Amino acid metabolism I
proteins
proteolysis
(digestion)
proteosynthesis
specific synthesis
(nonessencial AA)
amino acids
pool
common metabolic pathways
transamination
deamination decarboxylation
keto acids
NH3
biogenic amines
ketone bodies
glukose
TCA
CO2, H2O, ATP
UC
urea
H2N-C-NH2
O
specific pathways
↓
hormones
neurotransmiters
koenzymes
porphyrins
purines
pyrimidines
creatine
physiologically important
nitrogenous compounds
Protein digestion - proteolytic enzymes (proteinases/proteases)
↓Phe ↓
↓ Tyr ↓
endopeptidasses
stomach
Trypsin
Chymotrypsin
Elastase
Carboxypeptidases
Lys ↓, Arg ↓
Phe ↓, Tyr ↓, Trp ↓, Met ↓, Leu ↓
Ala ↓, Gly ↓,Ser ↓
A: ↓, Ala ↓ Ile, ↓ Leu, ↓Val
B: ↓ Lys, ↓ Arg
brush border
membrane enzymes
absorption - enterocyte (active transport)
portal blood
liver
exopeptidases
small
intestine
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 of
a short peptide → openning of an active site
pepsinogen
HCl
pepsin + 41 AA
trypsinogen enteropeptidase trypsin + 6 AA
trypsinogen
3. Inhibition: protein inhibitors
→ interaction with proteinases active sites
substrate
(polypeptide)
trypsin
Proteinases inhibitors
• pancreatic trypsin-inhibitor: small protein → very tight binding at the active
site of trypsinu ⇒ inactive complex (half life: several months)
↓
protection of intestinal walls and cells
against 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
↑
smokers
slow secretion from liver low blood level
↓
destruction of alveolar walls
(degradation of connective structures by elastase)
↓
emphysema
Protein turnover
Dietary proteins
100 g/day
equivalent amount of nitrogenous
metabolites (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:
<2h
< 2- 200 h
> 200 h
several months,
years
digestive enzymes, ornithine dekarboxylase, HMG-CoA-reductase
most proteins
hemoglobin, acetylcholine receptor
collagen, structural proteins
Intracellular protein degradation
1.
Lysosomal proteases (cathepsins, collagenase,
dipeptidases..): most extracellular proteins, long-lived
proteins
2. Proteasomes: abnormal proteins, short-lived proteins
Ubiquitination
cytosolic protein ubiquitin – kiss of death
- binding with a protein predestines it for degradation in
proteasome = oligomer – supramolecular structure, several
subunits = proteinases
ubiquitin-COOH + H2N-Lys-protein
mark for degradation
Signals for degradation:
1.oxidation: Lys, Trp, His, Cys
2. PEST-sequence: Pro, Glu, Ser, Thr
3. NH3-end of AA: Arg, Lys, Asp, Phe
Catabolism of amino acids – common reactions
Pyridoxal phosphate – unique role in amino acid metabolism - cofactor of
enzymes catalyzing different kinds of AA transformations
in the active site held by noncovalent interactions or
linked covalently to ε-amino group of lysine
a derivative of vitamin B6
3
2
pyridoxine
pyridoxal phosphate (PLP)
1
Schiff base
AA - PLP
essential intermediate of AA metabolism
1. Decarboxylation
common reactions of amino acid metabolism
2. Transamination
3. Transformation of a carbon chain → specific for each amino acid
1. Decarboxylation
biogenic amines; enzymes - decarboxylases
H
R-C-COOH
NH2
PLP
R-CH2-NH2 + CO2
neuromediators
histidine → histamine: mediator
serine → ethanolamine → phospholipids
cysteine → cysteamine → CoA-SH
Trp → 5-OH-Trp → serotonine
glutamic acid→ GABA
Polyamines – spermidine, spermine – initial substrates ornithine and methionine
stabilization of RNA, DNA, membranes - regulation of cell growth
and proliferation, regeneration of tissues
2
ornithine
decarboxylase
putrescine
2. Transamination → keto acids; enzymes – aminotransferases
H
R1-C-COOH
NH2
! reaction reversible – involved both in catabolism and
biosynthesis of aminoacids
H
PLP
+ R2-C-COOH
R1-C-COOH + R2-C-COOH
NH2
O
O
Example: alanine aminotransferase
-
-
alanine
2-oxoglutarate
pyruvate
glutamate
= α-ketoglutarate
exclusive acceptor of amino group in transaminase
reaction in AA catabolism AK
-NH2 of any AA incorporated
into molecule of glutamate
1
1
1
Mechanism of
transamination
1
2
2
2
2
ALT, AST – important aminotransferases/transaminases
ALT – alanine aminotransferase:
