07_Metabolism of aminoacids

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Transcript 07_Metabolism of aminoacids

PROTEIN
METABOLISM:
NITROGEN CYCLE;
DIGESTION OF
PROTEINS
Red meat is an important dietary
source of protein nitrogen
Protein digestion
Digestion in Stomach
Stimulated by food acetylcholine, histamine and gastrin
are released onto the cells of the stomach
The combination of acetylcholine, histamine and gastrin
cause the release of the gastric juice.
Mucin - is always secreted in the stomach
HCl - pH 0.8-2.5 (secreted by parietal cells)
Pepsinogen (a zymogen, secreted by the chief cells)
Hydrochloric acid:
 Creates optimal pH for
pepsin
 Denaturates proteins
 Kills most bacteria and
other foreign cells
Pepsinogen (MW=40,000) is activated by the enzyme pepsin
present already in the stomach and the stomach acid.
Pepsinogen cleaved off to become the enzyme pepsin
(MW=33,000) and a peptide fragment to be degraded.
Pepsin partially digests proteins by cleaving the peptide bond
formed by aromatic amino acids: Phe, Tyr, Trp
Digestion in the Duodenum
Stimulated by food secretin and cholecystokinin regulate
the secretion of bicarbonate and zymogens trypsinogen,
chymotrypsinogen, proelastase and procarboxypeptidase
by pancreas into the duodenum
Bicarbonate changes the pH to about 7
The intestinal cells
secrete an enzyme
called
enteropeptidase
that acts on
trypsinogen cleaving
it into trypsin
Trypsin converts chymotrypsinogen into chymotrypsin,
procarboxypeptidase into carboxypeptidase and
proelastase into elastase, and trypsinogen into more
trypsin.
Trypsin which cleaves peptide bonds between basic
amino acids Lys and Arg
Chymotrypsin cleaves the bonds between aromatic amino
acids Phe, Tyr and Trp
Carboxypeptidase which cleaves one amino acid at a
time from the carboxyl side
Aminopeptidase is secreted by the small intestine and
cleaves off the N-terminal amino acids one at a time
Most proteins are completely digested to free amino acids
Amino acids and sometimes short oligopeptides are
absorbed by the secondary active transport
Amino acids are transported via the blood to the cells of
the body.
The ways of entry and using of amino
acids in tissue
The sources of amino acids:
1) absorption in the intestine;
2) protein decomposition;
3) synthesis from the carbohydrates and lipids.
Using of amino acids:
1) for protein synthesis;
2) for synthesis of other nitrogen containing
compounds (creatine, purines, choline, pyrimidine);
3) as the source of energy;
4) for the gluconeogenesis.
PROTEIN TURNOVER
Protein turnover — the degradation and
resynthesis of proteins
Half-lives of proteins – from several minutes to many
years
Structural proteins – usually stable (lens protein
crystallin lives during the whole life of the organism)
Regulatory proteins - short lived (altering the amounts
of these proteins can rapidly change the rate of
metabolic processes)
How can a cell distinguish proteins that are meant
for degradation?
GENERAL WAYS OF AMINO
ACIDS METABOLISM
The fates of amino acids:
1) for protein synthesis;
2) for synthesis of other nitrogen containing
compounds (creatine, purines, choline,
pyrimidine);
3) as the source of energy;
4) for the gluconeogenesis.
The general ways of amino acids degradation:
 Deamination
 Transamination
 Decarboxilation
The major site of amino acid degradation - the liver.
Deamination of amino acids
Deamination - elimination of amino group from amino
acid with ammonia formation.
Four types of deamination:
- oxidative (the most important for higher animals),
- reduction,
- hydrolytic, and
- intramolecular
Reduction deamination:
R-CH(NH2)-COOH + 2H+  R-CH2-COOH + NH3
amino acid
fatty acid
Hydrolytic deamination:
R-CH(NH2)-COOH + H2O  R-CH(OH)-COOH + NH3
amino acid
hydroxyacid
Intramolecular deamination:
R-CH(NH2)-COOH  R-CH-CH-COOH + NH3
amino acid
unsaturated fatty acid
Oxidative deamination
L-Glutamate dehydrogenase plays a central role in amino acid
deamination
In most organisms glutamate is the only amino acid that has
active dehydrogenase
Present in both the cytosol and mitochondria of the liver
Transamination of amino acids
Transamination - transfer of an amino group from
an -amino acid to an -keto acid (usually to
-ketoglutarate)
Enzymes: aminotransferases (transaminases).
-amino acid -keto acid
-keto acid -amino a
There are different transaminases
The most common:
alanine aminotransferase
alanine + -ketoglutarate  pyruvate + glutamate
aspartate aminotransferase
aspartate + -ketoglutarate  oxaloacetate + glutamate
Aminotransferases funnel -amino groups from a
variety of amino acids to -ketoglutarate with
glutamate formation
Glutamate can be deaminated with NH4+ release
Mechanism of transamination
All aminotransferases require the
prosthetic group pyridoxal
phosphate (PLP), which is derived
from pyridoxine (vitamin B6).
