Download c) acidic amino acids

Chapter 7 Catabolism of Proteins
Nutritional Function of Proteins

Functions:
Structural
Catalytic,
Transport action
Signaling and hormonal functions
Source of energy (16.7kJ/g)
Nutritional Requirement of Proteins
Nitrogen Balance
Proteins contain about 16% nitrogen
Intake N = losses N
Intake N > Losses N
Intake N < Losses N

Nutritional Quality of Proteins

Essential Amino Acids
cannot be synthesized by the body and must be obtained
from diet

Eight nutritional essential amino acids
Tryptophan
Phenylalanine
Lysine
Threonine
Valine
Leucine
Isoleucine
methionine
Nutritional Quality of Proteins

Non-essential amino acids
synthesized in the body
synthesized by the transamination of a-keto acids

Tyrosine and cysteine
synthesized in the body by using essential amino acids
from phenylalanine and methionine respectively
semi-essential
Digestion of Dietary Proteins

Dietary proteins are digested in the
stomach and intestine
Digestion of Protein in the Stomach

The digestion of protein.
Protein is broken down
into amino acids by the
enzymes pepsin (secreted
by the stomach) and
trypsin and peptidase (in
the small intestine).
Table 1.
Phases of Digestion and Absorption of Protein
and its Degradative Products
Phase of
Digestion
Location
Agents
Outcome
1. Gastric
Digestion
stomach
stomach acid
denaturation
pepsin
large peptide fragments +
some free amino acids
2. Pancreatic
Proteases
lumen of small
Intestine
trypsin, chymotrypsin,
elastase, and
carboxypeptidases
free amino acids and
oligopeptides – 2 to 8
amino acids
3. Brush Border
Surface
brush border
surface of intestine
endopeptidases and
aminopeptidases
free amino acids and
di-/ tripeptides
4. Absorption
intestinal epithelial
cell brush border
membrane
transport systems
uptake into epithelial cell
dipeptidases
tripeptidases
free amino acids from
di-/tripeptides;
facilitated diffusion
amino acids transported
into capillaries
5. Cleavage of
epithelial cell –
di-/tripeptides cytoplasm
transport to
capillaries
contraluminal
membrane
Plasma
CO2
HCO3-
Gastric Parietal Cell
Lumen of the
Stomach
carbonic anhydrase
CO2 +
H2CO3
H2O
H+
H+
HCO3-
K+
ATP
Cl-
Cl-
ADP + Pi
Cl-
H+,K+-ATPase
Production of gastric acid and its secretion
Dietary
Protein
Phase 1- Gastric digestion
Figure 2. Gastric digestion
of dietary protein.
Gastric Chief
Cells
Pepsinogen
denaturation by stomach acid
autoactivation
(intramolecular
cleavage)
Pepsin
hydrolysis by pepsin
autocatalysis
large peptide fragments
free amino acids
aa
aa
Pyloric sphincter
aa
aa
Duodenum
Acid from parietal cells denatures protein to be more susceptible to pepsin cleavage .
Pepsinogen activated to pepsin by autoactivation and autocatalysis by pepsin.
Large peptide fragments/some amino acids pass through the pyloric sphincter to the duodenum
Phase 2- Digestion by pancreatic proteases
Duodenal
Endocrine
Cell
CCK-PZ
free amino acids
from gastric
digestion
Duodenal
Endocrine
Cell
CCK-PZ
Trypsinogen
Enteropeptidase
Bloodstream
(hydrolysis)
Trypsin
Pancreatic
Acinar
Cell
Mucosal
Epithelial
Cells
Figure 3. Secretion, activation and action of
pancreatic proteases and brush border
endopeptidases and aminopeptidases
Phase 2- Digestion by pancreatic proteases
Duodenal
Endocrine
Cell
CCK-PZ
free amino acids
from gastric
digestion
Trypsinogen
Enteropeptidase
(hydrolysis)
Duodenal
Endocrine
Cell
CCK-PZ
Secretin
Bloodstream
autocatalysis
Trypsin
Pancreatic
Acinar
Cell
Mucosal
