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 Aminopeptidases digest
lumen display a wide array of proteins from the amino-
specificity. terminal end.

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 PROTEIN METABOLISM

• Amino acid catabolism is part of the larger process of the

metabolism of nitrogen-containing molecules.

• Nitrogen enters the body in a variety of compounds present in

food, the most important being amino acids contained in
dietary protein. Nitrogen leaves the body as urea, ammonia,
and other products derived from amino acid metabolism.

• The role of body proteins in these transformations involves

two important concepts: the amino acid pool and protein
turnover.
 Amino acid pool

Free amino acids are present throughout the body, for

example, in cells, blood, and the extracellular fluids. For
the purpose of this, envision all these amino acids as if
they belonged to a single entity, called the amino acid
pool.

This pool is supplied by three sources:

1) amino acids provided by the degradation of body
proteins,
2) amino acids derived from dietary protein, and
3) synthesis of nonessential amino acids from simple
intermediates of metabolism.
 Amino acid pool
Conversely, the amino pool is depleted by three
routes:
1) synthesis of body protein,

2) amino acids consumed as precursors of essential

nitrogen-containing small molecules, and

3) conversion of amino acids to glucose, glycogen,

fatty acids, ketone bodies, or CO2 + H2O.
 Protein turnover

• Most proteins in the body are constantly being synthesized and

then degraded, permitting the removal of abnormal or
unneeded proteins.

• For many proteins, regulation of synthesis determines the

concentration of protein in the cell, with protein degradation
assuming a minor role.
 DIGESTION OF DIETARY PROTEINS

• Proteins are generally too large to be absorbed by the

intestine.

• Hydrolysis yield di- and tripeptides as well as individual

amino acids, which can be absorbed.

• Proteolytic enzymes responsible for degrading proteins

are produced by three different organs: the stomach, the
pancreas, and the small intestine.
Digestion of proteins by gastric secretion

1. Hydrochloric acid:
• Stomach acid is too dilute (pH 2–3) to
hydrolyze proteins.
• The acid, secreted by the parietal cells,
functions instead to kill some bacteria and to
denature proteins, thus making them more
susceptible to subsequent hydrolysis by
proteases .
Digestion of proteins by gastric secretion

2. Pepsin:
• This acid-stable endopeptidase is secreted by the chief
cells of the stomach as an inactive zymogen (or
proenzyme), pepsinogen.
• Zymogens contain extra amino acids in their
sequences that prevent them from being catalytically
active.
• Pepsinogen is activated to pepsin , either by HCl, or
autocatalytically by other pepsin molecules that have
already been activated.
• Pepsin releases peptides and a few free amino acids
from dietary proteins.
Digestion of proteins by pancreatic enzymes
 

1. Specificity:
• Each of these enzymes has a different specificity for the
amino acid R-groups adjacent to the susceptible peptide
bond.
• Ex: trypsin cleaves only when the carbonyl group of the
peptide bond is contributed by arginine or lysine.
• These enzymes, like pepsin described above, are
synthesized and secreted as inactive zymogens.
2. Release of zymogens:
• The release and activation of the pancreatic zymogens is
mediated by the secretion of cholecystokinin and secretin,
two polypeptide hormones of the digestive tract.
Digestion of proteins by pancreatic enzymes
 
3. Activation of zymogens:
• Enteropeptidase (formerly called enterokinase ) —an enzyme
synthesized by and present on the luminal surface of intestinal
mucosal cells of the brush border membrane—converts the
pancreatic zymogen trypsinogen to trypsin by removal of a
hexapeptide from the N-terminus of trypsinogen.
• Trypsin subsequently converts other trypsinogen molecules to
trypsin by cleaving a limited number of specific peptide bonds in
the zymogen.

4. Abnormalities in protein digestion:

• In individuals with a deficiency in pancreatic secretion (for
example, due to chronic pancreatitis, cystic fibrosis, or surgical
removal of the pancreas), the digestion and absorption of fat and
protein are incomplete. This results in the abnormal appearance
of lipids and undigested protein in the feces.
 Digestion of oligopeptides by enzymes of
the small intestine

 
The luminal surface of the intestine contains
aminopeptidase— an exopeptidase that repeatedly
cleaves the N-terminal residue from oligopeptides to
produce even smaller peptides and free amino acids.
 Absorption of amino acids and small
peptides

• Free amino acids are taken into the enterocytes by a Na+-

linked secondary transport system of the apical membrane.
• Di- and tri- peptides, however, are taken up by a H+-linked
transport system.
• The peptides are hydrolyzed in the cytosol to amino acids
that are released into the portal system by facilitated
diffusion.
• Only free amino acids are found in the portal vein after a
meal containing protein.
• Amino acids are either metabolized by the liver or released
into the general circulation.
 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 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 e-
amino 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 -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 N-
terminal 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 1
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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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What is the fate of amino acids released on protein
digestion?

• Nitrogen Removal is the first step in the degradation

of amino acids.
• Any amino acids not needed as building blocks are
degraded to various compounds, depending on the
type of amino acid and the tissue from which it
originates.
• The major site of amino acid degradation in
mammals is the liver.
• The resulting α-ketoacids are then metabolized so
that the carbon skeletons can:
1.enter the metabolic mainstream as precursors of
glucose
2. or as citric acid cycle intermediates.
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 Digested proteins

Amino Acids

Degradation in the
liver
NH4+ -ketoacids
enter the metabolic
The amino group must be
mainstream as precursors
removed, as there are no
to glucose or citric acid
nitrogenous compounds in
cycle
energy-transduction pathways
intermediates

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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 -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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 Exception

• the α-amino groups of serine and threonine

can be directly converted into NH 4+ .
• These direct deaminations are catalyzed by
serine dehydratase and threonine
dehydratase, in which pyridoxal phosphate
(PLP) is the prosthetic group.

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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 A phenolic hydroxyl group

slightly basic that is slightly acidic

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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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Peripheral Tissues Transport Nitrogen to the
Liver

• Muscle uses branched-chain amino acids:

- leucine,
- Valine,
- Isoleucine
 as a source of fuel during prolonged
exercise and fasting.

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How is the nitrogen processed in these
other tissues?
• As in the liver
- the first step is removal of nitrogen from the
amino acid.
- However, muscle lacks the enzymes of the
urea cycle (the set of reactions that prepares
nitrogen for excretion).
 in muscle, the nitrogen must be released in a
form that can be absorbed by the liver and
converted into urea.
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Nitrogen is transported from muscle to the liver
in two principal transport forms:

 Alanine and glutamine.

- Glutamate is formed by transamination
reactions,

- Nitrogen is then transferred to pyruvate to

form alanine, which is released into the blood.

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• The liver takes up the alanine and converts it
back into pyruvate by transamination.
• The pyruvate can be used for
gluconeogenesis,
and the amino group eventually appears as
urea.

This transport is referred to as the glucose–

alanine cycle.

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• It is reminiscent of the Cori cycle. However, in
contrast with the Cori cycle, pyruvate is not
reduced to lactate,
• Thus more high-energy electrons are
available for oxidative phosphorylation in
muscle.

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Glutamine synthase catalyzes the synthesis of
glutamine from glutamate and NH4+ in an
ATP-dependent reaction:
The nitrogen atoms of glutamine can be converted
into urea in the liver

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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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