NITROGEN METABOLISM

Prof. Dr. Nazamid Saari

Department of Food Science
Universiti Putra Malaysia

Learning outcomes
Recognize how amino acids/proteins are
turned into metabolic energy and the
chemical processes involved
Predict the energy content and value of the
chemical compounds
Identify its roles to human/animal as well as in
food production

Nitrogen balance and amino acid

metabolism

Nitrogen excretion

INTRODUCTION
The biosynthesis of proteins requires a
continuous source of amino acids.
Amino acids are generated by the digestion of
proteins in the intestine or by the degradation
of proteins within the cells.
Cellular proteins are constantly being
degraded and resynthesized.
The short lived proteins usually play important
metabolic roles.

Metabolic relationships of amino

acids

Cont.
The pools (=amino acid available for metabolic
process) of free amino acid in animals are
derived from a combination of dietary sources
and de novo synthesis.
Amino acids are important precursors of a
variety of biological molecules as well as
providing the building blocks for polypeptide
and protein synthesis. In addition, amino acid
carbons can be oxidized for energy production
after removal of their amino group.

Digestion of Dietary Proteins

Protein digestion begins in the stomach.
The primary enzyme involved in proteolytic digestion is
pepsin which catalyzes the nonspecific hydrolysis of peptide
bonds at an optimal pH of 2.
In the lumen of the small intestine, the pancreas secretes
zymogens of trypsin, chymotrypsin, elastase ect
This battery of proteolytic enzymes breaks the proteins
down into free amino acids as well as dipeptides and
tripeptides.
The free amino acids as well as the di- and tri-peptides are
absorbed by the intestinal mucosa cells which subsequently
are released into the blood stream where they are absorbed
by other tissues.

Protein digestion

Turnover of Cellular Proteins

Cellular proteins are continually being
synthesized and degraded cell.
Functional proteins are distinguished from old
proteins and are marked for degradation by the
attachment of an Ubiquitin tag.
Ubiquitin is a small protein found in all
eukaryotic cells. Ubiquitin is attached to the
terminal -amino group of lysine residues
marking these proteins for degradation.
Three enzymes are involved in the tagging of a
protein. E1, The ubiquitin activating enzyme E2,
ubiquitin conjugating enzyme E3,ubiquitin
protein ligase.

Cont.
Once a protein is marked for degradation,
proteasome executes the proteolysis using ATP
to hydrolyze the peptide bonds of proteins.
The proteasome has a sedimentation coefficient
of 26S and is composed of 2 subunits, a 20S
proteasome which contains all of the catalytic
machinery to digest proteins and a 19S regulatory
subunit.
The substrate proteins are degraded in a
processive manner until the entire protein has
been reduced to peptides of 7 to 9 residues.

Ubiquitin Related Protein

Degradation
Ubiquitin is a small
protein(8.5 kD = 76
amino acids)
Highly conserved among
all Eukaryotes.
When covalently
attached to a protein,
ubiquitin marks that
protein for destruction

Tagging of Proteins
The carboxyl-terminal glycine of ubiquitin
covalently attaches to e-amino group of
lysine residues on target protein
Requires ATP hydrolysis
Three enzymes involved: 1) E1, ubiqutiin
activating protein, 2) E2, Ubiquitin
conjugating enzyme, 3) E3, ubiquitinprotein ligase.

Protein Ubiquitination

Multiple Ubiquitins can be polymerized to each other.

What determines whether a protein

will become ubiquinated?
E3 enzyme are readers of Nterminal amino acid residues
N-terminal amino acids
determine stability of protein
Also proteins rich in proline,
glutamic acid, serine and
threonine (PEST sequences)
often have short lives.
Other specific sequences (e.g.
cyclin destruction box) target
proteins for ubiquitination

Ubiquitinated Proteins are Degraded

by the 26S Proteosome
The 26S proteosome is
a large protease
complex that
specifically degrades
ubiquinated proteins
2 major components
20S proteosome core,
19S cap.
Proteolysis occurs in
20S domain
Ubiquitin recognition
occurs at 19S domain

26S Proteosome
ATP dependent
process.
Protein is unfolded
as it enters 20S
domain.
Ubiquitin not
degraded, but
released and
recycled.

Cont.
The peptide products are further degraded by
cellular proteases to yield the individual
amino acids.

What is the fate of these amino acids?

The amino acids that are produced are either
utilized for the biosynthesis of newer proteins or
degraded.
In mammals, amino acids are degraded in the
liver by deamination of amino acids to form ketoacids.
The - ketoacids are metabolized and the
remaining carbon skeletons enters the
metabolic mainstream as precursors for
gluconeogenesis or as citric acid cycle
intermediates.