Epithelial
Cells
HCO3neutralizes
acid
Figure 3. Secretion, activation and action of
pancreatic proteases and brush border
endopeptidases and aminopeptidases
Phase 2- Digestion by pancreatic proteases
Duodenal
Endocrine
Cell
CCK-PZ
free amino acids
from gastric
digestion
Trypsinogen
Duodenal
Endocrine
Cell
CCK-PZ
Secretin
Enteropeptidase
(hydrolysis)
Bloodstream
autocatalysis
Trypsin
Chymotrypsinogen
Proelastase
Procarboxypeptidases
catalysis
Mucosal
Epithelial
Cells
Pancreatic
Acinar
Cell
HCO3neutralizes
acid
Chymotrypsin
Elastase
Carboxypeptidases
Figure 3. Secretion, activation and action of
pancreatic proteases and brush border
endopeptidases and aminopeptidases
Figure 3. Secretion, activation
and action of pancreatic
proteases and brush border
endopeptidases and
aminopeptidases Duodenal
Endocrine
Cell
CCK-PZ
Phase 2- Digestion by pancreatic proteases
Phase 3- Digestion at the brush border
free amino acids
from gastric
digestion
Trypsinogen
Enteropeptidase
(hydrolysis)
autocatalysis
Duodenal
Endocrine
Cell
CCK-PZ
Secretin
Bloodstream
Trypsin
Chymotrypsinogen
Proelastase
Procarboxypeptidases
catalysis
Mucosal
Epithelial
Cells
HCO3neutralizes
acid
Chymotrypsin
Elastase
Carboxypeptidases
brush border endo-/aminopeptidases hydrolyze products;
amino acids, di-/tripeptides absorbed by epithelial cells
Pancreatic
Acinar
Cell
amino acids
dipeptides
tripeptides
. Summary of the gastric and pancreatic digestive proteases
Protease
Source
Proenzyme
Activation
Specificity
stomach
Protease
family
aspartate
Pepsin (endo-)
pepsinogen
autoactivation/
H+; pepsin
aromatic
(tyr,phe, trp)
acidic (glu)
Trypsin (endo-)
pancreas
serine
trypsinogen
enteropeptidase
trypsin
basic (arg, lys)
Chymotrypsin
(endo-)
pancreas
serine
chymotrypsinogen
trypsin
bulky aromatic
(trp, phe, tyr, met)
Elastase
(endo-)
pancreas
serine
proelastase
trypsin
small neutral
R groups
(gly, ser, ala)
Carboxypeptidase A
(exo-)
pancreas
zinc
procarboxypeptidase A
trypsin
aromatic
(tyr, phe, trp)
hydrophobic
(val, leu, ile)
Carboxypeptidase B
(exo-)
pancreas
zinc
procarboxypeptidase B
trypsin
basic
(arg, lys)
Amino acids Na+ Di-, tripeptides
Intestinal Epithelium
Dipeptides, tripeptides
LUMEN OF INTESTINE
Phase 4 - Absorption
Brush border
Dipeptidases,
tripeptidases
Amino acids
3Na
Na+ 2K+
contraluminal membrane
ATP
ADP + Pi
3Na+ 2K+
= Na+-dependent co-transport
= Na+,K+-ATPase
Figure 4. Absorption of amino acids and di- and tripeptides from the intestinal lumen
BRUSH BORDER TRANSPORT SYSTEMS
a)
b)
c)
d)
e)
neutral amino acids (uncharged aliphatic and aromatic)
basic amino acids and cystine (Cys-Cys)
acidic amino acids (Asp, Glu)
imino acids (Pro)
dipeptides and tripeptides
Amino acids
Na+
Intestinal Epithelium
Di-, triLUMEN OF INTESTINE
peptides
Phase 4 Absorption
Dipeptides, tripeptides
Dipeptidases,
tripeptidases
Phase 5
3Na+ 2K+
Amino acids




Brush border
ATP

contraluminal membrane
ADP + Pi
Phase 5
 capillaries
3Na+ 2K+
= Na+-dependent co-transport
= Na+,K+-ATPase
= facilitated diffusion
Figure 4. Absorption of amino acids and di- and tripeptides from the intestinal lumen
Putrefaction


Decomposition of amino acids and proteins by
bacteria
Most ingested proteins are absorbed from the
small intestine
95% of total dietary proteins

Undigested proteins
pass into the large intestine

Bacterial activity occurs
Putrefaction

Bacteria putrefaction produces some
nutritional benefits,
Vitamin K, Vitamin B12, Folic acid