Ping-pong kinetic mechanism
First step: the amino group of
amino acid is transferred to
pyridoxal phosphate, forming
pyridoxamine phosphate and
releasing ketoacid.
Second step: -ketoglutarate
reacts with pyridoxamine
phosphate forming glutamate
Ping-pong kinetic mechanism of aspartate transaminase
aspartate + -ketoglutarate  oxaloacetate + glutamate
Decarboxylation of amino acids
Decarboxylation – removal of carbon dioxide from
amino acid with formation of amines.
amine
Usually amines have high physiological activity
(hormones, neurotransmitters etc).
Enzyme: decarboxylases
Coenzyme – pyrydoxalphosphate
Significance of amino acid decarboxylation
1. Formation of physiologically active compounds
GABA –
mediator of
nervous
system
glutamate
histidine
gamma-aminobutyric acid (GABA)
histamine
Histamine – mediator of inflammation, allergic reaction.
2. Catabolism of amino acids during the decay
of proteins
Enzymes of microorganisms (in colon; dead organisms)
decarboxylate amino acids with the formation of
diamines.
ornithine
putrescine
lysine
cadaverine
AMMONIA METABOLISM
The ways of ammonia formation
1. Oxidative deamination of amino acids
2. Deamination of physiologically active amines and nitrogenous
bases.
3. Absorption of ammonia from intestine (degradation of
proteins by intestinal microorganisms results in the ammonia
formation).
4. Hydrolytic deamination of AMP in the brain (enzyme –
adenosine deaminase)
Ammonia is a toxic substance to plants and animals
(especially for brain)
Normal concentration: 25-40 mol/l (0.4-0.7 mg/l)
Ammonia must be removed from the organism
Terrestrial vertebrates
synthesize urea (excreted by
the kidneys) - ureotelic
organisms
Birds, reptiles synthesize uric
acid
Urea formation takes place in
the liver
Peripheral Tissues Transport Nitrogen to the Liver
Two ways of nitrogen transport from peripheral
tissues (muscle) to the liver:
Glutamate is
1. Alanine cycle. Glutamate is
not deaminated
in peripheral
formed by transamination reactions
tissues
Nitrogen is then transferred to pyruvate to
form alanine, which is released into the blood.
The liver takes up the alanine and converts it back
into pyruvate by transamination.
The glutamate formed in the liver is deaminated
and ammonia is utilized in urea cycle.
2. Nitrogen can be transported as glutamine.
Glutamine synthetase catalyzes the synthesis of
glutamine from glutamate and NH4+ in an ATPdependent reaction:
THE UREA CYCLE
Urea cycle - a cyclic pathway of urea synthesis
first postulated by H.Krebs
The sources of
nitrogen atoms in
urea molecule:
- aspartate;
- NH4+.
Carbon atom
comes from CO2.
The free ammonia is coupling with carbon dioxide to
form carbamoyl phosphate
Two molecules of ATP are required
Reaction takes place in the matrix of liver
mitochondria
Enzyme: carbamoyl phosphate synthetase (20 % of
the protein of mitochondrial matrix)
Carbamoyl
ornithine
phosphate
donates
carbamoyl
group
to
The product - citruilline
Enzyme: ornithine carbamoyltransferase
Reaction takes place in the mitochondrial matrix
Citrulline leaves the matrix and passes to the cytosol
In the cytosol citrulline in the presence of ATP
reacts with aspartate to form argininosuccinate
Enzyme: argininosuccinate synthetase
Argininosuccinate is cleaved to free arginine and
fumarate
Enzyme: argininosuccinate lyase
The fumarate enters the tricarboxylic acid cycle
Arginine is hydrolyzed to generate urea and ornithine
Enzyme: arginase (present only in liver of ureotelic
animals)
Ornithine is transported back into the mitochondrion
to begin another cycle
Urea is excreted (about 40 g per day)
The
urea
cycle
The Linkage between Urea Cycle, Citric Acid Cycle
and Transamination of Oxaloacetate
Fumarate formed in urea cycle enters citric acid cycle
and is converted to oxaloacetate.
Fates of oxaloacetate:
(1) transamination to aspartate,
(2) conversion into glucose,
(3) condensation with acetyl CoA to form citrate,
(4) conversion into pyruvate.
SPECIFIC WAYS OF AMINO ACID
CATABOLISM
After removing of amino group the carbon skeletons of amino
acids are transformed into metabolic intermediates that can be
converted into glucose, fatty acids, ketone bodies or oxidized
by the citric acid cycle.
The carbon skeletons of 20 fundamental amino acids
are funneled into seven molecules:







pyruvate,
acetyl CoA,
acetoacetyl CoA,
-ketoglutarate,
succinyl CoA,
fumarate,
oxaloacetate.
