Protein Turnover and

Amino Acid Catabolism

 Protein Turnover and Amino Acid Catabolism- Overview

§ Cells need to renew their proteins.

§ Damaged or unneeded proteins must be degraded

Constant degradation and resynthesis occur in the cells.

A complex mechanism for the turnover of proteins has evolved.

Damaged or unneeded proteins are marked for destruction by

the covalent attachment of a small protein, ubiquitin .

Polyubiquitinated proteins are degraded by a large complex

called the proteasome.
 Protein Turnover and Amino Acid Catabolism- Overview

Proteins are degraded to their basic building blocks, i.e. amino acids
• by digestion in intestines
• by protein turnover in the cells

â a-amino group of the amino acid is removed

â carbon skeleton is converted into a major metabolic intermediate

(acetyl CoA, acetoacetyl CoA, pyruvate, or one of the intermediates of the CAC)

â Amino groups of surplus amino acids are converted into urea

urea
through the urea cycle
 Protein Turnover and Amino Acid Catabolism- Overview
Sources of aa’s:

â Dietary amino acids from digestion in intestine

â From degradation of damaged or unneeded proteins

Essential amino acids cannot be synthesized by the body !

All 8 must be acquired by diet!

All are present in milk, eggs, meat

and soybean products!
(Most plant foods do not contain all 8)
 digestion and absorption of proteins

âstarts in the acidic environment of stomach, pH 2

âpepsin, a non specific protease digests proteins in stomach.
âdigestion continues in intestine â peptidases secreted by pancreas.
âsingle aa’s & di- and tri-peptides are released to blood.
âthese can be taken up by cells for formation of new proteins.

Pepsin in stomach
further
digestion of associated with
proteins the intestinal epithelium
 Cellular Proteins Are Degraded at Different Rates

Protein turnover—the degradation and resynthesis of proteins—

takes place constantly in cells.

proteins may be very stable or short lived

Protein turnover â regulation of protein activity

â getting rid of damaged proteins
should not be
accumulated / aggregated
Some protein half lives:
11 minutes â Ornithine decarboxylase
120 days â hemoglobin (half life of red blood cell)
Human life â crystallin
 Protein Turnover Is Tightly Regulated

Ubiquitin, a small (8.5-kd) protein present in all eukaryotic cells,

is the tag that marks proteins for destruction

Ubiquitin, is highly conserved from

Human to yeast (only 3 differ out of 76 residues)

Ubiquitin, attaches via the carboxyl terminal

glycine residue to the e-amino group of
several lysine residues on its targets.
an isopeptide bond forms

Ubiquitin, has an extended carboxylterminal,

which is activated & linked to proteins targeted for destruction
 isopeptide bond: an amide bond
that is not present on the main chain of a protein;
it is between the C-terminus of one protein
and NH3 group of a Lys residue on another protein.
 Three enzymes mediate ubiquitin mediated degradation

ubiquitin-activating enzyme, or E1
ubiquitin-conjugating enzyme, or E2
ubiquitin-protein ligase, or E3

First, the C-terminal carboxylate group of ubiquitin becomes linked

to a sulfhydryl group of E1 by a thioester bond.

Second, activated ubiquitin is shuttled to a sulfhydryl group of E2.

Finally, E3 catalyzes the transfer of ubiquitin from E2 to

an e -amino group of a target protein
 linked to a Cys
“adenylated”

transferred to transferred to
a Lys on target a Cys on E2
protein by E3
http://en.wikipedia.org/wiki/File:Ubiquitylation.svg
 Why Lys – a good choice for Ubiquination?

1) they are positively charged

so they are more likely to be accessible to the ubiquitin system.
2) they have a long side chain,
again making them very likely to be on the outer region of a
protein, and accessible to ubiquitin system.
 The Kiss of Death = Ubiquitinylation

Ubiquitin chains linked

by lysine 48 of ubiquitin
mediate recognition of
ubiquitinated proteins
by the proteasome.

Ubiquination rxn is processive

The attachment of a single molecule of ubiquitin is only a weak
signal for degradation. Chains of four or more ubiquitin
molecules are particularly effective in signaling degradation
Eukaryotes have one or a small number of distinct E1 enzymes,
all eukaryotes have many distinct E2 and E3 enzymes.