Toxic for human
Amines, phenol, indole, H2S





Production of Amines
Production of phenol
Production indole
Production of H2S
Production of Ammonia
Page 209
Degradation of Protein in
Cells
The half-life of proteins is determined by
rates of synthesis and degradation
dC
Rate of Turnover =
dt
= KS - KDC
A given protein is synthesized at a constant rate KS
A constant fraction of active molecules are destroyed per
unit time
KS is the rate constant for protein synthesis; will
vary depending on the particular protein
C is the amount of Protein at any time
KD is the first order rate constant of enzyme degradation,
i.e., the fraction destroyed per unit time, also
depends on the particular protein
Steady-state is achieved when the amount of protein
synthesized per unit time equals the amount being destroyed
dC
= 0 KDC = KS
dt
t 1/2 =
0.693
KD
C
Protein
concentration
(enzyme activity)
Stop protein synthesis,
measure rate of decay
Hours after stopping synthesis
Steps in Protein Degradation
Transformation to a degradable form
(Metal oxidized, Ubiquination, N-terminal residues, PEST sequences)
Lysosomal Digestion
ATP
26S Proteasome digestion
AMP + PPi
Proteolysis to peptides
KFERQ
7  type, 7  type
subunits
8 residue fragments
Ubiquination
N-end rule:
PEST:
DRLKF: 2-3 min
AGMSV: > 20 hr
Rapid degradation
Glycine at C terminal of Ubiquitin
Ubiquitin
COOATP
Ubiquitin activating enzyme
HS
AMP + PPi
E1
O
C S
Activation
of Ubiquitin
Ubiquitin conjugating enzyme
20 or more per cell
NH3+
E1
HS
E2
3
HS E1
C
S
Ubiquitin
ligase
E2
O
C N
O
C
Poly Ubiquitin
Ubiquination
Page 211
SH
NH3+
H3N+
O
3
E2
ATP
NH
AMP + PPi
Degraded
protein
E3
O
N C
O
N C
Ubiquitinspecific proteases
(26S proteasome)
+ Ubiquitin
Amino Acid Catabolism

Deamination of Amino Acids
removal of the a-amino acids
Oxidative Deamination
Non-oxidative Deamination
Transamination
Oxidative Deamination
Only a few amino acids can be
deaminated directly. Glutamate
Dehydrogenase catalyzes a

major reaction that effects net
removal of N from the amino
acid pool .
Glutamate Dehydrogenase is
one of the few enzymes that can
utilize either NAD+ or NADP+
as electron acceptor.
Oxidation at the -carbon is
followed by hydrolysis, releasing
NH4+.
At right is summarized the
role of transaminases in
funneling amino N to
glutamate, which is
deaminated via Glutamate
Dehydrogenase, producing
NH4+.

Non-oxidative Deamination
Serine Dehydratase catalyzes:
serine à pyruvate + NH4
Transamination
Transaminase enzymes
(aminotransferases)
catalyze the reversible
transfer of an amino
group between two keto acids.
Example of a
transaminase
reaction
A
nis shown at right.
•Aspartate donates its
amino group,
becoming the -keto
acid oxaloacetate.
-Ketoglutarate
accepts the amino
group, becoming the
amino acid glutamate.

In another example
shown at right,
alanine becomes
pyruvate as the
amino group is
transferred to ketoglutarate.

Transaminases equilibrate amino groups
among available -keto acids. This permits
synthesis of non-essential amino acids,
using amino groups derived from other
amino acids and carbon skeletons
synthesized in the cell. Thus a balance of
different amino acids is maintained, as
proteins of varied amino acid contents are
synthesized.
Mechanism of Transamination
The prosthetic group of the transaminase
enzyme is pyridoxal phosphate (PLP), a
derivative of vitamin B6.

In the "resting" state,
the aldehyde group of
pyridoxal phosphate
is in a Schiff base
linkage to the eamino group of an
enzyme lysine
residue.



The -amino group of a
substrate amino acid
displaces the enzyme
lysine, to form a Schiff
base linkage to PLP.
The active site lysine
extracts a proton,
promoting
tautomerization (shift of
the double bond),
followed by
reprotonation with
hydrolysis.


