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Protein and amino acid
degradation
Chemistry 256
Proteins don’t last forever; in fact,
some are quite short-lived
• Thus there must be a
mechanism for the cell to
recycle the “broken” protein’s
amino acids and not have to
synthesize new amino acids,
which is energetically more
expensive.
• Shortest-lived enzymes tend to
be the ones that catalyze
reactions at metabolic control
points.
Ways to mark proteins for degradation
• In the cell, there are a couple of ways to target damaged or
unwanted proteins:
1. Non-selectively, the protein interacts with a lysosome, with
its numerous proteases. The process can be selective when
energy levels are low and “KFERQ” proteins are targeted.
2. Selectively, the protein is ubiquinated – that is, it is
covalently bonded to ubiquitin. This allows recognition by a
proteasome, which degrades the protein.
Lysosomes are vesicular structures
Lysosomes maintain an internal pH of 5 and contain about
50 hydrolytic enzymes that work optimally at that pH (are
inactive at cytosolic pH).
The “food vacuole” shown in
the diagram probably contains
proteins captured by the cell by
endocytosis; generally, the
process of which proteins are
degraded is nonselective,
except in the case of starvation,
in which case proteins with the
sequence KFERQ (lys-phe-gluarg-gln) or similar are removed
selectively for energy purposes
from certain cells.
Ubiquitin marks proteins for degradation
without the use of a lysosome
• Ubiquitin is a 76-residue
protein, highly conserved over
many species.
• Uses ATP and ubiquitinactivating and ubiquitinconjugating enzymes, as well
as a ligase, to covalently link
ubiquitin to the protein at a
lysine side chain.
• Need at least four ubiquitins
for protein to be efficiently
degraded by the 26S
proteasome = polyubiquitin
“N-end rule”
• “destabilizing” N-terminus residues = asp, arg,
leu, lys, phe – proteins with these N-terminus
residues have a half-life of 2 to 3 minutes.
• “stabilizing” N-terminus residues = ala, gly,
met, ser, thr, val – proteins with these Nterminus residues have a half-life of greater
than 10 hours in prokaryotes, greater than 20
hours in eukaryotes (these proteins are
basically never ubiquitinized).
Proteasome = degrades ubiquinated proteins
• Multiprotein complex – the 20S proteolytic part and
the two 19S “end caps” which recognize the
ubiquinated proteins.
The caps are
also ATPases,
which provides
the free energy
to break up the
proteins.
The 20S proteasome is not found in eubacteria; self-compartmentalized proteases are
• E. coli’s Clp protease has D7 symmetry
Hollow space in
the center is the
active site;
design is to
keep protease
from
indiscriminate
destruction (no
ubiquitin signal)
Top view
Side view
Transamination = transfer of amino
acid amine to an α-keto acid
• Once free amino acids are made (by proteasomes, pepsin,
trypsin, whatever) and transported in the bloodstream, they
are transaminated in the cell to yield…a different α-keto acid
and a different amino acid.
• So why bother? The goal is to get to glutamate, which can be
deaminated, and the resulting nitrogen excreted safely.
The recovery of carbon and nitrogen
from amino acids
• The amine
enters the
urea cycle,
and the
nitrogen
eventually
excreted.
• The rest of
the acid
skeleton is
recycled in
a number
of ways.
Schiff base (aka “imine”)
H
OH
A
R
R
C R
C
R
N
N
R
+
H2O
R
H
:B
Imines are created when a carbon of a ketone or aldehyde is
attacked by a (nucleophilic) amine; as shown above, the
inter-mediate is an amine with an alcohol on the α-carbon,
which will eventually rearrange into a C=N bond. This final
product is the imine, or Schiff base. Note the similarity of the
first step to hemiketal or hemiacetal formation.
The lysine of the transaminase enzyme forms a Schiff
base with the coenzyme pyridoxal-5’-phosphate (PLP)
• PLP is derived
from vitamin B6.
• Snell,
Braunstein and
Meztler (1954)
showed the
mechanism of
transaminase is
“Ping-Pong”.
Mechanism of transamination
Note
enzyme is
displaced by
the amino
acid but
Schiff base
remains.
Thus the
“ammonia”
is carried
safely.
Selectivity of the transimination
In the previous
diagram,
note that bonds a, b
or c can be cleaved
(in this diagram, the
C-X, C-Y and C-Z
bonds to the αcarbon). Which one
gets cleaved is a
function of which
one lies parallel to
the π-bond of the
imine that is about
to form.
