Amino Acid

Metabolism I
©Department of Medical and Clinical Biochemistry
UPJŠ in Košice, Faculty of Medicine
Amino acid metabolism
Outline

• Degradation of proteins endogenous and

from the diet
• Proteolytic enzymes
• Amino acid pool
• Catabolism of Amino acids
• The fate of ammonia
• The Urea cycle
• Metabolic degradation of C-skeleton of AAs
Degradation of Proteins
Dietary - exogenous

• Proteolysis - complete degradation of

proteins to free AAs:
– Requires series of enzymes with different
specificity: hydrolytically cleaved peptide bond of
proteine by enzymes - proteases
– Not only in the digestive tract, but also in every
cell → in lysosomes, in cytoplasm (proteasome)
by special proteases
Degradation of Proteins
Proteolysis
• Exogenous (dietary) • Endogenous (body)
proteins: proteins:
– Lumen GIT – Intracelular protease
– Stomach – pepsín – Two routes:
– Small intestine – pancreatic
• Lysosomes
protease (napr. trypsin,
chymotrypsin) • Ubiquitin-proteasomes
Protein-Ub

cytosolic
peptidase
Ub + short peptides AA
Protein Degradation
Endogenous - Turnover
• Protein turnover is the breakdown and re-
synthesis of body proteins:
– e.g. old tissues, damaged proteins, recycling enzymes
and hormones
• Turnover of protein is not constant - half lives of
proteins - vary from minutes to infinity
− “normal” proteins: 100-200 hrs
− short-lived proteins: minutes - hrs, regulatory proteins,
enzymes that catalyze committed steps, transcription
factors
− long-lived proteins: days - years, special cases (e.g.
dentin, elastin, collagen, crystallins)
Protein Degradation
Endogenous
Two Sites for Protein Degradation:
• Lysosomal processes: 10-20 %
– Receptor mediated endocytosis and phagocytosis e.g.:
o extracellular proteins, cell organelles
o some intracellular proteins
• Ubiquitin/Proteasome Pathway: 80-90 %
− Most intracellular proteins e.g.:
o transcription factors, cell cycle cyclins, virus coded proteins,
improperly folded proteins, damaged proteins
• Example:
– Cystic fibrosis is due to the accelerated degradation of chloride
transporter
Protein Degradation
Endogenous - lysosomes

• Lysosomes
− Basal degradation: non-
selective, not depend on ATP
− Proteins with longer half-life
− Lysosomal protease -
catepsins
− Degradation under starvation:
selective for “KFERQ”
(proteins Lys, Phe, Glu, Arg, Gln)
Protein Degradation
Endogenous - proteasomes
• Proteasomes
− Large (26S) multiprotein complex (28 subunits)
− „Waste bin" - a hollow, cylindrical body composed of 28
polypeptides (4 cyclic heptamers, 4  7 = 28)
− Process need ATP
− There are various specific proteases that cleave the
labeled protein into short peptides (8 AAs)
− Degrades ubiquitinated proteins
Protein Degradation
Cytosolic degradation in proteasomes
• Conjugation of ubiquitin (Ub) to a protein is a
major, non-lysosomal proteolytic pathway
• After selective labeling of the protein by conjugation
of the Ub chain K48 (4 or more Ub in the chain) its
degradation occurs in the proteasome, removing for
example:
− abnormal proteins, proteins with short half-lives (e.g.
regulation molecules, transription factors (p53), cyclins)
• Discovery of ubiquitin-dependent
proteasomalprotein degradation: 2004, Nobel Prize
in Chemistry – A. Hershko, A. Ciechanover, I. Rose
Protein Degradation
Endogenous - ubiquitin

• Small peptide that is a “tag”

− 76 amino acids, highly conserved,
termostable
− C-terminal Gly - isopeptide bond with
the ε-amino group of Lys residues on
the substrate - „kiss of death“
− Attached as monoubiquitin or
polyubiquitin chains
− Ub is not degraded, releases
unchanged
Protein Degradation
Exogenous
• Proteolysis = peptide hydrolysis
– facilitated nucleophilic attack of water on peptide bond by
enzyme - protease
• Four mechanistic categories of proteases:
– serine proteinases
• chymotrypsin family
• subtilisin family
– cysteine proteinases (e.g. papain, caspsases)
– aspartic proteinases (e.g. pepsin)
– metallo proteinases (e.g. thermolysin)
• Unlike proteasome, most proteases are specific
Protein Degradation
Proteolytic enzymes
Pepsin

• Endopeptidases (proteinases) hydrolyse the

peptide bond inside a chain, e.g.:
− Pepsin (pH 1.5-2.5) – peptide bond derived fromTyr,
Phe, bonds between Leu, Glu
− Trypsin (pH 7.5-8.5) – bonds between Lys, Arg
− Chymotrypsin (pH 7.5-8.5) – bonds between Phe, Tyr
• Exopeptidases – split the peptide bond at the end of a
protein molecule:
− aminopeptidase,
− carboxypeptidases,
− dipeptidases
Protein Degradation trypsin