What was an amino acid
leaves as an -keto acid.
The amino group
remains on what is now
pyridoxamine
Phosphate (PMP).
A different -keto acid
reacts with PMP, and the
process reverses, to
complete the reaction.
Purine Nucleotide Cycle


The activity of L-glutamate dehydrogenase
is low in the skeletal muscle and heart.
In this tissues
purine nucleotide cycle
Figure 9-7 page 216
Metabolism of One Carbon Units

One carbon units are one carbon containing
groups produced in catabolism of some
amino acids.
Methyl (-CH3), methylene (=CH2), formyl (O=CH-)
and formimino (HN=CH-)
tetrahydrofolate (FH4)

One carbon units are carried by
tetrahydrofolate (FH4), a reduced form of
folic acid.
tetrahydrofolate (FH4)

FH4 is formed in
reduction of folic acid
catalyzed by
dihydrofolate reductase.
The four hydrogens are
added to the four atoms
of folic acid in positions
5 to 8. The N5 and N10
nitrogen atoms of FH4
participate in the
transfer of one carbon
groups

Production of One Carbon Units

Either glycine or serine
can act as methylene
donor, giving N5,N10methyleneTHF. This
behaves as "virtual
formaldehyde" H2C=O in
reactions.
The oxidation level can be
changed to methyl or
methenyl by reduction or
oxidation; methenylTHF
can be hydrolyzed to
formylTHF.
Production of One Carbon Units
from Histidine

N5-formiminotetrahydrofolate,
produced in the
pathway for
degradation of
histidine


In the pathway of
histidine degradation,
conversion of Nformiminoglutamate
to glutamate involves
transfer of the
formimino group to
tetrahydrofolate
(THF), yielding N5formimino-THF.
Adenosylmethionine (SAM)


S-adenosylmethionin (SAM)
is the major donor of methyl
group. FH4 can carry a
methyl group on its N5 atom,
but its transfer potential is
too low for most biosynthetic
methylation.
The activated methyl donor is
SAM, which is synthesized by
the transfer of an adenosyl
group from ATP to the sulfer
atom of methionine. The Sadenosylhomocysteine is
formed when the methyl
group of SAM is transferred
to an acceptor.

Conversion of One Carbon Units
Figure 9-13
Metabolism of Methionine,
Cysteine and Cystine


Sulfur-containing amino acids
Methionine is an essential amino acid
Methionine cycle and
methylation

In methionine cycle,
the adenosyl group
of ATP is
transferred to a
sulfur atom of
methionine by
methionine
adenosyltransferase
to form Sadenosylmethionine
(Sam)

Methionine cycle
and methylation

All phosphates of ATP
are lost in this reaction.
The sulfonium ion of
methionine is highly
reactive and the methyl
group of SAM is good
leaving group. SAM
then transfers the
methyl group to some
acceptors for their
methylation by
methyltransferase.
Methionine cycle and
methylation


The resulting Sadenosylhomocysteine
is cleaved by
adenosylhomocysteinase
to produce
homocysteine and
adenosine.
Homocysteine accepts a
methyl group from N5methyl-FH4 to
regenerate methionine.
Methionine cycle and
methylation


This reaction is catalyzed
by homocysteine
methyltransferase, which
requires vitamin B12 as a
cofactor. This is the only
reaction known that uses
methyl-FH4 as a methyl
group donor.
The net result of the
reaction is donation of a
methyl group and
regeneration of
methionine to complete
the methionine cycle.
Methionine cycle and
methylation

Person with elevated serum levels of
homocysteine have a high risk for coronary heart
disease and arteriosclerosis. The molecule basis
of the action of homocysteine has not been
clearly identified. It appears to damage cells of
blood vessels and to increase the growth of
vascular smooth muscle. Treatment with vitamin
B12, folic acid and vitamin B6 is effective in
reducing homocysteine level in some people.
Creatine and Creatine
Phosphate


Glycine, areginine
and methionine
participate in
synthesis of creatine
Transfer of guanidine
group from arginine
to glycine forms
guanidoacetate
catalyzed by
transamidinase in
kidney
Creatine and
Creatine Phosphate



Synthesis of creatine is
completed by methylation f
guanidoacetate in the liver.
This reaction is catalyzed by
guanidoacetate
methyltransferase.
SAM serves as a donor of a
methyl group.
Storage of “high energy”
phosphate from ATP,
creatine converts to creatine
phosphate particularly in
cardiac and skeletal muscle
catalyzed by creatine kinase
(CK)
Creatine and Creatine
Phosphate


This reaction is reversible
and creatine phosphate can
readily convert ADP to ATP
in muscle to meet the energy
requirement. The amount of
creatine in the body is related
to muscle mass.
Creatinine is derived from
dephosphorylation of creatine
phosphate and also formed by
hydrolysis of creatine
nonenzymatically.