alanine + 2-oxoglutarate
PLP
pyruvate + glutamate
AST – aspartate aminotransferase:
aspartate + 2-oxoglutarate
PLP
oxaloacetate + glutamate
Localization: mainly liver, muscle, kidney
ALT – 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 of the 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)
Deamination: removal of -NH2 as NH3
1. Direct deamination → serine, threonine
H
H2O
HO
serine (threonine) dehydratase
2
CH2-C-COOH
OH NH2
CH2= C-COOH
NH2
OH
H
NH3
2. Oxidative deamination – other amino acids
L-amino acid oxidases, cofactor FMN
- liver, kidney, low activity – little value
for AA metabolism
D-amino acid oxidases, cofactor FAD
- ?deamination of D-amino acids
of bacterial and plant origin
+ CH2=C-COOH
OH
CH3-C-COOH
O
Oxidative deamination of glutamate - kee reaction for removal of nitrogen
from amino acids
amino acid +α-ketoglutarate → keto acid + glutamate -a product of transamination
oxidative deamination
NH3 + α-ketoglutarate
! Glutamate dehydrogenase (GDH) (NAD+/ NADP+)
(high amount – liver mitochondria – used in diagnostics of liver
diseases)
NAD+
HOOC-CH2-CH2-C-COOH
glutamate
NADH+H+
NH2
H2O
HOOC-CH2-CH2-C-COOH
O
NH
HOOC-CH2-CH2- C-COOH + NH3
O
H2
α−ketoglutarate
Ammonia - NH3
• sources:
1. Deamination of amino acids (glutamate) – all tissues
2. Hydrolysis of glutamine - kidney, small intestine, liver
3. Bacterial degradation of proteins and urea (urease) in the intestine
4. Catabolism of purines, pyrimidines, catecholamines - liver, brain
• detoxification:
1. Urea synthesis - !liver! (exclusively) → kidney → urine
2. Synthesis of glutamine – extrahepatic tissues
3.Formation of NH4+ - kidney → urine
NH3 + HOOC-CH-CH2-CH2-COOH
glutamate
NH2
muscle, brain
NH4+
kidney
H+
glutamine
synthetase
urine
HOOC-CH-CH2-CH2-C
NH2 glutamine NH2
NH3 + glutamate
kidney, liver
ad 2, 3
O
glutaminase
glutamine
Ammonia metabolism - overview
Urea synthesis = Ureagenesis: major pathway of NH3 detoxification
Localization: liver ⇒ mitochondria, cytosol of periportal hepatocytes
O
H2N-C-NH2
precursors:
aspartate
NH3
oxidative deamination
of glutamate (GDH)
transamination of
oxaloacetate (AST)
CO2
TCA cycle
glutamate
NAD+
glutamate
NADH+H+
GDH
NH3 + α-ketoglutarate
AST
aspartate
oxaloacetate
α-ketoglutarate
Energy needs of ureagenesis: 4 ATP
Proteins
↓
↓
amino acids
aminotransferase
α-ketoglutarate
α-keto acid
NAD+
glutamate
glutamate degydrogenase
aspartate aminotransferase
α-ketoglutarate
NADH+H+
CO2
NH3
oxaloacetate
α-ketoglutarate
aspartate
citrulline
carbamoyl phosphate
ornithine
urea
urea
cycle
arginine
argininosuccinate
fumarate
Urea cycle – formation of urea
- NH3 enters intu the cycle after formation
of carbamoyl phosphate
- carbamoyl phosphate formation from
NH3, CO2, ATP catalyzes carbamoyl
phosphate synthetase I
– enzyme located in hepatocyte
mitochondria only !
-carbamoyl phosphate is also precursor
of pyrimidine nucleotides – formation in
cytosol – catalyzed by carbamoyl
phosphate 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 delivery
into 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 enzymes
hyperammonemia – 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 of bile 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 for CNS) – suggested mechanisms
Brain: NH3 + α−ketoglutarate
GDH
glutamate
NADPH+H+ NADP+
decreased mitochondrial level → inhibition of citric acid cycle
(substrate depletion)
→ decreased ATP synthesis
NH3 + glutamate
glutamine
x
depletion of glutamate (neurotransmitter)
(excess of NH3 inhibits glutaminase)
alteration of nerve impuls transmission
Fate of ammonia in tissues
brain + most tissues (also muscle)
Liver
amino acid
amino acid
transamination
transamination
glutamate
glutamate
GDH
GDH
NH3
NH3
urea cycle
glutamine synthetase
urea
glutamine
Kidney
glutamine
glutaminase
urea
glutamate + NH3
urine
Glucose-alanine cycle
- connection of muscle glycolysis and
liver gluconeogenesis – analogy to Cori cycle
- kee role of ALT
- important in starvation
- gluconeogenesis from AA - dominant
- uses also alanine released during breakdown
of contractile proteins