Chapter 17

---2:
Amino Acid Oxidation and
the Production of Urea
Nitrogen Excretion and the Urea
Cycle
 the ammonia in the
mitochondria of
hepatocytes is converted
to urea via the urea cycle.
This pathway was
discovered in 1932 by
Hans Krebs and a medical
student associate, Kurt
Henseleit. Urea production
occurs almost exclusively
in the liver.
Urea Is Formed in the Liver

 Using thin slices of liver suspended in a

buffered aerobic medium, Krebs and
Henseleit found that the rate of urea
formation from ammonia was greatly
accelerated by adding any one of three α-
amino acids: ornithine, citrulline, or
arginine.
ornithine and citrnlline can serve as successive precursors
of arginine. Note that citrulline and ornithine are
nonstandard amino acids that are not found in proteins.
The Production of Urea from Ammonia
Involves Five Enzymatic Steps

 The urea cycle begins inside the mitochondria of

hepatocytes, but three of the steps occur in the
cytosol; the cycle thus spans two cellular
compartments .
 The first amino group to enter the urea cycle is
derived from ammonia(Deamination) .together
with HCO3- produced by mitochondrial
respiration, to form carbamoyl phosphate in the
matrix
 HCO3−
ATP
ADP
Carbamoyl Phosphate
O
Synthase (Type I) catalyzes
a 3-step reaction, with HO C OPO32−
carbonyl phosphate and NH3 carbonyl phosphate
carbamate intermediates. Pi
O
Ammonia is the N input.
H2N C O−
The reaction, which
ATP carbamate
involves cleavage of 2 ~P
bonds of ATP, is essentially ADP
irreversible. O

H2N C OPO32−
carbamoyl phosphate
carbamoyl phosphate synthetase
I and II
 carbamoyl phosphate synthetase I. The
mitochondrial form of the enzyme is distinct from
the cytosolic (II) form, which has a separate
function in pyrimidine biosynthesis (Chapter 21).
Carbamoyl phosphate synthetase I is a
regulatory enzyme; it requires N-acetylglutamate
as a positive modulator (see below). Carbamoyl
phosphate may be regarded as an activated
carbamoyl group donor.
 H O H

N-ac
glutamat
H3N+ C COO− H3C C N C COO−
H
CH2 CH2

CH2 CH2

COO− COO−

Carbamoyl Phosphate Synthase has an absolute

requirement for an allosteric activator N-acetylglutamate.
This derivative of glutamate is synthesized from acetyl-
CoA & glutamate when cellular [glutamate] is high, signaling
an excess of free amino acids due to protein breakdown or
dietary intake.
urea cycle
 The carbamoyl phosphate now enters the urea
cycle, which entails four enzymatic steps.
Carbamoyl phosphate donates its carbamoyl
group to ornithine to form citrulline and release Pi
in a reaction catalyzed by ornithine
transcarbamoylase. The citrulline is released
from the mitochondrion into the cytosol.
 cytosol
mitochondrial matrix

carbamoyl phosphate
Pi
ornithine citrulline

ornithine citrulline
urea aspartate
arginine argininosuccinate
fumarate
For each cycle, citrulline must leave the mitochondria, and
ornithine must enter the mitochondrial matrix.
An ornithine/citrulline transporter in the inner
mitochondrial membrane facilitates transmembrane fluxes of
citrulline & ornithine.
mitochondrial matrix and the cytosol.
17
 O

H 2N C NH2
urea

Most terrestrial land animals convert excess nitrogen to

urea, prior to excreting it.
Urea is less toxic than ammonia.
The Urea Cycle occurs mainly in liver.
The 2 nitrogen atoms of urea enter the Urea Cycle as
NH3 (produced mainly via Glutamate Dehydrogenase)
and as the amino N of aspartate.
The NH3 and HCO3− (carbonyl C) that will be part of urea
are incorporated first into carbamoyl phosphate.
The Citric Acid and Urea Cycles Are Linked