Creatinine has no function and is excreted
in urine. The amount of creatinine
eliminated by an individual is constantly
from day to day. When a 24 hours urine
sample is requested, the amount of
creatinine in sample can be used as a gross
determining test to know renal function.
Cysteine and Cystine

Conversion of
Cysteine To Cystine
two molecules of
cysteine are linked by a
disulfide bond to form
cystine. The major
catabolic pathway of
cystine is conversion of
cysteine catalyzed by
cystine reductase. The
disulfide bond of cystine is
important to maintain the
conformation and
function of proteins
Synthesis of Taurine



Cysteine is the precusor of taurine. The
major oxidative metabolite of cysteine is
cysteine sulfinate, which is further
decarboxylation to form taurine.
Taurine is found rich in brain. It appears to
play role in brain development, but its
exact role is unknown
Figure. Page 229
Formation 3’-phosphoadenosine
5’phosphosulfate (PAPS)


Sulfate is produced mostly from
metabolism of cysteine. Catabolism of
cysteine produces pyruvate, NH3 and H2S.
Oxidation of H2S forms sulfate. Some
sulfate group for addition to biomolecules,
such as in biosynthesis of chondroitin
sulfates and keratan sulfate.
Figure. Page 229
Glutathione

Glutathione is the
tripeptide Gammaglutamylcysteinylgly
cine containing a
sulfhydryl group.
Glutathione has
several important
role.
serves as a transporter
in the gamma-glutamyl
cycle for amino acids
across cell membranes
protects erythrocytes
from oxidative damage
Glutathione cycles (Meister cycle)
figure.9-16

The enzyme gammaglutamyl transpeptidase,
located on the cell
membrane of kidneys
and other tissue cells,
catalyzes glutathion
(GSH) to transfer its
glutamyl group to amino
acid, then the gammaglutamyl-ammino acid is
transported inside of the
cell.
Glutathione cycles (Meister cycle)
figure.9-16


The gamma-glutamylamino acid releases amino
acid and 5-oxiproline.
This is the process for
amino acid transportation
into the cell.
The 5-oxiproline converts
to glutamate under the
action of enzyme and uses
ATP.
Glutathione cycles (Meister cycle)
figure.9-16



The 5-oxiproline converts to
glutamate under the action of
enzyme and uses ATP.
Glutamate and the other parts
of GSH, glycine and cysteine,
are regenerated GSH in
cytosol and 2 ATPs are used.
So 3 ATPs are required for
the transportation of each
amino acid.
The key enzyme of the
gamma-glutamyl cycle is
gamma-glutamyl
transpeptidase which is found
in high levels in the kidneys
Glutathione cycles (Meister cycle)
figure.9-16



Glutathion cycles between a
reduced form with a sulfhydryl
group (GSH) and an oxidized
form (GSSG), in which two
GSHs are linked by a disulfide
bond. GSH is reductant, its
sulhydryl group can be used to
reduce peroxides formed during
oxygen transport.
Glutathione plays a key role in
detoxification by acting with
hydrogen peroxide and organic
peroxide.
Glutathion peroxidase catalyzes
this reaction, in which GSH
converts to GSSG. Then GSSG is
reduced to GSH by glutathione
reductase, an enzyme containing
NADPH as a cofactor.
Metabolism of Aromatic Amino Acids


Formation of Tyrosine
from phenylalanine
First product in
degradation of
phenylalanine
Metabolism of Aromatic Amino Acids

Formation of
Tyrosine from
phenylalanine
first product in
degradation of
phenylalanine
Phenylalanine hydroxylase
Phenylketonuria (PKU)


Small amounts of
phenylalanine can
convert to phenylpyruvate
by transamination to
remove an amino group
in a healthy person.
If a genetic deficiency of
phenylalanine
hydroxylase occurs,
phenylketonuria is caused
Phenylalanine hydroxylase
Phenylketonuria (PKU)


