Download Protein Metabolism

Protein Metabolism
 Denotes the various biochemical processes
responsible for the synthesis of proteins and amino acids
the breakdown of proteins (and other large molecules,
too) by catabolism
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The Digestion and Absorption of
Dietary Proteins
• Pepsin nonspecific maximally
active at low pH of the
stomach.
• Proteolytic enzymes of the
pancreas in the intestinal
lumen display a wide array of
specificity.
Aminopeptidases digest
proteins from the aminoterminal end.
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Cellular Proteins Are Degraded at
Different Rates
• Some proteins are very stable, while others are short
lived.
– Altering the amounts of proteins important in metabolic
regulation can rapidly change metabolic patterns.
• Cells have mechanisms for detecting and removing
damaged proteins.
– A significant proportion of newly synthesized protein molecules
are defective because of errors in translation.
– Other proteins may undergo oxidative damage or be altered in
other ways with the passage of time.
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Ubiquitin Tags Proteins for Destruction
• How can a cell distinguish proteins that
are meant for degradation?
• Ubiquitin, a small (8.5-kd) protein present
in all eukaryotic cells, is the tag that
marks proteins for destruction.
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• The c-terminal glycine residue of ubiquitin
(Ub) becomes covalently attached to the eamino groups of several lysine residues on
a protein destined to be degraded.
• The energy for the formation of these
isopeptide bonds (iso because e- rather
than a-amino groups are targeted) comes
from ATP hydrolysis.
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• Three enzymes participate in the attachment of ubiquitin to
each protein:
– ubiquitin-activating enzyme, or E1
– ubiquitin-conjugating enzyme, or E2
– ubiquitin-protein ligase, or E3.
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• Chains of ubiquitin can be
generated by the linkage of
the e-amino group of lysine
residue 48 of one ubiquitin
molecule to the terminal
carboxylate of another.
• Chains of four or more
ubiquitin molecules are
particularly effective in
signaling degradation
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What determines whether a protein
becomes ubiquitinated?
1. The half-life of a cytosolic protein is determined to a
large extent by its amino-terminal residue “the Nterminal rule”.
– In yeast: if N terminus is methionine half-life > 20 hours,
whereas if N terminus is arginine half-life ≈ 2 minutes.
– A highly destabilizing N-terminal residue such as arginine
or leucine favors rapid ubiquitination, whereas a
stabilizing residue such as methionine or proline does not.
– E3 enzymes are the readers of N-terminal residues.
2. Cyclin destruction boxes are amino acid sequences
that mark cell-cycle proteins for destruction.
3. Proteins rich in proline, glutamic acid, serine, and
threonine (PEST sequences).
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The Proteasome Digests the Ubiquitin-Tagged Proteins
• A large protease complex called the proteasome or
the 26S proteasome digests the ubiquitinated
proteins.
– In eukaryotes, they are located in the nucleus and the
cytoplasm.
– The degradation process yields peptides of about 7-8
amino acids long, then further degraded into amino acids
and used in synthesizing new proteins.
• This ATP-driven multisubunit protease spares
ubiquitin, which is then recycled.
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Protein Degradation Can Be Used to
Regulate Biological Function
Example:
E3
P P
I-kB
P
P
NF-kB
P
P
E3
Inflammation
initiates the
expression of a
number of the
genes that take
part in this
response
Ub
Ub
Ub
NF-kB
P
P
Ub
Ub
proteosome
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Digested proteins
Amino Acids
NH4+
Degradation in the
liver
The amino group must be
removed, as there are no
nitrogenous compounds in
energy-transduction pathways
a-ketoacids
enter the metabolic
mainstream as precursors to
glucose or citric acid cycle
intermediates
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The fate of the a-amino group
• The a-amino group of many aas is transferred to aketoglutarate to form glutamate.
• Glutamate is then oxidatively deaminated to yield
ammonium ion (NH4+).
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• Aminotransferases (transaminases) catalyze the
transfer of an a-amino group from an a-amino
acid to an a-keto acid.
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Example:
• Aspartate aminotransferase:
• Alanine aminotransferase:
• These transamination reactions are reversible and can
thus be used to synthesize amino acids from a-ketoacids,
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• The nitrogen atom that is transferred to a-ketoglutarate
in the transamination reaction is converted into free
ammonium ion by oxidative deamination.
– This reaction is catalyzed by glutamate dehydrogenase.
• This enzyme is unusual in being able to utilize either
NAD+ or NADP+ at least in some species.
• The reaction proceeds by dehydrogenation of the C-N
bond, followed by hydrolysis of the resulting Schiff base.
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• Glutamate dehydrogenase and other enzymes required for
the production of urea are located in mitochondria.
• This compartmentalization sequesters free ammonia, which
is toxic.
• In most terrestrial vertebrates, NH4+ is converted into urea,
which is excreted.
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Pyridoxal Phosphate Forms Schiff-Base
Intermediates in Aminotransferases
• All aminotransferases
contain the prosthetic
group pyridoxal phosphate
(PLP), which is derived from
pyridoxine (vitamin B6).
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Pyridoxal phosphate derivatives can form
stable tautomeric forms
The most important functional group allows PLP to
form covalent Schiff-base intermediates with amino
acid substrates
a pyridine ring that is
slightly basic
A phenolic hydroxyl group
that is slightly acidic
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• The aldehyde group of PLP usually forms a Schiff-base linkage with
the e-amino group of a specific lysine residue of the enzyme.
• The a-amino group of the amino acid substrate displaces the eamino group of the active-site lysine residue.
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The Urea Cycle
• Some of the NH4+ formed in the breakdown of amino
acids is consumed in the biosynthesis of nitrogen
compounds.
• In most terrestrial vertebrates, the excess NH4+ is
converted into urea and then excreted.
• The urea:
– One nitrogen atom is transferred from aspartate.
– The other nitrogen atom is derived directly from free NH4+ .
– The carbon atom comes from HCO3-.
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The Urea Cycle Reactions
1. Formation of Carbamoyl Phosphate: catalyzed by
carbamoyl phosphate synthetase.
• The consumption of two molecules of ATP makes the
synthesis essentially irreversible.
• The carbamoyl group of carbamoyl phosphate has a high
transfer potential because of its anhydride bond.
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2.
Carbamoyl is transferred to ornithine to form citrulline.
–
•
The reaction is catalyzed by ornithine transcarbamoylase.
Ornithine and citrulline are amino acids, but they are not
used as building blocks of proteins.
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3. Citrulline is transported to the cytoplasm where it
condenses with aspartate to form argininosuccinate
–
–
The reaction is catalyzed by argininosuccinate synthetase.
The reaction is driven by the cleavage of ATP into AMP and PPi, and
by the subsequent hydrolysis of PPi.
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4. Argininosuccinase cleaves argininosuccinate into arginine
and fumarate.
–
Thus, the carbon skeleton of aspartate is preserved in the form of
fumarate.
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5. Arginine is hydrolyzed to generate urea and ornithine in a
reaction catalyzed by arginase.
–
Ornithine is then transported back into the mitochondrion to begin
another cycle.
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• Mitochondrial reactions:
– The formation of NH4+ by glutamate
dehydrogenase.
– Its incorporation into carbamoyl phosphate
– Synthesis of citrulline
• Cytosolic reactions:
– The next three reactions of the urea cycle, which
lead to the formation of urea, take place in the
cytosol.
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