 The fumarate produced in the argininosuccinate lyase

reaction is also an intermediate of the citric acid cycle.
Fumarate enters the mitochondria, where the combined
activities of fumarase and malate dehydrogenase
transform fumarate into oxaloacetate . Because the
reactions of the urea and citric acid cycles are
inextricably intertwined, together they have been called
the "Krebs bicycle."
Fumarate produced in the cytosol by argininosuccinate lyase of the urea cycle
enters the citric acid cycle in the mitochondrion and is converted in several
steps to oxaloacetate. Oxaloacetate accepts an amino group from glutamate
by transamination, and the aspartate thus formed leaves the mitochondrion
and donates its amino group to the urea cycle in the argininosuccinate
synthetase reaction.
 COO− COO−

COO− CH2 COO− CH2

CH2 CH2 CH2 CH2

HC NH3+ + C O C O + HC NH3+

COO− COO− COO− COO−

aspartate α-ketoglutarate oxaloacetate glutamate

Aminotransferase (Transaminase)
Fumarate is converted to oxaloacetate via Krebs Cycle
enzymes Fumarase & Malate Dehydrogenase.
Oxaloacetate is converted to aspartate via transamination
(e.g., from glutamate).
Aspartate then reenters Urea Cycle, carrying an amino
group derived from another amino acid.
The Activity of the Urea Cycle Is
Regulated
 The flux of nitrogen through the urea cycle varies
with the composition of the diet. When the diet is
primarily protein, the use of the carbon skeletons
of amino acids for fuel results in the production of
much urea from the excess amino groups. During
severe starvation, when breakdown of muscle
protein supplies much of the metabolic fuel, urea
production also increases substantially, for the
same reason.
The reaction catalyzed by
carbamoyl phosphate
synthetase I. The
formation of carbamoyl
phosphate in the
mitochondrial matrix is
strongly stimulated by the
allosteric effector N-
acetylglutamate . Note that
the terminal phosphate
groups of two molecules of
ATP are used to form one
molecule of carbamoyl
phosphate: two activation
steps occur in the
carbamoyl phosphate
synthetase I reaction.
 Figure 17-14
Synthesis of N-
acetylglutamate,
the allosteric
activator of
carbamoyl
phosphate
synthetase I, is
stimulated by high
concentrations of
arginine. Increasing
arginine levels
signal the need for
more flux through
the urea cycle.
long term regulation
 These changes in demand for urea cycle activity
are met in the long term by regulation of the rates
of synthesis of the urea cycle enzymes and
carbamoyl phosphate synthetase I in the liver. All
five enzymes are synthesized at higher rates
during starvation or in animals on very high-
protein diets than in well-fed animals on diets
containing primarily
The Urea Cycle Is Energetically
Expensive

 The urea cycle brings together two amino

groups and HCO3- to form a molecule of
urea, which diffuses from the liver into the
bloodstream, thence to be excreted into
the urine by the kidneys. The overall
equation of the urea cycle is
 2NH4+ + HCO3- + 3ATP4- + H2O urea
+ 2ADP3- + 4Pi2- + AMP2- + 5H+
3 ATP
 The synthesis of one molecule of urea requires
four high-energy phosphate groups. Two ATPs
are required to make carbamoyl phosphate, and
one ATP is required to make argininosuccinate. In
the latter reaction, however, the ATP undergoes a
pyrophosphate cleavage to AMP and
pyrophosphate, which may be hydrolyzed to yield
two Pi.
 It has been estimated that, because of the
necessity of excreting nitrogen as urea
instead of ammonia, ureotelic animals lose
about 15% of the energy of the amino
acids from which the urea was derived.
Hereditary deficiency of any of the Urea Cycle
enzymes leads to hyperammonemia - elevated
[ammonia] in blood.
Total lack of any Urea Cycle enzyme is lethal.
Elevated ammonia is toxic, especially to the
brain.
If not treated immediately after birth, severe
mental retardation results.
Postulated mechanisms for toxicity of high [ammonia]:
1. High [NH3] would drive Glutamine Synthase:
glutamate + ATP + NH3  glutamine + ADP + Pi
This would deplete glutamate – a neurotransmitter &
precursor for synthesis of the neurotransmitter GABA.