E3 is the most evolutionarily diverse component key for

recognizing a variety of different substrates

Furthermore, E2–E3 complex allow for more finely tuned

substrate discrimination.
 What determines whether a protein becomes ubiquitinated?

N-terminal rule

The half-life of a cytosolic protein is determined to a large extent

by its amino-terminal residue

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.

E3 enzymes are the readers of N-terminal residues.

Other destruction signals exist â proteins rich in PEST sequences,
cyclin destruction boxes etc. P-proline
E-glutamic acid
S-serine
degron T-threonine
 The Proteasome Digests the Ubiquitin-Tagged Proteins

The executioner â A large protease complex called the

proteasome or the 26S proteasome digests the ubiquitinated proteins.

Composed of two subunits:

§ 20S subunit with catalytic activity,
§ 19S regulatory subunit.

• ATP driven
• multi-subunit protease
• reserve Ub
20S proteasome consists of 28 homologous subunits arranged in
four rings of 7 subunits like a barrell.
b subunits on the interior are the ones actively processing
proteins for degradation whereas ubiquitin is recycled.
 The 20S proteasome is a sealed barrel.

Access to its interior is controlled by a 19S regulatory complex

ATP hydrolysis
likely assists to unfold
the substrate and induce
conformational changes
in the 20S proteasome
to pass substrate
to the center of the complex.
19S regulatory complex has 3 functions:

1) binds specifically to polyUb- chains

to prevent the degradation of non-ubiquinated proteins

2) an isopeptidase in this 19S unit cleaves of

intact Ub molecules from the tagged protein
so, they can be re-used

3) the tagged protein is unfolded and directed into the catalytic core.
substrates are degraded in a
processive way
without releasing the degraded
intermediates
 The Ubiquitin Pathway and the Proteasome
Have Prokaryotic Counterparts

Ubiquitin pathway and the proteasome â present in all eukaryotes.

Homologs of the proteasome are found in prokaryotes,

â physiological roles unknown.

Ubiquitin's molecular ancestors identified in prokaryotes.

their role is
not in protein modification
but in coenzyme biosynthesis

eukaryotic mechanism for protein modification evolved

from a preexisting prokaryotic pathway for coenzyme biosynthesis
 Protein Degradation Can Be Used to Regulate Biological Function

Protein degradation
by the ubiquitin-proteasome system is
central to cell homeostasis and survival.

Defects in this process

are associated with diseases such as
cancer and neurodegenerative disorders.

The balance between the synthesis and degradation of

key regulatory proteins determines the activities of
several cellular signaling pathways.
One example of a protein that is regulated by the
ubiquitin-proteasome system is NF-kB.
The first step in amino acid degradation is the removal of nitrogen

The major site of amino acid degradation in mammals is the liver.

First, deamination and excreting nitrogen:

there are no nitrogenous compounds

in energy-transduction pathways

Then, remaning a-ketoacids that result

from the deamination of amino acids are metabolized so that
the carbon skeletons can be used as precursors for
§ glucose
§ CAC intermediates
§ acetyl CoA
 alpha-amino groups are converted into ammonium ions
by the oxidative deamination of glutamate

a-amino group of many amino acids is transferred to

a-ketoglutarate (to form glutamate) by aminotransferases

a-ketoglutarate â forms glutamate

glutamate â oxidatively deaminated to yield ammonium ion, NH4+

Aminotransferase reaction using

α-ketoglutarate as the amino group
acceptor.
Transamination Oxidative Deamination

ammonium
ion
aminotransferase

Urea
(in liver)

Urea Cycle
 Aspartate aminotransferase

Aspartate + a-ketoglutarate D oxaloacetate + glutamate

w Aspartate donates its amino group, becoming the a-keto acid oxaloacetate.
w a-Ketoglutarate accepts the amino group, becoming the amino acid glutamate.

These reactions are reversible and

can be used to synthesize amino acids!!
 Alanine aminotransferase

Alanine + a-ketoglutarate D pyruvate + glutamate

w Alanine becomes pyruvate as the amino group is transferred to a-ketoglutarate.