Proteolytic enzymes
• Classification of proteinases → endopeptidases
hydrolysis within the peptide chain:
1. serine proteinases – Ser or His residues in active sites,
pH optimum 7-9
– trypsin, chymotrypsin, thrombin, plasmin
2. cysteine proteinases – Cys residue in active site,
pH optimum 4-7
– calpain, caspases
3. aspartic proteinases – two Asp residues in active site,
pH optimum below 5
– pepsin, gastricsin, rennin, HIV proteinase
4. metaloproteinases – metal ion important for catalysis,
pH optimum 7-9
– matrix metaloproteinases, collagenase, gelatinase
Protein Degradation
Proteolytic enzymes
• Classification of exopeptidases - cleave off a single
amino acid one after another:
1. aminopeptidases – cleave off a single amino acid
from amino terminus
2. dipeptidyldipeptidases – cleave off dipeptide from
amino terminus
3. carboxypeptidases – cleave off a single amino acid
from carboxy terminus
4. dipeptidases – hydrolyse dipeptides in two single
amino acids
• Individual proteases differ in their substrate specifity
• For complete degradation of a protein, enzymes
from each of the above-named groups are required
Protein Degradation
Digestion proteins from diet

• The digestion of proteins:

– begins in the stomach
o denatures proteins and activates
enzyme pepsin that hydrolyze
the peptide bonds
– moves into the small intestine
o is completed in the small
intestine by trypsin and
chymotrypsin to form amino
acids
Protein Degradation
Summary
• Digestion of proteins from diet by proteolytic enzymes

+
H3N CH-CO-NH-CH- COO−
CH-CO-NH-CH-CO-NH-CH-CO-NH-
R R R R
proteins food Phe,Tyr

pepsine trypsine chymotrypsine elastase

+H N
3 CH-CO-NH-CH-CO-NH-CH-CO-NH-
CH-CO-NH-CH-CO-NH-
stomach
R R R R
polypeptides Arg Phe,Tyr,Trp, Gly, Ala,
and aminoacids Met, Leu
pancreas trypsine,
liver chymotrypsine,
elastase, karb oxy-

karboxypeptidase B Arg
small oligopeptides
and aminoacids Lys
intestine
aminopeptidase +H N
3 CH-CO-NH-CH- COO−
CH-CO-NH-CH-CO-NH-CH-CO-NH-

amino acids R R R R
Ala,Val
karboxypeptidase A
Leu,Ile
Protein Quality
BV – Biological Value
• The amount of endogenous proteins (g) produced from
100 g of exogenous proteins (%)
– daily intake of proteins: 0.8 g/kg BVanim. > BVplant
• Quality of proteins depend on: Wheat: deficit Lys, Met, Trp, Thr,
Leguminous plant: deficit Met, Cys
− Digestibility: animal vs. plant
− AAs composition: essential, limiting AAs
− Their (AAs) relationship
• Is reflected in the AA score: content of individual essential
AAs in food
− Four AA are likely to be limiting: Lys, Met/Cys, Thr, Trp
milk seat
wheat egg whey
soja
Protein Quality
BV – Biological Value

Protein BV (%)
Egg white 100
Whey 100
Egg 96
Casein 80
Beef 77 • contains ~ 12 % high
quality protein (lacto
Pork 70
albumin, lactoglobulins)
Oatmeal 60 • also contains vitamins
Wheat flour 53 B-complex and lactose
Leguminous plants 46
Gelatin 25
Protein Nutrition
Protein-Energy Malnutrition (PEM)
• Acute PEM when one is
recently deprived of
food
– children are thin for their
weight
• Chronic PEM from long
term food deprivation
– children are short for
their age
Protein Nutrition
Protein-Energy Malnutrition (PEM)
• Marasmus - inadequate energy and protein
over a long period of time
– often seen at 6-18 months of age
– look like little old people
• Kwashiorkor - “the evil spirit that infects the
first child when the second child is born”
– sudden deprivation at 18 mon to 2 years
• Marasmus-kwashiorkor mix: edema of
marasmus with wasting of kwashiorkor
Protein Degradation
Nitrogen balance - NB
• Nitrogen balance: NB = Ninput − Noutput
– normal state - balanced NB
– positive NB - growth, pregnancy, reconvalescence
– negative NB - metabolic stress, starvation, low-value
proteins
• Increased protein catabolism:
– dreaded complication of the underlying disease,
– increased risk of infection,
– coagulopathy,
– decreased regeneration of blood elements,
– reduced enzyme synthesis,
– disorders in wound healing
Protein Degradation
Nitrogen balance - NB
• Our bodies maintain a nitrogen balance in the cells
so that the amount of protein we break down is equal
to the amount that is reused:
– diets high in protein, however, have a positive nitrogen
balance because a high-protein diet supplies more nitrogen
than we need, e.g. during growth or when building new
tissue;
– the body cannot store nitrogen, so the excess is excreted
as urea, putting an extra demand on the liver and kidneys;
– diets that do not provide sufficient protein have a negative
nitrogen balance, a condition that occurs during, e.g.
starvation and fasting, if burned, fever, injury, infection.
 Protein Metabolism
Amino Acid Pool

Tissue proteins nucleotide, hem…

energy

Proteins Excretion as
AAs pool urea and NH4+
from food

• The amount of nitrogen taken up by food is normally in

equilibrium, excluding its equivalent amount
• About 80 % of the excreted nitrogen is in the form of urea
Protein Metabolism
Amino Acid Pool

• Proteins are degraded and resynthesized

continuously
− several times more protein is turned over daily within
the body (endogenous) than is consumed (exogenous)
• AAs consumed in excess or unable to be used are
not stored, they are:
− degraded into urea, uric acid, and creatinine
− lost in feces or sweat
− converted into body proteins e.g. enzymes, hair, nails
Amino Acids
„AA pool“ ~ 100 g/d

• Source of organically bound nitrogen:

~ 80 % in the muscle
~ 10 % in the liver
~5% in the kidney
~5% in the circulation (25 % Gln)
proteins from diet proteosynthesis
exogenous
body proteins AA pool N-containing comp.
endogenous
biosynthesis de novo degradation
(E,Glc,fat)
Amino Acids
Energy

• Energy is extracted from AAs in conditions

such as fasting or starvation:
– if AAs remain the only source of energy for a long
period of time, the breakdown of body proteins
eventually leads to a destruction of essential
body tissues;
– in anorexia, the loss of protein decreases muscle
mass and may severely weaken the heart muscle
and impair heart function
Amino acids
Catabolism
Amino acids

deamination α-oxo acid

Carbon skeleton

Glukóza
Glucose Keto bodies
Ketone látky

detoxication formation of common metabolic products

Urea
Amino Acids
Classification

• Proteinogenic AAs from metabolic point of

view:
1) biosynthesis in a human body
− nonessential (are synthesized)
− essential (must be present in a diet)
2) degradation within cells
− glucogenic (Glc can be formed from their C-skeleton)
− ketogenic (= AAs degraded to acetyl-CoA)
− know which are purely ketogenic
Amino Acids
Essential

• Required in diet
• Humans incapable of forming requisite
carbon skeleton - Isoleucine - Ile
• During the growth period: - Leucine - Leu
– Arginine - Arg - Lysine - Lys
– Histidine - His - Methionine - Met
- Phenylalanine - Phe
• In metabolic stress: - Threonine - Thr
– Alanine - Ala - Tryptophan - Try
– Glutamine - Gln - Valine - Val
 Amino Acids
Catabolism – basic reactions

transamination a−keto acid

+ new amino acid

NH2 deamination a−keto acid

α
R C H

COOH decarboxylation biologically

active amines
Amino Acids
Catabolism – basic reactions
• Transamination
– an α-amino group is transferred from an AA to an α-keto
acid, usually α-ketoglutarate, oxalacetate, pyruvate
– a new AAs (Glu, Asp, Ala), a new α-keto acid are
produced and native forms of enzyme.
• The enzymes for the transfer of amino groups are known as
transaminases or aminotransferases.
Catabolism AAs
Enzymes

• Clinically important transaminases:

– ALT – alanine aminotransferase,
• clinical marker for irreversible liver damage
– AST – aspartate aminotransferase
• clinical marker for irreversible myocardial damage
Catabolism AAs
Basic reactions
• Deamination (elimination of amino group)
– shift amino group to ammonia or amino group of Asp
• Oxidative deamination
− conversion Glu by glutamate dehydrogenase and
coenzymes NAD(P)+ in mitochondria → a-
iminoglutarate, which is hydrolysed to 2-oxoglutarate
and ammonia
• Build in atoms of N ammonia and Asp to urea for
elimination from organism – urea cycle
– change the a-oxoacids (C-skeleton of AAs after
deamination) to standard metabolic intermediates
Catabolism AAs
Reactions – Summary
R-CH-COOH FAD FADH2
NH2
decarboxylation
NADH R-C-COOH
CO2 H2O NH3
R-CH2-NH2 R1-CO-COOH NH
NAD+
R1-CH-COOH
NH2 NH3
transamination R-CO-COOH
R-CO-COOH R-CH2-COOH
reduced deamination
CO2
CO2
R-COOH R-COOH
aerobic decarboxylation aerobic deamination
Catabolism AAs
Basic steps
hydrolyse
proteins Amino Acid + 2-oxoglutarate
Nitrogen intake
transamination

Motion of nitrogen 2-oxoglutarate + glutamete

deamination

Detoxication in NH3
Detoxication
extrahepatal tissues
in the liver

glutamín (Gln) urea

Nitrogen out

• Transamination
• Oxidative deamination of Glu
– detoxication of ammonia
Catabolism AAs
Detoxication of ammonia
• Excess nitrogen produced by AAs
degradation is excreted in the form of:
− urea – most terrestrial vertebrates
− ammonia – bacteria, aquatic vertebrates or
amphibian larvae NH4+
Cation of ammonia
− uric acid – birds and land reptiles
Amino Acids NH4+

N
N

NH3
Sources of ammonia
transamination (ALT)  Glu
cytosol

• Cell localization of
Glu + NH3 → Gln GDH
Glu
selected conversion NH3 mitochondria

transamination
Urea (AST)
synthesis

• The main sources of ammonia in the body:

− oxidative deamination of Glu – in the cells of the
most tissues
− bacterial fermentation in the large intestine –
ammonia by diffusion passes into the portal blood
Amino Acids NH4+

N
N

Sources of ammonia NH3

• Sources of ammonia in the body:

− oxidation deamination some AAs
H2O
R CH COOH R C COOH R C COOH
NH2 NH O
FAD FADH2 iminokyselina
iminoacid
NH3
• Gly
catalase
katalasa
• degradation of D-amino acid
H2O + O2 H2O2 O2 • by product H2O2

− desaturation deamination of His

N N
CH CH COOH CH CH COOH
N H NH2
- NH3 N Urocanic acid
H H
Amino Acids NH4+

N
N

Sources of ammonia NH3

• Sources of ammonia in the body:

− oxidation deamination of Lys
lyzyloxidase(Cu2+): Lys + O2 → NH3 + allyzine + H2O
− dehydratation deamination of Ser
− oxidation deamination biogenic amines
monoaminoxidasa
monoaminooxidase H2O
R CH2 NH2 R CH NH R CH O
biogenníamine
biogenic amin imín
imin
imine aldehyd
aldehyde
FAD FADH2
NH3
H2O2 O2
kyselina
acid
 NH4+ N
Amino Acids N
NH3
Sources of ammonia
• Other sources of ammonia in the body:
− nonenzymatic karbamylation of proteins
Prot-NH2 + NH2-CO-NH2 → NH3 + Prot-NH-CO-NH2
− catabolism of pyrimidine nucleotides
cytozine/uracil → NH3 + CO2 + β-alanine
tymin → NH3 + CO2 + β-aminoizobutyrate
− deamination of purine nucleotides
Asp + IMP → AMP + fumarate + NH3
− synthesis of heme
4 porphobilinogen → 4 NH3 + uroporphyrinogen
Amino Acids NH4+

N
N

Sources of ammonia NH3

• Other sources of ammonia in the body:

− hydrolysis of Gln in kidney is released ammonia in
the form NH4+ which is excreted by urine
− Gln is considered to be a non-toxic transport form of
ammonia
COOH COOH
H2N CH H2N CH
CH2 H2O CH2
+ NH3
CH2 CH2
C C
O NH2 O OH

glutamin
glutamín
Glutamine glutamátacid
Glutamic
Amino Acids NH4+

N
N

Sources of ammonia NH3

• Increased ammonia production under pathological

conditions:
− GIT bleeding  increase of NH3 in portal blood
− uroinfection – bacterial urease catalyzes the hydrolysis of
urea
H2N-CO-NH2 + H2O → 2 NH3 + CO2

NH3 + H2O  NH4+ + OH-

alkaline urine (pH up to 8)  phosphate stones

Ammonia NH4+

N
N

NH3
Properties
• Ammonia is an endogenous metabolic toxin
– Toxic is especially for CNS:
o react with α-ketoglutarate, thus reducing its availability
for the citrate cycle (CC)  collapse CC and
consequently synthesis of ATP
– Liver damage or congenital metabolic disorder
increases ammonia concentration
o can appear a shiver, irrational speech, blurred vision,
coma, death
• Normal concentration of ammonia in the blood is
30-60 µmol/L
Ammonia NH4+ N

N
Properties NH3

• How to limit its formation in the human body (e.g. in

case of liver failure)?
– low protein diet
– probiotics – living micro-organisms, support fermentation
processes at the expense of putrid (lactobacilly,
bifidobacteria)
– prebiotics – indegistible food ingredients that selectively
stimulate the growth of probiotics (lactulose, oligofructose,
inulin, fiber)
– intestinal antibiotics – locally acting (neomycin,
metronidazole), extreme solution, short-term
 H
Ammonia NH2 – C – COOH
Detoxification
R
• Ammonia detoxification – three ways
produced three products: Urea, Glu, Gln
• Procesess detoxication include:
1. Removing the amino group
2. Transfer to the liver
3. Entry into the mitochondria
4. Preparation of nitrogen for entry into the most
important way of detoxication - urea cycle (UC)
The Urea Cycle
• N and C come from NH4+, HCO3-
(carbamoyl-P), and the a-NH2 of Glu
and Asp
• Breakdown of Arg (guanidino group) in
the urea cycle releases two N and one
C as urea
NH2
C O
NH2
The Urea Cycle

• Urea is synthesized in the liver

• By blood is transported to the kidneys
• From organisms is diluted by urine
• Urea cycle:
– explain Hans Krebs and Kurt Henseleit
in the 1932
– is linked to CC (1937) by fumarate
The Urea Cycle
AAs transported nitrogen
1. Glutamate
– transmits one amino group inside the cells :
transaminases → to α-ketoglutarate transmits amino
group and produce glutamate
– Glutamate dehydrogenase → reversible reaction
2. Glutamine
– transmits two amino groups between cells →
release them in the liver
3. Alanine
– transmits amino group from tissues (muscles) to the
liver: glucose-alanine cycle
The Urea Cycle
AAs transported nitrogen glutamate
Glutamine synthetase

Transfer inside cells

γ-glutamyl-P

Glutamine synthetase

Transfer between cells

glutamine
glutaminase
liver mitochondria

In the liver

glutamate
The Urea Cycle
Enzymes

1. Carbamoylphosphate synthase
2. Ornithine-carbamoyltransferase
3. Argininosuccinate synthetase
4. Argininosuccinate lyase
5. Arginase
The Urea Cycle
1. Carbamoylphosphate synthase

• CPS I ( in the mitochondria) is the rate –limiting

enzyme in the UC
• Catalyse condensation and activation of NH4+
and HCO3- (+ 2 ATP)
– CPS II (is located in the cytosol)- participates in
the synthesis of pyrimidine nucleotides
2 ATP 2 ADP + 1 P O
O
C
CO2 + NH4+ H2N O P O
CPS I
O
carbamoylphosphate
The Urea Cycle
2. Ornithine-carbamoyltransferase
ornithine transcarbamoylase - OTC
• Carbamoyl group is transferred from phosphate to the
side chain amino group of ornithine, resulting in the
production of citrulline and Pi (in the mitochondria)
– citrulline is transported out of the mitochondria into the cytosol
where the remaining reactions occur
CH2CH2CH2CHCOOH CH2CH2CH2CHCOOH
NH2 NH2 NH NH2

ornitin C O
ornithine
NH2 citrulline
citrulin

O
O O
C
H 2N O P O HO P O
O O
carbamoyl
karbamoyl
The Urea Cycle
3. Argininosuccinate synthetase
• The condensation of citrulline with aspartic acid results
in the formation of argininosuccinate
– Source of second atom of nitrogen in the urea
– The energy for argininosuccinate synthesis is provided by the
hydrolysis of ATP to AMP → two molecules of Pi
• Enzyme with lowest activity – regulation of UC
AMP + PP
ATP

