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Amino acid metabolism I
proteinsproteins
amino acidspool
proteosynthesis specificspecificsynthesissynthesis((nonessencialnonessencialAA)AA)
keto acids NH3
CO2, H2O, ATP physiologicallyphysiologicallyimportantimportantnitrogenousnitrogenouscompoundscompounds
specific pathways↓
hormonesneurotransmiters
koenzymesporphyrins
purinespyrimidines
creatine
biogenic amines
proteolysis(digestion)
common metabolic pathways
TCAUC
urea
glukose ketone bodies
H2N-C-NH2
O
transamination deamination decarboxylation
↓Phe ↓↓ Tyr ↓
Lys ↓, Arg ↓Phe ↓, Tyr ↓, Trp ↓, Met ↓, Leu ↓Ala ↓, Gly ↓,Ser ↓A: ↓, Ala ↓ Ile, ↓ Leu, ↓ValB: ↓ Lys, ↓ Arg
endopeptidasses
exopeptidases
Protein digestion - proteolytic enzymes (proteinases/proteases)
stomach
smallintestine
brush bordermembrane enzymes
absorption - enterocyte (active transport)portal blood liver
TrypsinChymotrypsinElastase
Carboxypeptidases
Specificity and activation of pancreatic proteases
Trypsin = activator of all digestive proteolytic enzymes in the small intestine
Proteinases (proteases)
1. Synthesis and secretion: proenzymes (zymogenes)
2. Activation: cleavage of several peptide bonds or/and removal ofa short peptide→ openning of an active site
pepsinogen pepsin + 41 AA
trypsinogen trypsin + 6 AA
3. Inhibition: protein inhibitors→ interaction with proteinases active sites
HCl
enteropeptidase
trypsin
trypsinogen
substrate(polypeptide)
• pancreatic trypsin-inhibitor:small protein → very tight binding at the activesite of trypsinu ⇒ inactive complex (half life: several months)
↓protection of intestinal walls and cellsagainst proteolytic cleavage
• α1-antitrypsin (α1-antiproteinase, antielestase):plasma protein – irreversible binding at the active site of trypsin and elastase→ protection of tissues against digestion by elastase (trypsin)
genetic mutants↑
smokerssmokers
low blood level↓
destruction of alveolar walls(degradation of connective structures by elastase)
↓emphysema
Proteinases inhibitors
slow secretion from liver
Protein turnover
Dietary proteins 100 g/day equivalent amount of nitrogenousmetabolites (mainly urea) excreted
Body proteins 400 g/day constant degradation and resynthesis
AA – mostly metabolised in liver; Leu, Ile, Val pass without transformation –utilized in muscle and brain
glutamine, valine, alanine, glycine- most abundant AA in the blood circulation
biological half-life of proteins:
< 2 h digestive enzymes, ornithine dekarboxylase, HMG-CoA-reductase< 2- 200 h most proteins> 200 h hemoglobin, acetylcholine receptorseveral months, collagen, structural proteins
years
Intracellular protein degradation
1. Lysosomal proteases (cathepsins, collagenase,dipeptidases..):most extracellular proteins, long-lived
proteins2. Proteasomes:abnormal proteins, short-lived proteins
Ubiquitinationcytosolic protein ubiquitin –kisskiss ofof deathdeath- binding with a protein predestines it for degradation inproteasome= oligomer – supramolecular structure, severalsubunits = proteinases
ubiquitin-COOH + H2N-Lys-protein
mark for degradation
Signals for degradation:1.oxidation: Lys, Trp, His, Cys2. PEST-sequence: Pro, Glu, Ser, Thr3. NH3-end of AA: Arg, Lys, Asp, Phe
Catabolism of amino acids – common reactions
Pyridoxal phosphate– unique role in amino acid metabolism - cofactor ofenzymes catalyzing different kinds of AA transformations
in the active site held by noncovalent interactions orlinked covalently to ε-amino group of lysine