2. Depletion of glutamate & high ammonia level would drive

Glutamate Dehydrogenase reaction to reverse:
glutamate + NAD(P)+ α -ketoglutarate +
NAD(P)H + NH4+
The resulting depletion of α -ketoglutarate, an essential
Krebs Cycle intermediate, could impair energy metabolism
in the brain.
Treatment of deficiency of Urea Cycle enzymes
(depends on which enzyme is deficient):
 limiting protein intake to the amount barely
adequate to supply amino acids for growth,
while adding to the diet the α -keto acid
analogs of essential amino acids.
 Liver transplantation has also been used,
since liver is the organ that carries out Urea
Cycle.
 Table Nonessential and essential amino acids for
humans and the albino rat Nonessential Essential
Alanine Arginine* Asparagine Histidine Aspartate
Isoleucine Cysteine Leucine Glutamate Lysine
Glutamine Methionine Glycine Phenylalanine
Proline Threonine Serine Tryptophan Tyrosine
Valine * Essential in young, growing animals but
not in adulta.
Summary

 Ammonia is highly toxic to animal tissues.

Ureotelic animals (adult terrestrial amphibians
and all mammals) excrete amino nitrogen as
urea, formed in the liver by the urea cycle.
Arginine is the immediate precursor of urea.
Arginase hydrolyzes arginine to yield urea and
ornithine, and arginine is resynthesized in the
urea cycle.
Summary
 Ornithine is converted to citrulline at the
expense of carbamoyl phosphate, and an
amino group is transferred to citrulline
from aspartate, re-forming arginine.
Ornithine is regenerated in each turn of
the cycle.
Summary
 Several of the intermediates and byproducts of the urea cycle are
also intermediates in the citric acid cycle, and the two cycles are
thus interconnected.
 The activity of the urea cycle is regulated at the levels of enzyme
synthesis and allosteric regulation of the enzyme that forms
carbamoyl phosphate.
 The formation of the nontoxic urea and of solid uric acid has a
high ATP cost.
 Genetic defects in enzymes of the urea cycle can be
compensated for by dietary regulation.
Overview of Amino Acid Catabolism:
organ Relationships
Detoxification of Ammonia by the
Liver: the Urea Cycle
 Amino acid N flowing to liver as:
 Alanine& glutamine
 Other amino acids
 Ammonia (from portal blood)

 Urea
 chief N-excretory compound
 Flow of Nitrogen from Amino
Acids to Urea in Liver
 Amino acid flow from muscle to liver
 Alanine & glutamine
 Liver
 Transfers N to GLU
 GLN’ase
 Transaminases
 Transfers GLU-N to:
 ASP
 AST
 Transamination route
 NH3
 GDH
 Trans-deamination route
 Transfers N to urea
 28.2 Amino
Acid
Metabolism: An
Overview
 The amino acid
pool, the entire
collection of free
amino acids
throughout the
body, occupies a
central position
in amino acid
metabolism.
1. Protein Degradation
Dietary proteins are a vital source of
amino acids.

Discarded cellular proteins are another

source of amino acids.

40
1.2 Cellular Protein Degradation
 Cellular proteins are degraded at different rates.

 Ornithine decarboxylase has a half-life of 11

minutes.

 Hemoglobin lasts as long as a red blood cell.

 Υ-Crystallin (eye lens protein) lasts as long as

the organism does.

41
Turnover
 Amino acids used for synthesizing proteins are obtained by
degrading other proteins

 Proteins destined for degradation are labeled with

ubiquitin.

 Polyubiquinated proteins are degraded by proteosomes.

 Amino acids are also a source of nitrogen for other

biomolecules.