CH2CH2CH2CHCOOH CH2CH2CH2CHCOOH
NH NH2 NH NH2

C O C N CHCOOH
- H2O
NH2 NH2 CH2COOH
argininosuccinate
argininsukcinát
H2N CHCOOH
CH2COOH
aspartát
Asp
The Urea Cycle
4. Argininosuccinate lyase
• Enzyme cleaves nonhydrolytical the bond between the amino
group and the a-carbon of the aspartic acid moiety in
argininosuccinate
– The C-skeleton of Asp is released as fumarate and the amino group
becomes a part of the Arg side chain - is the last precursor of urea
• The fumarate conected UC with other pathways, including
CAC, gluconeogenesis (acrose phosphoenolpyruvate), and the
synthesis of Asp (acrose transamination oxalacetate)
CH2CH2CH2CHCOOH CH2CH2CH2CHCOOH
NH2 NH NH2
NH
C N CHCOOH C N H
NH2 arginin
Arg
NH2 CH2COOH

argininsukcinát
argininosuccinate
HOOC H
C fumarát
fumarate
C
H COOH
The Urea Cycle
5. Arginase
• The cycle is ompleted by removal of urea from the
side chain of argine (hydrolytic), resulting in the
regeneration of ornithine.
• Regenerated ornithine is transported from cytosole
(acrose mitochondrial membrane) to mitochondria
and the new cycle can started
CH2CH2CH2CHCOOH CH2CH2CH2CHCOOH
NH NH2 NH2 NH2
C N H ornitin
ornithine
H 2O
NH2
NH2
Arg
arginin C
O NH2
urea
 The Urea Cycle
Enzymes and reactions - Summary
Cell
Name Acronym Catalysed reaction localisation
Carbamoylphosphate NH4+ + HCO3- +2ATP →
CPS1 mitochondria
synthetase carbamoylphosphate + 2ADP+ Pi
Ornithine carbamoylphosphate + ornithine →
OTC mitochondria
transcarbamoylase citruline + Pi
Argininosuccinate citruline + Asp + ATP →
ASS cytoplasm
synthetase argininosuccinate + AMP + PPi
Argininosuccinase
ASL argininosuccinate → Arg + fumarate cytoplasm
(rgininosuccinate lyase)
Arginase ARG1 Arg → urea + ornithine cytoplasm

• Sumary reaction: 2 NH3 + CO2 + 3 ATP 

urea + 2 ADP + AMP + PPi + 2Pi
The Urea Cycle
Protone - production

CO2 + NH4 + Asp → urea + fumarate + H2O + 2 H+

HCO3− + NH4+ + OOC C H C H2 C OO

NH3
O
C
H2N NH2 + −OOC-CH=CH-COO− + H2O + 2 H+

• Urea - diamid of carbonic acid:

− not electrolyte, very good disolved in water
− diffuses easily through all membranes
− uniform distribution in body fluid
Urea O
HCO3−
Properties C
NH4+ H2N NH2 Asp

• Urea is formed immediately by cleavage of Arg

in UC
– Arg is immediate precursor of urea synthesis
• It is synthesized in the liver
– by blood is transported to the kidney
• It is excreted in the urine, depending on the
amount of proteins:
– normal diet: 330-600 mmol/d, respectively 20-35 g/d
– starvation: 150-200 mmol/d
– protein-free diet: 50-80 mmol/d
Urea
Blood serum

• BUN – blood urea nitrogen test

• Increased concentration:
− excretion disorders (renal failure)
− excessive protein breakdown in catabolic states
− for example: sepsis, burns, polytrauma, tumors, fever
• Decreased concentration:
O
– lack of protein in the diet
– production disorders (liver failure) C
H2N NH2
The Urea Cycle
Regulation
• The rate at which the UC operates is controlled by
short- and long-term regulatory mechanisms
– Short-term regulation – the rate of UC is limited by the
activity of CPS I which is allosteric activated by NAG (N-
acetylglutamate, which synthese is stimulated by Arg)
– Long-term regulation – the rate at which the enzymes of
the UC are synthesized depends on the amount of
protein diet
o example: an increase in protein content over several days results in
increased synthesis of all enzymes in the cycle, resulting in
increased excretion of urea;
o the process is reversed by a shift to a low-protein diet - durig
starvation, when protein degradation in tissues is accelerated, the
synthesis of UC enzymes is also induced
The Urea Cycle
Regulation
• UC enzymes are synthesized very quickly:
– during starvation
– on a high protein diet
• UC enzymes are slowly synthesized:
– with sufficient intake of saccharides and dietary fats
– on a protein-free diet
• CPS I: also hormonal – glucagon and glucocorticoids
• Arginine succinate synthetase – enzyme with the
lowest activity:
– is allosteric activated by N-acetylglutamic acid
The Urea cycle
Regulation
• Allosteric regulation

Regulatory enzyme Activation

Carbamoyl phosphate synthetase I N-acetylglutamate
(= mitochondrial)
N-acetylglutamate synthetase Arginine