pyridoxal phosphate (PLP)pyridoxine Schiff baseAA - PLP
3
21
essential intermediate of AA metabolism
a derivative of vitamin B6
1. Decarboxylation2. Transamination3. Transformation of a carbon chain→ specific for each amino acid
common reactions of amino acid metabolism
1. Decarboxylation biogenic amines; enzymes - decarboxylases
R-C-COOH R-CH2-NH2 + CO2
neuromediators
H
NH2
histidine → histamine: mediatorserine→ ethanolamine→ phospholipidscysteine → cysteamine→ CoA-SHTrp → 5-OH-Trp → serotonineglutamic acid→ GABA
PLP
2
putrescine
Polyamines– spermidine, spermine– initial substrates ornithine and methioninestabilization of RNA, DNA, membranes - regulation of cell growthand proliferation, regeneration of tissues
ornithinedecarboxylase
2. Transamination→ keto acids; enzymes – aminotransferases! reaction reversible – involved both in catabolism andbiosynthesis of aminoacids
H
NH2
R2-C-COOH
O
R2-C-COOH
H
NH2
R1-C-COOH PLP+ R1-C-COOH
O
+
Example: alanine aminotransferase
alanine 2-oxoglutarate pyruvate glutamate= α-ketoglutarate
-
--
-
exclusive acceptor of amino group in transaminase -NH2 of any AA incorporatedreaction in AA catabolism AK into molecule of glutamate
1 1
1
2
1
2
2
2
Mechanism oftransamination
ALT – alanine aminotransferase:
alanine + 2-oxoglutarate pyruvate + glutamate
AST – aspartate aminotransferase:
aspartate+ 2-oxoglutarate oxaloacetate+ glutamate
Localization: mainly liver, muscle, kidneyALT – cytosol, AST –cytosol and mitochondria!
Metabolic significance:ALT, AST – catabolism andi biosynthesis of alanine/aspartate,ALT – important for utilization of muscle protein AA for gluconeogenesis
(glucose-alanine cycle)AST – replenishment of oxalacetate for CC (anaplerotic reaction),
transport of oxalacetate across mitochondrial membrane (gluconeogenesis),important for the function of malate dehydrogenase shuttle and urea syntesis(formation of aspartate as a donor of one nitrogen atom ofthe urea)
Diagnostic value:ALT, AST – markers of liver injury (hepatocellular necrosis),AST – blood level increases at myocardial infarction (not used as a marker in the present
IM diagnostics)
ALT, AST – important aminotransferases/transaminases
PLP
PLP
Deamination: removal of -NH2 as NH3
1. Direct deamination→ serine, threonine serine (threonine) dehydratase
2. Oxidative deamination– other amino acids
L-amino acid oxidases, cofactor FMN
- liver, kidney, low activity – little valuefor AA metabolism
D-amino acid oxidases, cofactor FAD
- ?deamination of D-amino acidsof bacterial and plant origin
CH2-C-COOH
NH2OH
H
CH2= C-COOH
NH2
NH3CH3-C-COOH
O
+ CH2=C-COOH
OH
H2O H2O
OHH
Oxidative deamination of glutamate - kee reaction for removal of nitrogenfrom amino acids
amino acid+α-ketoglutarate→ keto acid + glutamate -a product of transamination
NH3 + α-ketoglutarate
! Glutamate dehydrogenase (GDH) (NAD+/ NADP+)(high amount – liver mitochondria – used in diagnostics of liver
diseases)
HOOC-CH2-CH2-C-COOH
HOOC-CH2-CH2-C-COOH HOOC-CH2-CH2- C-COOH + NH3
NAD+
NADH+H+
H2ONH2
NHO
H2
O
α−ketoglutarate
glutamate
oxidative deamination
Ammonia - NH3• sources:
1. Deamination of amino acids (glutamate) –all tissues2. Hydrolysis of glutamine -kidney, small intestine, liver3. Bacterial degradation of proteins and urea (urease) in theintestine4. Catabolism of purines, pyrimidines, catecholamines- liver, brain
• detoxification:1. Urea synthesis -!liver! (exclusively) → kidney→ urine2. Synthesis ofglutamine – extrahepatic tissues3.Formation ofNH4