LIVER

PYRIDOXAL PHOSPHATE

Three stages of amino acid catabolism

1. Deamination (removal of the -amino
group and transport to the liver)
2. Urea synthesis (to excrete nitrogen; occurs
only in the liver)
3. Conversion of the carbon skeleton to one of
seven metabolic intermediates

Deamination/Transamination By Transaminase/Amino
Transferase (common name)

Cont..Transamination of Amino Acid

There are three main transaminases or Amino transferases,
all requiring Pyridoxal-P derived from vitamin B6
(pyridoxine) via phosphorylation as a cofactor:
Glutamate aminotransferase (third most active in liver):
amino acid + 2-oxoglutarate/-ketoglutarate
2oxoacid/-keto acid + glutamate
Alanine aminotransferase (second most active in liver): ala
+ 2-oxoglutarate/-ketoglutarate
pyruvate +
glutamate
Aspartate aminotransferase (most active in liver): asp + 2oxoglutarate/-ketoglutarate
oxalacetate +
glutamate

ContTransamination of Amino Acid

The various aminotransferases in the liver all
funnel excess N to glutamate and aspartate.
Glutamate can then be deaminated by
Glutamate dehydrogenase to give ammonia,
contributing up to 1/2 of the N in urea.

Ammonia production

Cont.
Most transaminases share a common
substrate and product (glutamate and
oxoglutarate) with glutamate dehydrogenase,
and this permits a combined nitrogen
excretion pathway for individual amino acids
that is commonly described as TRANSDEAMINATION. This process demonstrates the
central roles of glutamate in the overall
control of nitrogen metabolism

Amino group transport

Urea Cycle
Every amino acid contains at least one amino
group. Amino acid catabolism generates
ammonia which is sensitive to brain tissue.
Therefore every amino acid degradation pathway
has a key step where the amino group is
removed.
Cells get rid of excess ammonia by the reductive
amination of ketoglutarate to form glutamate
by glutamate dehydrogenase and the conversion
of glutamate into glutamine by glutamine
synthetase

Cont.
-Ketoglutarate + NH4+ + NADH
Glutamate + NH4+ +ATP

Glutamate + NAD+
Glutamine + ADP + Pi

Glutamate is a neurotransmitter. Glutamate is also the

precursor for -aminobutyrate (GABA) which is another
important neurotransmitter. High concentrations of ammonia
deplete the concentration of glutamate which produces a
similar decrease of GABA which impairs brain function.

UREA CYCLE

Urea Cycle

Cont
step1 ornithine transcarbamylase catalyzes
carbamoyl phosphate to transfer the
carbamoyl group to ornithine (non-standard
aa) to form citrulline (non-standard aa) takes
place in mitochondria; citrulline transported
out of mitochondria in exchange for ornithine
source of first N in urea

Cont.
step 2 argininosuccinate synthetase
condenses citrulline with aspartate as source
of second N in urea to form arginosuccinate
requires hydrolysis of ATP to PPi and then to
2Pi takes place in cytoplasm step 3 carbon
skeleton of aspartate removed as fumarate by
argininosuccinase arginine is produced
takes place in cytoplasm

Cont.
step 4 urea is formed from arginine by
arginase and ornithine regenerated ornithine
is transported

Urea/TCA cycle coupling (Krebs

bicycle)

cont
The urea cycle and the tricarboxylic acid cycle are
coupled together through fumarate and
aspartate. Thus unless the fumarate released
when arginosuccinate is cleaved can be cycled
through the TCA cycle to oxaloacetate, the urea
cycle will be slowed or inhibited. Fumarate is
the precursor to oxaloacetate Oxaloacetate
can: be transaminated to aspartate and feed
back into urea cycle condense with AcCoA and
feed into citric acid cycle proceed into
gluconeogenesis be converted to pyruvate

Amino acid carbons

Glucogenic (Aspartic acid, glutamic acid, asparagine,
glutamine, histidine, proline, arginine, glycine, alanine,
serine, cysteine, methionine, valine) and Ketogenic
(leucine and lysine) Amino Acids. Both Glucogenic and
Ketogenic (phenylalanine, tyrosine, tryptophan,
isoleucine, and threonine)
The carbon skeletons of amino acids are metabolized,
resulting in intermediates which are central to either
carbohydrate or lipid metabolism. Those which are
metabolized to yield potential substrates for
gluconeogenesis are termed glycogenic, those which
yield acetate or acetoacetate are termed ketogenic.
Some amino acids yield both kinds of intermediate.

Amino acid carbon metabolism

Cont.
This panel represents central carbon
metabolism and the points at which various
amino acid structures feed into it. Note that
some amino acids may feed different
metabolic products into this scheme at two
different points if the carbon skeleton is
metabolized to produce two different kinds of
fragments (i.e. some amino acids can be both
glycogenic and ketogenic)

Metabolic intermediates

Summary
Synthesis of UREA requires energy input as
follow:
CO2+NH4+ + 3ATP + aspartate +2H2O ---Urea + 2ADP +2Pi +AMP +PPi + Fumarate
Formation of one molecule of UREA requires the
energy from cleavage of 4 phosphoanhydride
bonds

cont.
Step 1: 2 ATP---2ADP + Pi
Step 3: ATP---AMP + Ppi
Followed by
. Ppi + H2O ---2Pi