• Enzyme induction by protein rich diet

• By metabolic changes during starvation
• Urea synthesis is inhibited by acidosis
– HCO3- is saved
The Urea Cycle
Genetic deficiencies
• Deficiencies in the UC enzymes occur at a frequency of
about 1 in 25 000 live births
– inherited defect in each of the enzymes of the UC have been
described (Table)
• Because of the high toxicity of ammonia, neonatal
hyperammonemia must be treated immediately to avoid
brain damage
– these disorders are associated with mental retardation, convulsions,
coma, and death
Disease Defective enzyme Product accumulated
Hyperammonemia type I CPS I NH4+, Gln, Ala
Hyperammonemia type II Ornithine transcarbamoylase NH4+, Gln, orotic acid
Citrullinemia Argininosuccinate synthetase Citrulline
Argininosuccinic aciduria Argininosuccinate lyase Argininosuccinate
Argininemia Arginase Arg
The Urea Cycle
Summary

NH3 + HCO3− + aspartate + 3ATP + 2H2O →

urea + fumarate + 2ADP + AMP + 2Pi + PPi
The Urea (ornithine) cycle
Summary

• Detoxification pathway (NH3 is toxic for brain)

• Proceeds only in the liver
• Is localized in mitochondria /cytoplasm
• Regulation by CPS I (= mitoch.)
• Can acidify the organism (consumes HCO3-)
• Needs energy (3 ATP, but 4 energy rich bonds)
• Is connected with CC through fumarate
• Urea is end product of –NH2 metabolism (→ urine)
 NH4+ N

The Fate of Ammonia N

NH3

Three major reactions in all cells

• Carbamoyl-phosphate synthetase I
– two ATP required - one to activate bicarb, one
to phosphorylate carbamate
• Glutamate dehydrogenase (GDH)
– reductive amination of alpha-ketoglutarate to
form glutamate
• Glutamine synthetase
– ATP-dependent amidation of gamma-carboxyl
of glutamate to glutamine
 N

The Fate of Ammonia NH4+

N

Three major reactions in all cells NH3

1. Carbamoylphosphate synthetase I – CPS I

2 ATP 2 ADP + 1 P O
O
NH3 +
C
CO2 + NH4 H2N O P O
CPS I O
2. Glutamate dehydrogenase - GDH

GDH
2-oxoglutarate Glu
ATP ADP + P
3. Glutamine synthetase – GS COOH COOH
H2N CH H2N CH
CH2 CH2
+ NH3 Gln
CH2 - GS CH2
Glu H2O
C C
O OH O NH2
68 NH4+
Ammonia N
N

NH3
Detoxification
• Three product of ammonia detoxification:
urea, Glu, Gln
Characteristics Urea Glutamine Glutamate
Importance   
Chemistry diamid H2CO3 γ-amide Glu α-amino acid
Reaction urea synthesis Glu + NH3 red. amination 2-OG
Enzyme 5 enzymes cycle Gln-syntase GDH
Energy 3 ATP, 4 macroergic bonds 1 ATP 1 NADH
Cell localisation mitoch. + cytosol mitochondries mitochondries
Organ only liver liver, others mainly CNS
Amino acids
Gln, Ala - importance

• Gln and Ala: most represented AAs in post-

resorption phase circulation
– Gln (25 %), Ala (12 %) – released into the circulation
mainly from muscle tissue
• Ala: an important substrate for gluconeogenesis
• Gln: releases ammonia in tubular kidney cells
– Gln is an exclusive energy source for some cells
(enterocytes, fibroblasts, lymfocytes, macrophages)
Amino acids
Detoxication of ammonia
• Glutaminase catalyse hydrolytic cleavage the amide
group of Gln
COOH COOH
H2N CH H2N CH
CH2 H2O CH2
+ NH3
CH2 glutamináza CH2 glutaminasa glutamátdehydrogenasa
glutaminase glutaminase
glutamináza GDH
C
O
C
NH2 O OH Gln Glu 2-oxoglutarát
2-oxoglutarate

glutamin
Gln glutamát
Glu
NH 3 NH 3
+ +
H H
• In the kidney is from Gln release NH4+
+ +
NH 4 NH 4

Moč ~ 5)
urin (pH
Amino acids
Catabolism
Amino Acids

deamination α-oxoacid

Glucose Ketone bodies

detoxication the emergence of common metabolic products

Urea
Amino Acids
Catabolism
• Two steps in degrading amino acids:
– removed the α-amino group → to yield an α-keto acid,
which can be converted to an intermediate for other
metabolic pathways;
– breakdown and process carbon skeleton → used in
the citric acid cycle as well as for the synthesis of fatty
acids, ketone bodies, and glucose;
• Release of an amino group is also two step:
– transamination
– oxidative deamination
– most of the amino group from AAs are converted to urea
Amino Acids
Degradation of C-skeleton
• 20 AAs are degradet to seven products which are
intermediates in central pathways of metabolism
• N – transamination, deamination and urea
• C-skeletons – saccharides (glukoneogenesis) and
lipids (synthesis of FA)→ the AAs can be classified
into two major classes:
– Glucogenic: pyruvate, oxaloacetate, fumarate,
succinyl-CoA, a-ketoglutarate
– Ketogenic: acetyl-CoA, acetoacetyl-CoA
– with a few being both keogenic and glucogenic
Amino Acids
Degradation of C-skeleton