PKU is the most common autosomal disease. Over 170
mutations in the gene have been reported. The elevated
phenylpyruvate, phenyllacetate (reduction product of
phenylpyruvate) and phenylacetate (decarboxylation of
phenlpyruvate) excreted in urine give urine its
characteristic odor. The neurological symptoms and
light color of skin and eyes are generally toxic effects of
high levels of phenylpyruvate and low concentrations of
tyrosine. The conventional treatment is to feed the
effected infant a diet low in phenylalanine with dietary
protein restrictions.
Figure 9-17 Metabolism and major derivatives of
phenylalanine and tyrosine
Metabolism of Tyrosine

The first step in catabolism of
tyrosine is transamination
catalyzed by tyrosine
transaminase to produce phydroxyphenylpyruvate, which
converts to homogentisate by
oxidase. Homogentisate is then
cleaved to fumarate and
acetoacetate. Fumarate is used in
the TCA cycle for energy or for
gluconeogenesis. Acetoacetate can
convert to acetyl CoA for lipid
synthesis or energy.
Production of Dopamine, Epinephrine
and Norepinephrine


Some tyrosine is used as a
precursor of catecholamines
(term of dopamine,
epinephrine and
norepinephrine)
The first step in the
synthesis of catecholamines
is catalyzed by tyrosine
hydroxylase, which is an
enzyme dependent on
tetrahydrobiopterin.


The product of this reaction is dihydroxyphenylalanin, known as Dopa. A product
of decarboxylation of Dopa is dopamine, which is a neurotransmiter. Parkinson’s
disease is induced by decreasing production of dopamin.
The adrenal medulla converts dopamine to norepinephrine by dopamine
hydroxylase, which accepts a methyl group from S-adenosylmethionine to form
epinephrine.
Synthesis of Melanin
Figure 9-17



Tyrosine is precursor of melanin. Dopa is the
intermediate in the synthesis of both melanin and
epinephrine.
Different enzymes dydroxylate tyrosines in
melanocytes and other cell type. In pigment cell,
tyrosine is hydroxylated to form Dopa by tyrosinase, a
copper-containing enzyme.
Dopa forms dopamine then converts it to indo-5-6quinone. Melanin is polymers of these tyrosine
catabolites with proteins from the eyes and skin. There
are various types of melanin, which are all aromatic
quintines complexes giving color, colorless, yellow and
dark to the skin.
Albinism

Albinism results from a genetic lack of tyrosinase. Lack
of pigment in the skin makes a patient sensitive to
sunlight and increases the incidence of skin cancer in
addition to burns. Lack of pigment in eyes may induce
photophobia
Production of Thyroid Hormone
:
tetraiodothyronine, T4:

triiodothyronine,T3.
Tyrosine is the precursor of the thyroid hormone: T4
and T3. The thyroid hormone has importance in
regulating the general metabolism, development and
tissue differentiation. Iodination of tyrosine residues in
thyroglobulin forms T4 and T3
Metabolism of Tryptophan
Figure 9-18
Metabolism of Tryptophan
Figure 9-18
Trytophan

precursor of nicotinic acid, one of the B vitamins.
b
hydroxylation and decarboxylation forms 5hydroxytryptamine (5-HT, serotonin)
Melatonin is a derivative of tryptophan, N-acetyl-5methoxytryptamine. It is a sleep-inducing molecule and
is synthesized in the pineal gland and retina mostly at
night. Melatonin appears to function by inhibiting
synthesis and secretion of other neurotransmitters, such
as dopamine and GABA.
Degradation of Branched-Chain
Amino Acids (BCAAs)
Figure 9-19


Valine, isoleucine and leucine are
branched-chain amino acids (BCCAs).
BCAAs transaminases are present at a
much higher level in muscle than that in
liver

Valine converts to succinyl CoA. So it is a glucogenic amino acid.
Leucine converts to acetyl CoA and acetoacetate. Leucine is a
ketogenic amino acid. Isoleucine produces acetyl CoA and
succinyl CoA and is both glycogenic and ketogenic amino acid. All
these intermediates of BCAAs degradation are oxidation in the
TCA cycle to support energy in muscle.
Transport of Ammonia in Blood