+ - kidney → urine
HOOC-CH-CH2-CH2-COOH
NH2 NH2
O
NH2
HOOC-CH-CH2-CH2-CNH3 +glutamate glutamine
glutaminesynthetase
glutamineglutaminase
NH3 + glutamate
muscle, brain
kidney
H+NH4
+
ad 2, 3
urine
kidney, liver
Ammonia metabolism - overview
Urea synthesis = Ureagenesis:major pathway of NH3 detoxification
Localization: liver⇒ mitochondria, cytosol of periportal hepatocytes
precursors: NH3oxidative deamination
of glutamate (GDH)CO2
TCA cycle
aspartatetransamination ofoxaloacetate (AST)
glutamate
glutamateNH3 + α-ketoglutarate
aspartateα-ketoglutarate
oxaloacetate
NAD+
Energy needs of ureagenesis: 4 ATP
H2N-C-NH2
O
NADH+H+
GDH
AST
Proteins↓↓↓↓↓↓↓↓
amino acidsaminotransferase
α-keto acid
glutamate
α-ketoglutarate
glutamate degydrogenase
NAD+
NH3 aspartate
NADH+H+
α-ketoglutarate
oxaloacetate
α-ketoglutarate
aspartate aminotransferase
citrulline
argininosuccinate
fumaratearginineurea
ornithine
carbamoyl phosphate
ureaureacyclecycle
CO2
Urea cycle – formation of urea
- NH3 enters intu the cycle after formationof carbamoyl phosphate
- carbamoyl phosphate formation fromNH3, CO2, ATP catalyzescarbamoylphosphate synthetase I
– enzyme located in hepatocytemitochondriaonly !
-carbamoyl phosphateis also precursorof pyrimidine nucleotides – formation incytosol – catalyzed by carbamoylphosphate synthetase II,source of its NH2-group is glutamine(! not NH3)
Cooperation of urea cycle and citric acid cycle- utilization of fumarate, a side product of ureagenesis
oxaloacetate
Regulation of urea synthesis
1. A substrate delivery- low-protein diet - decreased AA intake decreased NH3 formation
low activity of urea cycle, decreased urea level in urine
- high-protein diet, fasting (degradation of muscle proteins) - increased AA deliveryinto liver increased activity of urea cycle
2. N-acetylglutamate– allosteric activator of carbamoyl phosphate synthetase I (essential for the enzyme activity)formation from glutamate – increased at higher AA delivery= at protein degradation -explanation of increased
ureagenesis in fasting
Genetic defects of urea cycle enzymeshyperammonemia – partial deficit can be treated by low-protein diet and
suplementation of food with arginine (precursor of ornithine)- significantly decreased activities of ureagenetic enzymes -incompatible with life
Hyperammonemia
1. liver cirrhosis (alcoholic), hepatitis, obstruction ofbile duct⇒ NH3 from GIT enters via portal blood→ directly into systemic circulation
2. genetic defects of enzymes of ureagenesis→ inhibition of urea synthesis→ NH3 cannot be detoxified⇒ vomiting, lethargy, coma , serious brain
damage, death
Ammonia toxicity (namely forCNS) –suggested mechanisms
Brain: NH3 + α−ketoglutarate glutamate
decreased mitochondrial level→ inhibition of citric acid cycle(substrate depletion)
→ decreased ATP synthesisNH3 + glutamate glutamine
depletion of glutamate (neurotransmitter)
(excess of NH3 inhibits glutaminase)
alteration of nerve impuls transmission
GDH
NADPH+H+ NADP+
x
Fate of ammonia in tissues
brain + most tissues(also muscle)
amino acid
transamination
glutamate
GDHNH3
glutamine synthetase
glutamine
Liver
amino acid
transamination
glutamate
GDHNH3
urea cycle
urea
Kidneyglutamine
glutaminase
glutamate + NH3 urine
urea
Glucose-alanine cycle
- connection of muscle glycolysis andliver gluconeogenesis – analogy to Cori cycle
- kee role ofALT
- important in starvation- gluconeogenesis from AA - dominant- uses also alanine released during breakdownof contractile proteins