of ammonium ion NH4 in vertebratesNH4 formed in the breakdown of amino acidsMost aquatic vertebrates(bony fishes)The larvae of ampihibiaBirdsSnakesLizardsMost terrestial vertebratesAmmonotelic animalsUricotelic animalsUreotelic animalsNH4 SharksAdult frogsUric acidUreaO CNH2NH227
The ureosynthetic cycleIn most terrestial vertebrates, the liver cells detoxify NH4 ions originatingin the catabolism of amino acids by the synthesis of urea.NH2NH2C OUreaUrea (carbamide, carbonic acid diamide) is a non-electrolyte,highly soluble in water. It diffuses easily across biologicalmembrane, and therefore it is evenly distributed in all biologicalfluids and excreted into urine.The ureosynthesis is one of the unique functions of the liver cells. Thesecells také up glutamine and ammonium ions from the blood, they can alsodeaminate glutamate or other amino acids.The carbon atom of urea is derived from hydrogencarbonate anion, one ofthe nitrogens comes from ammonium ion, the other is obtained fromaspartate.Ureosynthesis is an endergonic process – 3 ATPs are hydrolysed to2 ADPs, 1 AMP and 4 Pi in the synthesis of one molecule of urea.It is recommendable to remember, from the physiological standpoint, that the ureosynthesisis a proton-producing process which consumpts hydrogencarbonate, a buffer base.
The simplified pathway of ureosynthesis(the ornithine or Krebs-Henseleit cycle)ATP29
Urea is synthesized in five steps:1 Synthesis of carbamoyl phosphate in the mitochondrial matrix2 ATPHCO3– NH4 2 ADP PiH2N–COOO–P–O–O–Carbamoyl phosphate(a mixed anhydride,a high-energy compound)The reaction is catalysed by carbamoyl phosphate synthetase I which requiresessentially the allosteric activator – N-acetylglutamate.The cytosolic form of the enzyme – carbamoyl phosphate synthetase II – utilizes glutamineinstead of ammonia and catalyses the first step of the pyrimidine base synthesis.30
2 The transfer of carbamoyl to ornithineH2NC OH2N–COOO–P–O–O–Carbamoyl phosphate CH2–NH2CH2–NHCH2CH2CH2CH2 rnithineCitrullineThe reaction is catalysed by ornithine transcarbamoylase to formcitrulline.Both ornithine and citrulline are amino acids, but they are not used as buildingblocks of proteins.The next three reaction of the cycle také place in the cytoplasma.31
3 The transport of citrulline to the cytoplasmaand condensation with aspartateH2NATPH2NAMP 2 H2O 2 P2C OC partateArgininosuccinateThe reversible reaction is catalysed by argininosuccinate synthetase andis driven by the cleavage of ATP into AMP and diphosphate and by thesubsequent hydrolysis of diphosphate.32
4 The cleavage of argininosuccinate into arginine and fumarateH2NH2NCOOHC N–CHC NH2 2COOHCOOHArgininosuccinateHOOC CHCHCOOHFumarateArginineArgininosuccinase cleaves argininosuccinate into arginine and fumarate.The carbon skeleton of aspartate is preserved in the form of fumarate.The synthesis of fumarate by the urea cycle is important becauseit links the urea cycle and the citric acid cycle: Fumarate after hydratation tomalate may enter mitochondrion and be oxidized to oxaloacetate, which canundergo transamination to aspartate. .33
5 The hydrolysis of arginine generates urea and ornithineNH2NH2H2NUreaC NH2 H2O CH2–NHCH2CH2CH–NH2COOHArginineC OArginaseCH2–NH2CH2CH2CH–NH2COOHOrnithine34
35
The amino acid degradation The first step in degradation of many standard amino acids is the removal of the α-amino group, i.e. deamination or transamination. The product is mostly the corresponding 2-oxoacid (α-ketoacid) and α-amino group is released as ammonia or ammonium ion. Direct deamination of amino acids
amino acid recommendations based on amino acid levels, and functional vitamin and mineral cofactor recommendations based on amino acid metabolism. These nutrient need suggestions are synthesized depending on the patients’ amino acid results, taking into account the age/gender of the patient and the severity of abnormal findings.
Introduction to amino acid metabolism Overview The body has a small pool of free amino acids. The pool is dynamic, and is . require dietary sources of these amino acids. In general, the synthesis pathways for the essential amino acids are complex, and involve a large number of reactions.
The 3 general reactions Removal of the NH 3 group Removal of the Carboxyl group Transfer of Methyl groups Specific pathways for amino acid carbon metabolism One carbon metabolism Metabolic processing of amino acid nitrogen The Urea Cycle Phosphocreatine, an i mportant energy reservoir I. OVERVIEW
Titration of amino acid: When an amino acid is dissolved in water it exists predominantly in the . isoelectric form. Amino acid is an . amphoteric. compound It act as either an acid or a base: Upon titration with acid it acts as a BASE (accept a proton). Upon titration with base it acts as an ACID (donate a proton)
Numerous qualitative amino acid differences were also found. Of the twenty amino acids commonly occurring in proteins only alanine and tyrosine were not detected in any metamorphic stage of any genotype at either 16 G or 25 G« Th remaining eighteen commonly occurring amino acids oocurred in three general categories. Four amino
Amino Acid Metabolism Metabolism of the 20 common amino acids is considered from the origins and fates of their: (1) Nitrogen atoms (2) Carbon skeletons For mammals: Essential amino acids must be obtained
A-Disorders of nitrogen-containing compounds: 6-6-Disorders of glutathione metabolism 11-Disorders of phenylalanine 12-Disorders of tyrosine metabolism 13-Disorders of sulfur amino acid and sulfide metab. 14-Disorders of branched-chain amino acid metab. 15-Disorders of lysine metabolism 16-Disorders of proline and ornithine metabolism 18 .
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