1. pyruvate  Gly, Ala, Ser, Thr, Cys, Trp

2. oxaloacetate  Asp, Asn
3. a-ketoglutarate  Glu, Gln, Pro, Arg, His
4. succinyl-CoA  Val, Ile, Met, Thr
5. fumarate  Phe, Tyr glucogenic AAs
ketogenic AAs
6. acetyl-CoA  Ile
7. acetoacetyl-CoA  Leu, Lys, Phe, Tyr, Trp
Amino Acids
Degradation - glucogenic
• Metabolized to α-ketoglutarate,
pyruvate, oxaloacetate, fumarate, or
succinyl CoA

• Aspartate • Methionine • Alanine

• Asparagine • Valine • Serine
• Arginine • Glutamine • Cysteine
• Phenylalanine • Glutamate • Glycine
• Tyrosine • Proline • Threonine
• Isoleucine • Histidine • Tryptophan
Amino Acids
Degradation - ketogenic

• Metabolized to acetyl CoA or

acetoacetate

• Isoleucine • Lysine
• Leucine • Phenylalanine
• Threonine • Tyrosine
• Tryptophan
Degradation of C-skeleton
Ala, Cys, Gly, Ser, Thr*,(Trp) → pyruvate

• Ala – aminotransferase
reaction, form pyruvate
– very important in the transfer
N from muscle to the liver,
– the amino group is
transferred back to glutamate
(Asp + NH4+ - substrates of
urea synthesis)
Degradation of C-skeleton
Ala, Cys, Gly, Ser, Thr*,(Trp) → pyruvate

• Gly – Ser, Thr → 3-phosphoglycerate or pyruvate

– synthese kreatinine, porfrines, purine nucleotides
• Ser – glycine – CO2 and NH3
– Ser/Thr-dehydratase (PLP)
• Thr – Thr-dehydrogenase (acetyl-CoA, Gly and
aminoacetone → pyruvate)
– Ser/Thr-dehydratase (propionyl-CoA → CAC)
– Thr-aldolase (acetyl-CoA and pyruvate)
Degradation of C-skeleton
Ala, Cys, Gly, Ser, Thr*,(Trp) → pyruvate
COO−
H3N+-C-H

CH OH
CH3
Thr

HC=O
Alanine
COO− COO− CH3
Acetaldehyde
Acetaldehyde
+ +
H3N -C-H H3N -CH2

CH2 Gly

N Trp N5,N10-Methylene-THF
Serine H
Indole ring THF
products

COO− COO− COO−

H3N+-CH-CH3 H3N+-C-H H3N+-C-H

CH2 OH CH2 SH
Ala
Cysteine Ser Cys

COO−
C=O
CH3
Threonine pyruvate
 Degradation of C-skeleton
Ala, Cys, Gly, Ser, Thr*,(Trp) → pyruvate

Gly Thr Ser Cys Trp Hyp

COO− COO− gluconeogenesis glucose

H3N+-CH C=O
oxidation decarboxylation acetyl-CoA
CH3 CH3
alanine aminotransferase glycolysis lactate
Ala pyruvate
ALT
oxalacetate Asp
oxoacid amino acid
Degradation of C-skeleton
Asp, Asn → oxalacetate

• Asp is transaminated directly to oxalacetate

– Share to synthese of purine (too AMP, IMP)
– Decarboxylation provides -alanine (e.g. in
coenzyme A)
– Aspartate give –NH2 in synthese Arg and urea
• Asn is hydrolysed by asparaginase on Asp
and in the next step on oxalacetate
Degradation of C-skeleton
Asp, Asn → oxalacetate
COO− COO−
1
C=O H3N+-C-H
CH2 PLP CH2

COO− COO−
Oxalacetate Asp
Gln NH 3 Oxalacetate
2 3
PEP Glu
COO−
H3N+-C-H

Glucose CH2
CONH2
Asn

1 aspartate aminotransferase (AST)

2 asparagine syntetase
3 asparaginase
Degradation of C-skeleton
Glu,Gln,Arg*,His*,Pro → 2-oxoglutarate

• Transformed to glutamate, which is oxidated by

glutamate dehydrogenase on 2-oxoglutarate
• Glu – glutamine syntetase – glutamine
(detoxication of ammonia in brain, and transports
form of ammonia from cells by blood to the liver
and kidneys) - dekarboxylation GABA, syntese
glutatione ( - amid. bond)
• His – source circle N (THF), its decarboxylation to
results histamine
Degradation of C-skeleton
Glu,Gln,Arg*,His*,Pro → 2-oxoglutarate
Arg Pro His THF - tetrahydropholate
pyroglutamate
− −
COO NH 3 COO

C=O H3N+-C-H CO2 H3N+-CH2

2
CH2 CH2 CH2
PLP 1
CH2 CH2 PLP CH2
5
COO− COO− COO−
2-oxoglutarate Glu GABA

Cys NH3 NH 3
3 4 NH 3
−
COO
γ-Glu-Cys H3N+-C-H CHO COO−

Gly glutatione synthase CH2 CH2 CH2

GSH Glutathione CH2 CH2 CH2

glutatiónreduktáza O=C-NH2 COO− COO−

GS-SG Gln Semialdehyde succinte Succinate

1 transaminase 4 glutaminase PLP - pyridoxalphosphate

2 glutamate dehydrogenase 5 glutamate decarb oxylase
3 glutamine synthetase GABA - γ-aminobutiric acid
Degradation of C-skeleton
Glu,Gln,Arg*,His*,Pro → 2-oxoglutarate

