Protein Turnover and Amino Acid Catabolism. The Digestion and Absorption of Dietary Proteins Pepsin...
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Protein Turnover and Amino Acid 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 amino-terminal 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 that are 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 -amino groups of
several lysine residues on a protein destined to be degraded.
•The energy for the formation of these isopeptide bonds (iso
because- rather than -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 -amino group of lysine residue
48 of one ubiquitin molecule to the terminal
carboxylate of another.•Chains of four or more Chains of four or more
ubiquitin molecules are ubiquitin molecules are particularly effective in particularly effective in signaling degradationsignaling degradation
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What determines whether a protein becomes ubiquitinated?
.1The half-life of a cytosolic protein is determined to a large extent by its amino-terminal residue “the N-terminal rule.”
–A yeast protein with methionine at its N terminus typically has a half-life of more than 20 hours, whereas one with arginine at
this position has a half-life of about 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..2Cyclin destruction boxes are amino acid sequences that
mark cell-cycle proteins for destruction..3Proteins 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.•This ATP-driven multisubunit protease spares
ubiquitin, which is then recycled.•The 26S proteasome is a complex of two
components:–20S proteasome, which contains the catalytic
activity1–19S regulatory subunit .
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• The OH groups of these aas are converted into nucleophiles with the assistance of their own amino groups.
• These nucleophilic groups then attack the carbonyl groups of peptide bonds and form acyl-enzyme intermediates.
• ATP hydrolysis may assist the 19S complex to unfold the substrate and induce conformational changes in the 20S proteasome so that the substrate can be passed into the center of the complex
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Protein Degradation Can Be Used to Regulate Biological
Function
NF-BI-B
PPE3
PP
UbU
b
Ub
Ub
NF-kB
E3
PP
P P
Inflammation Ub
initiates the expression of a
number of the genes that take
part in this response
Example:
proteosome
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Digested proteins
Amino Acids
Degradation in the liver
NH4+ -ketoacids
enter the metabolic mainstream as precursors
to glucose or citric acid cycle intermediates
The amino group must be removed, as there are no nitrogenous compounds in energy-transduction pathways
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The fate of the -amino group
•The -amino group of many aas is transferred to -ketoglutarate to form glutamate.
•Glutamate is then oxidatively deaminated to yield ammonium ion (NH4
+).
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•Aminotransferases (transaminases) catalyze the transfer of an -amino group from an
-amino acid to an -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 -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
a pyridine ring that is slightly basic
A phenolic hydroxyl group that is slightly acidic
The most important functional group allows PLP to form covalent Schiff-base intermediates with amino acid substrates
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•The aldehyde group of PLP usually forms a Schiff-base linkage with the -amino group of a
specific lysine residue of the enzyme.
•The -amino group of the amino acid substrate displaces the -amino group of the active-site
lysine residue .
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-ketoglutarate
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•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-.
The Urea Cycle
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The Urea Cycle Reactions
.1Formation 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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.2Carbamoyl 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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.3Citrulline 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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.4Argininosuccinase cleaves argininosuccinate into arginine and fumarate.
•Thus, the carbon skeleton of aspartate is preserved in the form of fumarate.
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.5Arginine 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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