42
Protein Turnover Is Tightly
Regulated

The turnover of cellular proteins is a regulated
process requiring complex enzyme systems.
Proteins to be degraded are conjugated with ubi-
quitin, a small conserved protein, in a reaction
driven by ATP hydrolysis. A large, barrel-shaped
complex called the proteasome digests the
ubiquitinated proteins. The proteasome also
requires ATP hydrolysis to function.
 Ubiquitin:
Proteins are usually tagged for
selective destruction in proteolytic
complexes called proteasomes by
covalent attachment of ubiquitin, a
small, compact, highly conserved
protein.

ubiquitin PDB 1TBE

H
However, some proteins may be degraded H3N+ C COO−
by proteasomes without ubiquitination. CH2

An isopeptide bond links the terminal CH2

carboxyl of ubiquitin to the ε -amino CH2
group of a lysine residue of a "condemned" CH2
protein. + NH
3
The joining of ubiquitin to a condemned protein is
ATP-dependent.
Three enzymes are involved, designated E1, E2 &
E3. The ubiquitin pathway is a multi-step process
in which an ubiquitin molecule is activated by an
E1 enzyme, transferred to an E2 enzyme, and
then covalently attached to the protein substrate
either directly or in conjunction with E3 enzyme .
Proteins destined to be degraded are subject to
multiple rounds of ubiquitin attachment and are
then proteolyzed by the 26S proteasome.
 O

ubiquitin C S Cys E2 + H2N Lys protein to be degraded

E3 (Ubiquitin-Protein Ligase)
O

ubiquitin C N Lys protein to be degraded + HS Cys E2

A Ubiquitin-Protein Ligase (E3) then transfers activated

ubiquitin to the ε -amino group of a Lys residue of a protein
recognized by that E3, forming an isopeptide bond.
 destruction Primary structure of a protein
box targeted for degradation

H2 N COO−
chain of
ubiquitins

More ubiquitins are added to form a chain of ubiquitins.

The terminal carboxyl of each ubiquitin is linked to the ε -
amino group of a Lys residue of the adjacent ubiquitin.
A chain of 4 or more ubiquitins (linked via Lys29 or Lys48)
targets proteins for degradation in proteasomes.
 destruction Primary structure of a protein
box targeted for degradation

H2 N COO−
chain of
ubiquitins

Some proteins (e.g., mitotic cyclins involved in cell

cycle regulation) have a destruction box sequence
recognized by a domain of the corresponding
Ubiquitin Ligase.
 20 S Proteasome
(yeast) closed state
Proteasomes: α
Selective protein
degradation occurs β
in the proteasome,
a large protein β
complex in the
nucleus & cytosol α
of eukaryotic cells. two views PDB 1JD2

The proteasome core complex, with a 20S sedimentation

coefficient, contains 2 each of 14 different polypeptides.
 7 α -type proteins form each of the two α rings, at the
ends of the cylindrical structure.
 7 β -type proteins form each of the 2 central β rings.
 20 S Proteasome
(yeast) closed state
α

α
two views PDB 1JD2

The 20S proteasome core complex encloses a cavity with

3 compartments joined by narrow passageways.
Protease activities are associated with 3 of the β
subunits, each having different substrate specificity.
1. One catalytic β -subunit has a chymotrypsin-like
activity with preference for tyrosine or
phenylalanine at the P1 (peptide carbonyl)
position.
2. One has a trypsin-like activity with preference for
arginine or lysine at the P1 position.
3. One has a post-glutamyl activity with preference
for glutamate or other acidic residue at the P1
position.
Proteasome evolution:
Proteasomes are considered very old.
They are in archaebacteria, but not most eubacteria,
although eubacteria have alternative protein-
degrading complexes.
 The archaebacterial proteasome has just 2
proteins, α & β , with 14 copies of each.
 The eukaryotic proteasome has evolved 14
distinct proteins that occupy unique positions
within the proteasome (7 α -type & 7 β -type).