Download Principles of BIOCHEMISTRY

PROTEIN
METABOLISM:
NITROGEN CYCLE;
DIGESTION OF
PROTEINS
Red meat is an important dietary
source of protein nitrogen
The Nitrogen Cycle and Nitrogen
Fixation
• Nitrogen is needed for amino acids,
nucleotides, etc
• Atmospheric N2 is the ultimate source of
biological nitrogen
• Nitrogen fixation: biosynthetic process of
the reduction of N2 to NH3 (ammonia)
• Higher organisms are unable to fix nitrogen.
• Some bacteria and archaea can fix nitrogen.
Symbiotic Rhizobium
bacteria invade the roots of
leguminous plants and form
root nodules.
Nodules of Rhizobium
bacteria
Rhizobium bacteria fix
nitrogen supplying both
the bacteria and the
plants.
Archaea
The amount of
N2 fixed by
nitrogen-fixing
microorganisms
is about 60% of
Earth's newly
fixed nitrogen.
25% is fixed by
industrial processes
(fertilizer factories)
Lightning and ultraviolet
radiation fix 15%
Nitrogen-fixing bacteria possess nitrogenase complex
which can reduce N2 to ammonia
The nitrogenase complex consists of two proteins:
 reductase, which provides electrons
 nitrogenase, which uses these electrons to reduce N2 to NH3.
The transfer of electrons from the reductase to the
nitrogenase is coupled to the hydrolysis of ATP.
The high-potential
electrons come from
protein ferredoxin,
generated by
photosynthesis or
oxidative processes.
16 molecules of ATP are
hydrolyzed for each
molecule of N2 reduced.
• Nitrogenase reaction:
N2 + 8 H+ + 8 e- + 16 ATP 
2 NH3 + H2 + 16 ADP + 16 Pi
Reductase – dimer
containing Fe-S clusters
and ATP-binding site
The nitrogenase
component is an 22
tetramer.
Contains P cluster
(Fe-S) and FeMo
cofactor.
FeMo cofactor is the
site of nitrogen
fixation.
Ammonia in the presence of
water becomes NH4+ which can
be used by plants
NH4+ can be rapidly oxidized
by soil bacteria Nitrosomonas
and Nitrobacter to NO2- and
NO3- (nitrification)
Nitrosomonas
NO2- and NO3- are used by higher plants
Another soil bacteria can reverse the nitrification
process and convert NO2- and NO3- back to nitrogen
Nitrogen from plants and animals is recycled to soil
(excretion of nitrogen in the form of urea or uric acid;
decay of plants and animals) - nitrogen cycle
Assimilation of Ammonia
• Ammonia generated from N2 is assimilated into
amino acids such as glutamate or glutamine
A. Ammonia Is Incorporated into Glutamate
• Reductive amination of a-ketoglutarate by
glutamate dehydrogenase occurs in plants, animals
and microorganisms
This reaction establishes the stereochemistry of the
-carbon atom in glutamate. Only the L isomer of
glutamate is synthesized.
B. Glutamine Is a Nitrogen Carrier
• A second route in assimilation of ammonia is via
glutamine synthetase
All organisms have both enzymes: glutamate
dehydrogenase and glutamine synthetase.
Amino acids are used for the synthesis of proteins.
Animals and humans consume proteins.
Proteins undergo digestion in the stomach and intestine.
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
METABOLISM:
PROTEIN TURNOVER;
GENERAL WAYS OF
AMINO ACIDS
METABOLISM
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?
Ubiquitin - is the tag that
marks proteins for
destruction ("black spot" - the
signal for death)
Ubiquitin - a small (8.5-kd)
protein present in all
eukaryotic cells
Structure:
 extended carboxyl terminus
(glycine) that is linked to other
proteins;
 lysine residues for linking
additional ubiquitin molecules
Ubiquitin covalently
binds to -amino group
of lysine residue on a
protein destined to be
degraded.
Isopeptide bond is
formed.
Mechanism of the binding of ubiquitin to target protein
E1 - ubiquitin-activating enzyme (attachment of ubiquitin
to a sulfhydryl group of E1; ATP-driven reaction)
E2 - ubiquitin-conjugating enzyme (ubiquitin is shuttled
to a sulfhydryl group of E2)
E3 - ubiquitin-protein ligase (transfer of ubiquitin from
E2 to -amino group on the target protein)
Attachment of a single
molecule of ubiquitin - weak
signal for degradation.
Chains of ubiquitin are
generated.
Linkage – between -amino
group of lysine residue of
one ubiquitin to the terminal
carboxylate of another.
Chains of ubiquitin
molecules are more
effective in signaling
degradation.
What determines
ubiquitination of the
protein?
1. The half-life of a protein
is determined by its aminoterminal residue (Nterminal rule).
E3 enzymes are the readers
of N-terminal residues.
2. Cyclin destruction boxes
- specific amino acid
sequences (proline, glutamic
acid, serine, and threonine –
PEST)
Digestion of the Ubiquitin-Tagged Proteins
What is the executioner of the protein death?
A large protease complex proteasome or the
26S proteasome digests the ubiquitinated
proteins.
26S proteasome - ATP-driven multisubunit
protease.
26S proteasome consists of two components:
 20S - catalytic subunit
 19S - regulatory subunit
20S subunit
 resembles a barrel
 is constructed from 28 polipeptide chains which are
arranged in four rings (two  and two )
 active sites are located in  rings on the interior of the
barrel
 degrades proteins to peptides (seven-nine residues)
19S subunit
 made up of 20 polipeptide
chains
 controls the access to interior
of 20S barrel
 binds to both ends of the 20S
proteasome core
 binds to polyubiquitin chains
and cleaves them off
 possesses ATPase activity
 unfold the substrate
 induce conformational changes
in the 20S proteasome (the
substrate can be passed into the
center of the complex)
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
PROTEIN METABOLISM:
UTILIZATION OF AMMONIA
IONS; UREA CYCLE
The basic features of nitrogen
metabolism were elucidated
initially in pigeons
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.