Mechanism of transamination
• Enzyme
hangs
around to
act as a
base.
Mechanism of transamination
• Release of the
new α-keto acid.
• Then what? Do
the whole thing
again, but
backwards to
yield either
aspartate or
glutamate.
• Lysine is the only
acid that is not
transaminated.
Presence of transaminases in the
bloodstream is a bad sign
• Comes from damaged liver or muscle tissue.
• Serum glutamate-oxaloacetate transaminase
(SGOT) or serum glutamate-pyruvate
transaminase (SGPT) are measured.
Glutamate dehydrogenase deaminates
glutamate
• Activated by ADP and NAD+
• Inhibited by GTP and NADH
• The ammonium ion liberated is eventually
excreted through the urea cycle
Some amino acids break down readily
Even complex amino acids break down
to simpler amino acids
How to remove carbons:
Tetrahydrofolate (THF) is a one-carbon
carrier in amino acid degradation
The group that THF carries determines
the oxidation state of THF
The oxidation state will determine the potential of the
reduction needed to transfer the carbon group.
oxidation!
oxidation!
So an antibiotic strategy is to introduce a
molecule that looks like THF but will not
carry a carbon group!
• Hans Krebs &
Kurt Henseleit,
“Studies on urea
formation in the
animal
organism”
Hoppe-Seylers Z.
Physiol. Chem.
(1932).
• First metabolic
cycle elucidated.
• Purpose: excrete
excess nitrogen
by converting
ammonia into
less-toxic urea.
• Cost: 3 ATP
The urea cycle
Different organisms excrete the
nitrogen in different ways
Pre-urea cycle step occurs in mitochondrion
Multi-step synthesis
begins with ATP
reacting with
bicarbonate ion to
form
carboxyphosphate,
which reacts with
ammonia to yield
carbamate.
Urea cycle occurs
mostly in liver, and
some in kidneys.
Carbamoyl phosphate
synthetase catalyzes step 1
• Enzyme complex is like an
assembly line, with a 96 Å
long tunnel that allows the
substrate to move through
as it is processed.
• Protects intermediates with
short half-lives
(“channeling”).
• Also concentrates ammonia
to make carbamate.
Several different CPS enzymes
• CPS I is found in the mitochondrion and
catalyzes the precursor step to the urea cycle
• CPS II is found in the cytosol and catalyzes
pyrimidine metabolism
• CPS III is found in fish, and is considered to be
an evolutionary-link between glutaminedependent and ammonia-dependent CPS
enzymes
The urea cycle occurs in both mitochonrion
and cytosol
• Citrulline and ornithine
are the components
transported in and out of
mitochondrion.
• Cytochrome c, NADH,
ATP are necessary
reactants (Philip Cohen,
1946)
• Citrulline/aspartate step
(Sarah Ratner, 1949).
Fumarate is the major carbon product of
the urea cycle
Urea cycle regulated by substrate concentration
• The rate determining step of the urea cycle is
the first one – the synthesis of carbamoyl
phosphate – so the enzyme carbamoyl
phosphate synthase is activated by the
presence of N-acetylglutamate, which is
produced when there is an excess of
glutamate due to protein breakdown.
Amino acid degradation
• 10–15% of the amino acids
are broken down to CO2 and
H2O and the free energy
harvested.
• The rest enter the TCA cycle
in one of two ways:
• Glucogenic amino acids are
degraded to pyruvate or
other TCA intermediate that
will become glucose.
• Ketogenic amino acids are
degraded to acetyl-CoA or
acetoacetate and thus can
be made into fatty acids or
ketone bodies.
Some amino acids can never recovered
as glucose
• Leucine and lysine degrade to only acetyl-CoA
and/or acetoacetate, so can’t become glucose.
• Animals have no way of converting either
acetyl-CoA or acetoacetate back into a glucose
precursor.
Methionine leads to homocysteine
• Methionine’s breakdown leads through Sadenosylmthionine to homocysteine, which is a
cysteine with an extra methylene on the side chain.
• Excess levels of homocysteine have been correlated
to cardiovascular disease and the probable
mechanism is oxidative damage to arterial walls.
Tetrahydrofolate is derived from folic acid
(vitamin B9)
THF is a one-carbon carrier
• Note that R in the diagram below is the p-aminobenzoic acid
and the glutamate moieties.
• The one-carbon units can be methyl, methylene, formyl,
formimino, methenyl, and are covalently bonded to N-5
and/or N-10.
• Critical in formation of neural tubes, so folic acid is a critical
nutrient for moms.