At physiological pH, 98.5% exists as
ammonium ion (NH+4)
Only traces of NH3 are present
Even trace of NH3 are toxic to the nervous
system
NH3 is rapidly removed
Glutamine synthetase fixes
ammonia as glutamine

Formation of glutamine is catalyzed by glutamine
synthetase. Synthesis of the amide bond of glutamine is
accomplished at the expense of hydrolysis of one mole of
ATP to ADP and Pi.
Glutamine Synthetase

Hydrolysis of glutamine produces
glutamate and NH3 in the liver and kidneys

Glutamine supports an amide group for synthesis of
asparagine from aspartate by asparagine synthetase.
Since certain tumors such as leukemic cells seem to
lose this ability and exhibit abnormally high
requirements for asparagine and glutamine, hydrolysis
of asparagine is catalyzed by asparaginase. So,
exogenous asparaginase and glutaminase had been
tested as antitumor agents
Alanine-glucose cycle
Figure 9-8






Muscles generate over half of the total metabolism pool of amino
acids. The ammonia produced in catabolism of amino acids in
muscle is accepted by pyruvate to form alanine, which is released
into the blood.
Alanine appears to be the vehicle of ammonia for transport in the
blood
The liver takes up the alanine and converts it back into pyruvate
by transamination
The resulting pyruvate can be converted to glucose by the
gluconeogenesis pathway and an amino group eventually appears
as urea.
Glucose formed in gluconeogenesis is released into the blood and
taken up by muscles.
Glycolysis of glucose produces pyruvate, which is then
resynthesized alanine. This is called alanine-glucose cycle
Formation of Urea (Urea Cycle)

Urea Cycle

The urea cycle takes place partly in the
cytosol and partly in the mitochondria, and
the individual reactions are as follows
Urea Cycle

carbamyl phosphate synthetase 1 [CPS1]
This liver mitochondrial enzyme converts the ammonia
produced by glutamate dehydrogenase into carbamyl
phosphate (=carbamoyl phosphate) which is an unstable
high energy compound. It is the mixed acid anhydride of
carbamic acid and phosphoric acid, and requires two
molecules of ATP to drive its synthesis.
Urea Cycle
CPS1 is an allosteric enzyme and is
absolutely dependent up on Nacetylglutamic acid for it activity
Urea Cycle

CPS1 deficiency results in hyperammonemia. The
neonatal cases are usually lethal, but there is also a less
severe, delayed-onset form. Ammonia-dependent CPS1
is present only in the liver mitochondrial matrix space. It
should be distinguished from a second cytosolic
glutamine-dependent carbamyl phosphate synthetase
[CPS2] which is found in all tissues and is involved in
pyrimidine biosynthesis. Carbamyl phosphate synthesis
is a major burden for liver mitochondria. This enzyme
accounts for about 20% of the total protein in the matrix
space. Glutamate dehydrogenase is also present in very
large amounts.
Urea Cycle

The next reaction also takes place in the liver
mitochondrial matrix space, where ornithine is
converted into citrulline
ornithine transcarbamylase [OTCase]
Urea Cycle