These reactions are reversible and

can be used to synthesize amino acids!!
 There are multiple transaminase enzymes which vary in substrate specificity.
Some show preference for particular amino acids or classes of amino acids as
amino group donors, and/or for particular a-keto acid acceptors.

Aspartate + a-ketoglutarate D oxaloacetate + glutamate

Alanine + a-ketoglutarate D pyruvate + glutamate

glutamate D a-ketoglutarate

NAD+â NADH
Transaminase
Glutamate (NADP+ â NADPH)
dehydrogenase H2O â NH4+

Transaminases funnel amino N to glutamate,

which is deaminated by Glutamate dehydrogenase, producing NH4+.
Coenzymes:
Glutamate dehydrogenase
is unusual in that it
can use either NAD+ or NADP+
as a coenzyme.

NAD+ is used primarily in oxidative

deamination
(the simultaneous loss of ammonia
coupled with the oxidation of
the carbon skeleton - A),

and NADPH is used in

reductive amination (the
simultaneous gain of ammonia
coupled with the reduction of the
carbon skeleton - B).
Dehydrogenation
of C-N bond

Hyrdolysis of
Schiff base

Glutamate dehydrogenase
 Glutamate dehydrogenase
can use both
(NAD+ â NADH) or (NADP+ â NADPH)
is in mitochondria.
This helps compartmentalization of toxic ammonia!

rapid removal of ammonium ion drives it forward.

regulation:
Ø allosterically inhibited by GTP and ATP
Ø allostericaly activated by GDP and ADP

so, low energy charge favors amino acid catabolism!

 Transamination mechanism :
1) the external aldimine loses a proton
to form a quinonoid intermediate.
a new
Schiff-base 2) reprotonation of this intermediate at
enzyme
active site
linkage the aldehyde C atom yields a
ketimine
3) this intermediate is hydrolyzed to
generate the a-ketoacid product and
pyridoxamine phosphate.

The prosthetic group of

transaminase is pyridoxal
phosphate (PLP),
a derivative of vitamin B6.
 Transaminaton Oxidative deamination

exceptions:
serine and threonine can be directly deaminated.
 Peripheral Tissues Transport Nitrogen to the Liver

Amino acid degradation

can also take place in tissues other than the liver.

For example:
During prolonged exercise and fasting muscle uses amino acids
as a source of fuel but no urea enzymes in muscle!

Liver deaminates amino acids

and processes the nitrogen by urea cycle.

How does the nitrogen get from the muscle to the liver?

Two forms: alanine and glutamine

Glutamine in converted into urea in the liver.
Glucose-Alanine Cycle:
reminiscent of Cori cycle
 Glutamine in converted into urea in the liver. Notice that the C atoms of
urea and uric acid are
(a) Overview of highly oxidized; the
organism discards C only
catabolism after extracting most of
of amino groups its available E of oxidation.
(shaded) in
vertebrate liver.

(b) Excretory forms

of nitrogen.
Excess NH4 is
excreted as
ammonia
(microbes, bony
fishes),
urea (most
terrestrial
vertebrates), or uric
acid (birds and
terrestrial reptiles).
 ammonium ion is converted into urea
in most terrestrial vertebrates
Some NH4+ is re-used in biosynthesis of nitrogen compounds.

In most terrestial vertebrates, ammonium ion (NH4+) is excreted.

â they are called “ureotelic”
Urea is synthesized by Urea Cycle.

1 nitrogen atom is from aspartate

1 nitrogen is from NH4+
The carbon is from HCO3-
+3 ATP
In mitochondria

fate of oxaloacetate

1. Transamination to aspartate
2. Glucose by gluconeogenesis
3. Combine with acetyl coA to form Citric acid by CAC.
4. Conversion into pyruvate

The Urea Cycle, Gluconeogenesis and Transamination of OAA

are linked by Fumarate and Aspartate.

overall reaction for the Urea Cycle:

CO2 + NH4+ + 3 ATP + aspartate + H2O ª urea + 2 ADP + 2 Pi + AMP + PPi + fumarate
Carbon atoms of degraded amino acids pyruvate the seven
emerge as major metabolic intermediates acetyl CoA metabolites
acetoacetyl CoA to which
The carbon skeletons of the diverse set of 20 fundamental a-ketoglutarate all amino acid
amino acids are funneled into only seven molecules: succinyl CoA metabolism
fumarate converges.
oxaloacetate