Fates of carbon
skeleton of
amino acids
Glucogenic vs ketogenic amino acids
• Glucogenic amino acids (are degraded to
pyruvate or citric acid cycle intermediates) can supply gluconeogenesis pathway
• Ketogenic amino acids (are degraded to acetyl
CoA or acetoacetyl CoA) - can contribute to
synthesis of fatty acids or ketone bodies
• Some amino acids are both glucogenic and
ketogenic
Pyruvate as an Entry Point into Metabolism
Oxaloacetate as an Entry Point into Metabolism
Aspartate and asparagine are converted into oxaloacetate
aspartate + -ketoglutarate  oxaloacetate + glutamate
Asparagine is hydrolyzed to NH4+ and aspartate, which
is then transaminated.
-Ketoglutarate as an Entry Point into
Metabolism
Succinyl Coenzyme A Is a Point of Entry
for Several Nonpolar Amino Acids
Methionine Degradation
S-adenosylmethionine (SAM) - a common methyl donor in
the cell
Homocysteine (< 15 μmol/L)
Hyperhomocysteinemia can results in:
•Vascular diseases, endothelial
dysfunction, atherosclerosis,
thrombophilia
•Skeletal anomalies
•retardation of mental development
•Ectopic lens
•Alzheimer's disease
•Kidneys insufficiency
•Colorectal cancer
Homocysteine
The Conversion of Branched-Chain Amino Acids
branchedchain
dehydrogen
ase
The degradative pathways of valine and isoleucine resemble
that of leucine.
Isoleucine yields acetyl CoA and propionyl CoA
Valine yields CO2 and propionyl CoA.
Degradation of Aromatic Amino Acids
Acetoacetate, fumarate, and pyruvate — are common intermediates.
Molecular oxygen is used to break an aromatic ring.
PA
hydrotetrahy +O
xylase 2
drobiopteri
n
homogenti
sate
oxidase
Tryptophan degradation requires several oxygenases
Pyruv
INBORN ERRORS OF AMINO ACIDS METABOLISM
Alcaptonuria - inherited disorder of the
tyrosine metabolism caused by the
absence of homogentisate oxidase.
 homogentisic acid is accumulated and
excreted in the urine
 turns a black color upon exposure to air
 In children:
 urine in diaper may
darken
 In adults:
 darkening of the ear
 dark spots on the on the
sclera and cornea
 arthritis
Alcaptonuria
Accumulation of oxidized homogentisic acid
pigment in connective tissue (ochronosis)
Arthritis of the spine is a complication
of alkaptonuria ochronosis
Aortic valve stenosis in alcaptonuria
Urine turns a black color upon exposure to air
Phenylketonuria is caused by an absence or deficiency
of phenylalanine hydroxylase or of its
tetrahydrobiopterin cofactor.
Phenylalanine accumulates in all body fluids and converts
to phenylpyruvate.
Defect in
myelination of nerves
The brain weight is
below normal.
Mental and physical
retardations.
The life expectancy
is drastically
shortened.
Diagnostic criteria:
 phenylalanine level in
the blood
 FeCl3 test
 DNA probes
(prenatal)
Phenylalanine
Albinism –
genetically
determined lack or
deficit of enzyme
tyrosinase
Tyroxine
Tyrosine
Melanin
tyrosinase
DOPA
Dopamine
Tyrosinase in
melanocytes
oxidases tyrosine
to DOPA and
DOPA-chinone
Norepinephrine
Epinephrine
Symptoms of albinism:
• inhibition of production
or lack of melanin in skin,
hair, eyes
• increased sensitivity to
sunlight
• increased risk of skin
cancer development
• sun burns
• photophobia
• decrease
of
vision
acuity
• strabismus, nystagmus
Maple syrup urine disease - the disorder of the
oxidative decarboxylation of -ketoacids derived
from valine, isoleucine, and leucine caused by the
missing or defect of branched-chain dehydrogenase.
The levels of branched-chain amino
acids and corresponding -ketoacids
are markedly elevated in both blood
and urine.
The urine has the odor of maple syrup
The early symptoms:
 lethargy
 ketoacidosis
 unrecognized disease leads to
seizures, coma, and death
 mental and physical retardation
SYNTHESIS OF NITRIC OXIDE
(NO) FROM ARGININE
• Nitric oxide (.N=O) is a gas which can
diffuse rapidly into cells, and is a
messenger that activates guanylyl cyclase
(GMP synthesis)
• NO relaxes blood vessels, lowers blood
pressure, and is a neurotransmitter in the
brain
• Nitroglycerin is converted to NO
and dilates coronary arteries in
treating angina pectoris
Conversion of arginine to NO via
nitric oxide synthase
SPECIFIC WAYS OF AMINO ACID
SYNTHESIS
•Plants and microorganisms can make all 20
amino acids
•Humans can make only 11 of the 20 amino acids
(“nonessential” amino acids)
•Nonessential amino acids for mammals are
usually derived from intermediates of
glycolysis or the citric acid cycle
•The others are classed as "essential" amino
acids and must be obtained in the diet
A deficiency of
even one amino
acid results in a
negative
nitrogen
balance.
In this state,
more protein is
degraded than
is synthesized.
The pathways for the biosynthesis of amino acids are
diverse
Common feature: carbon skeletons come from
intermediates of
 glycolysis,
 pentose phosphate pathway,
 citric acid cycle.
All amino acids
are grouped
into families
according to the
intermediates
that they are
made from