Glutamine
Glutamate

Proline

Arginine

a−ketoglutarate

Histidine
Degradation of C-skeleton
Met*, Ile*, Val* → succinyl-CoA
• Ile*, Leu*, Val* Branched-Chain Aas (BCAA)
→ across propionyl-CoA
• Catabolism Val, Ile, Leu begins in skeletal
muscle where the concentration of BCAA
transaminase is high
– BCAA is not expressed to a significant extent in
the liver
• Ile*, Val* – branched chain – transamination
+ oxid. decarboxylation + dehydrogenation
(FAD, =) – NADH a FADH2 (ATP)
Degradation of C-skeleton
Met*, Ile*, Val* → succinyl-CoA

Propionyl-CoA • Metabolic acidosisis and

Propionyl-CoA carboxylase the excretion of organic
Requiring acids in the urine
biotin

Vitamin B12
Methylmalonyl-CoA mutase
Degradation of C-skeleton
BCAA
• All 3 (Ile, Val, Leu) are essential
– after meal, their blood levels are high (~ 70% of all AAs) because
the liver does not use them (lack of aminotransferases)
– they are most utilized in muscle and CNS
– favorably affect catabolic states (infusions)
• A genetic defect in BCAA dehydrogenase is responsible
for MSUD - Maple Syrup Urine Disease
– the symptoms (vomiting, lethargy, severe brain damage) appear
early in infancy, and death often occurs by 1 year of age
– as the name implies, the urine has a chracteristic odor similar to
that of burnt sugar
– it is autosomal recesive disorder, occurs in about 1 in 185 000
newborns
Degradation of C-skeleton
BCAA

Leucin
Leucine Isoleucin
Isoleucine Valin
Valine
B12 B12

Acetoacetate
acetoacetát Succinyl-CoA
sukcinyl-CoA Succinyl-CoA
sukcinyl-CoA
acetyl-CoA
Acetyl-CoA acetyl-CoA
Acetyl-CoA

ketogenní
ketogenic ketogenní
ketogenic glukogenní
glucogenic
glukogenní
glucogenic
Degradation of C-skeleton
Leu*, Lys* → acetoacetate and acetyl-CoA
• Leu – branched-chain (as Val, Ile – 3 steps) – biotine
– is strictly ketogenic, its C-skeleton can either be
oxidized to CO2 and H2O or used for the synthesis of
ketone bodies or fatty acids
HOOC CH2 OH 2 1
H2O 3
C CH C S CoA HOOC CH2 C CH2 C S CoA
H3C O CH3 O

3-hydroxy-3-methylglutaryl-CoA

• Lys – a few different roads – dominate

(HMG-CoA)
reaction 2-
oxoglutarate with Lys → sacharopine
Degradation of C-skeleton
Leu*, Lys* → acetoacetate and acetyl-CoA
Degradation of C-skeleton
Trp* → Ala and acetyl-CoA
• Very complicated degradation – kynureninase
(need PLP) cleave 3-hydroxykynurenine to
Ala and 3-hydroxyantranilate
• From 3-hydroxyantranilate to result nikotinic
acid, nikotinamide (NAD+, NADP+)
• Precurzor of serotonine and melatonine
• From serotonine - alkaloids
Degradation of C-skeleton
Phe*,Tyr → fumarate and acetoacetate
• For polypeptide chain
• Hydroxylation of Phe involving enzyme
phenylalanine hydroxylase (Fe3+ and cofactor
biopterine – BH4)
• Prekurzor of hormones – adrenaline,
noradrenaline, iodination of thyronine -
hormones thyroid – thyroxine and
triiodthyronine
Degradation of C-skeleton
Phe*,Tyr → fumarate and acetoacetate

hydroxylation

Phenylalanine
 Amino Acids
Genetic defect in transport
• Genetic disease arising from defects in each of the AAs
transport systems reported in next Table
Transport system Amino acids transported Genetic disease
Neutral AAs Ala, Gly, Ser, Thr, Val, Leu, Ile, Hartnup disease
Phe, Tyr, Trp, His, Cys, Met,
citrulline
Acidic AAs Glu, Asp Dicarboxylic aminoaciduria
Dibasic AAs Lys, Arg, cystine, ornithine Cystinuria
Imino acids and Gly Pro, OH-Pro, Gly Joseph´s syndrome

• Most of these conditions were initially described as defects in

the renal tubular transport systems in which specific group of
AAs were excreted in large amounts in the urine.
• Both the absorptiom of AAs from the intestine and reabsorption
from the glomerular filtrate are impaired.
AAs Metabolism
Summary
Endogenous
proteins Urea Glutamin
Intracellular
Exogenous degradation

proteins
GIT
Pool AAs NH3  NH4+

Synthesis of FA, TAG

Nonessential AAs
Amines CC
Neurotransmiters
Purins/pyrimidins Glucose
Porphyrins
Creatine CO2 + energy
NO etc.
 Gly Ser Ala Cys Hyp Ile Leu Trp

ylkynurenine acetaldehyd metylacet- HMG-CoA formylkynurenine

pyruvate acetyl-CoA
Thr Ala
xyantranilate CO2 propionate 3-hydroxyantranilate
CO2
!
glucose
acetyl-CoA acetacetyl-CoA glutaryl-CoA Lys

PEP
fumarylacetacetate Tyr Phe
oxaloacetate citrate
Asp
Asn Gln ornithine Arg
malate isocitrate
Citrate
CO2
cycle
semialdehyd Pro
fumarate 2-oxoglutarate Glu glutamate
CO2
Asp succinate succinyl-CoA FIGLU
Phe
Tyr propionyl-CoA urokanate His
methylmalonyl-CoA

Val Ile HSer Met

Thank you for attention