Amino acids are

grouped into 2 classes,
based on whether or not their
carbon skeletons
can be converted to glucose

ketogenic
amino acids
give rise to ketone
bodies and fatty acids

glucogenic
amino acids
The net synthesis of glucose from
these amino acids is feasible
because these CAC intermediates
and pyruvate can be converted
into phosphoenolpyruvate and
then into glucose.
Who is who?

exclusively ketogenic
amino acids
Amino acids, when deaminated, yield a-keto acids that, directly or via
additional reactions, feed into major metabolic pathways (e.g., CAC).
Carbon skeletons of glucogenic amino acids are degraded to:
§ pyruvate, or
§ a 4-C or 5-C intermediate of CAC. These are precursors for gluconeogenesis.

Glucogenic amino acids are

the major carbon source for gluconeogenesis when glucose levels are low.
They can also be catabolized for energy,
or converted to glycogen or fatty acids for energy storage.

Carbon skeletons of ketogenic amino acids are degraded to:

w acetyl-CoA, or
w acetoacetate.

Acetyl CoA, & its precursor acetoacetate, can not yield net production of
oxaloacetate, the gluconeogenesis precursor.
For every 2C acetyl residue entering CAC, 2C leave as CO2.
Carbon skeletons of ketogenic amino acids can be catabolized for energy in CAC, or
converted to ketone bodies or fatty acids.
They cannot be converted to glucose.
Pyruvate is an entry point into metabolism
for a number of amino acids

The 3-C a-keto acid pyruvate

is produced from
alanine, cysteine, glycine, serine, & threonine.

Serine is deaminated to pyruvate

glycine, which is also product of threonine
via Serine Dehydratase.
catabolism, is converted to serine by a
reaction involving tetrahydrofolate

alanine deamination
via Transaminase
directly yields
pyruvate.
 Oxaloacetate is an entry point into metabolism for
aspartate & asparagine
The 4-C Krebs Cycle intermediate oxaloacetate is
produced from aspartate & asparagine.
Aspartate transamination yields oxaloacetate.
Aspartate is also converted to fumarate in Urea Cycle.
Fumarate is converted to oxaloacetate in CAC.

Asparagine loses the

amino group from its R-
group by hydrolysis
catalyzed by
Asparaginase.
This yields aspartate,
which can be converted
to oxaloacetate, e.g., by
transamination.
Succinyl CoA is an entry point into metabolism
for several non-polar amino acids

The 4-C Krebs Cycle intermediate succinyl-CoA is produced from

isoleucine, valine, & methionine.
Propionyl-CoA, an intermediate on these pathways, is also
a product of b-oxidation of fatty acids with an odd number of C atoms.

Propionyl-CoA is carboxylated to methylmalonyl-CoA.

a-ketoglutarate
The 5-C Krebs Cycle intermediate a-ketoglutarate is
produced from arginine, glutamate, glutamine, histidine, & proline.

Glutamate deamination via Transaminase

directly yields a-ketoglutarate.
Glutamate deamination by Glutamate Dehydrogenase
also directly yields a-ketoglutarate.
 Leucine the branched-chain amino acids yield
Isoleucine acetyl CoA, acetoacetate, or propionyl CoA
Lysine
Tryptophan
form acetyl CoA and acetoacetyl Coa directly
without pyruvate serving as an intermediate

phenylalanine and tyrosine also give rise

to acetoacetate during their catabolism
Therefore, there are a total of six
ketogenic amino acids.
Aromatic amino acids phenylalanine & tyrosine are
catabolized to fumarate and acetoacetate.
Summary of the metabolism
of amino acids in humans.
Genetically determined
enzyme deficiencies are
summarized in white boxes.
Nitrogen-containing
compounds derived from
amino acids are shown in
small, yellow boxes.
Classification of amino acids
is color coded:
red = glucogenic
brown = glucogenic and ketogenic
green = ketogenic

Compounds in BLUE ALL CAPS are

the seven metabolites to which all
amino acid metabolism converges.