Citrulline is transported out of the mitochondria into
cytosol by the mitochondrial inner membrane transport
system. Once in the cytosol, citrulline condenses with
aspartate and the reaction is driven by ATP. In this way
aspartate contributes the second nitrogen atom to urea,
the first having come from glutamate
Urea Cycle
Production of arginino-succinate is an
energetically expensive process, since the
ATP is split to AMP and pyrophosphate.
The pyrophosphate is then cleaved to
inorganic phosphate using pyrophosphatase,
so the overall reaction costs two
equivalents of high energy phosphate per
mole.
Urea Cycle
Elimination of fumarate from
succinate then yields arginine.
arginino-succinate lyase
arginino-
Urea Cycle
Fumarate can be converted into
oxaloacetate under catalysis of some
enzymes as in the TCA cycle. Oxaloacetate
can be converted to aspartate by
transamination. The aspartate is then
reutilized in the urea cycle
Urea Cycle
Cleavage of arginine by arginase to produce urea
regenerates ornithine, which is then available for
another round of the cycle.
Urea Cycle
Since humans can not metabolize urea, it is
transported to the kidneys for excretion.
Some urea that enters the intestinal tract is
cleaved by bacteria urease, the resulting
ammonia being absorbed and treated by the
liver
Note that of the two nitrogen atoms of urea, one comes
from carbamoyl phosphate, being ultimately derived
from ammonia. The other nitrogen is derived from the
a-amino group of aspartate which in turn is obtained
from transamination of oxaloacetate with glutamate.
The formation of one molecule of urea requires the
hydrolysis of four high-energy phosphate groups from 3
molecules of ATP
The overall reaction is as follows:
2NH3 + CO2 +3ATP + 3H2O -> H2N-CO-NH2 +2ADP + AMP +4Pi
Urea Cycle (review)
1. Occurs in the liver mitochondria and cytosol
2. Starts with carbamoyl-PO4
3. Ends with arginine
4. Requires aspartate
5. Requires 3 ATPs to make one urea
Synthesis of Carbamoyl-PO4
NH4+ + HCO3- + 2 ATP
O
H2N
O
C ~ O-P-O
+ 2 ADP + Pi
O
High energy bond
Carbamoyl phosphate Synthetase I
Citrulline
Carbamoyl-PO4
Urea
Cycle
Ornithine
+
NH3
CH2
CH2
CH2
HC
COOH3N
Arginine
H2O
O
C
H2 N
NH2
Urea
Aspartate
ATP
Argininosuccinate
NH2
+
H2N=C
NH
CH2
CH2
CH2
HC
COOH3N
Reactions of Urea Cycle
COOH3N+-C-H
O
CH2
CH2
CH2
+ NH
3
COOH3N+-C-H
+
H2N
C
CH2
CH2
CH2
NH
OPO3
Carbamoyl-PO4
O=C
Ornithine
CH2
CH2
CH2
NH
O=C
NH2
H3N+-C-H
COO+
Citruline
COO-
COOH3
+ OPO3=
NH2
Mitochondria
N+-C-H
Cytosol
ATP
CH2
+
H-C-NH3
COO-
L-Aspartate
ADP + Pi
CH2
CH2
COO- CH
2
CH2
NH
H-C-N =C
COO- NH2
Argininosuccinate
Cytosol
COOH3N+-C-H
COOCH2
CH2
CH2
COO-
CH2
CH2
NH
CH2
CH2
NH
CH
H2N+ =C
H-C-N =C
COO-
COOH3N+-C-H
NH2
+
HC
COOFumarate
NH2
L-Arginine
COO-
COO-
COO-
CH2
CH2
CH2
H-C-NH3
C=O
H C-OH
COO-
COO-
+
L-Aspartate
Oxaloacetate
COOL-Malate
COOH3N+-C-H
COOH3N+-C-H
CH2
H2O
CH2
CH2
CH2
NH
CH2
CH2
+ NH3
NH2
Ornithine
H2N+ =C
O
+
C
H2N
NH2
Urea
L-Arginine
Return to Mitochondria
Nitric Oxide


Arginine also serves as a direct precursor of nitric oxide (NO). The
free-radical gas NO is the potent muscle relaxant and short-lived
signal molecule. Nitric oxide is formed by the catalysis of the cytosol
enzyme nitric oxide synthase (NOS), which is a very complex
enzyme with five cofactors: NADPH, FAD, FMN, heme and
tetrahydrobiopterin.
The substrate in the reaction is arginine and products are citrulline
and NO. Oxygen is required in the complex reaction. NO plays an
important role in many physiologic and pathologic processes
Decarboxylation of Amino Acids
Decarboxylation of amino acids forms amine.
This reaction is catalyzed by decarboxylase,
which contains pyridoxal phosphate as a
cofactor. Amines always have potential
physiological effects.
GABA
gamma-Aminobutyric acid (GABA) is formed by
pyridoxal phosphate-dependent enzyme, L-glutamate
decarboxylase, which is principally present in brain
tissue. GABA functions as inhibitory neurotransmitter.
GABA, catalyzed by gamma-aminobutyrate
transaminase, forms succinate and semialdehyde, which
may be oxidized to form succinate and via TCA cycle to
form CO2 and H2O
Histamine
Decarboxylation of histidine forms histamine, a reaction
catalyzed by histidine decarboxylase. Histamine has
many physiological roles, including vasodilation and
constriction of certain blood vessels. An overreaction of
histamine can lead to bronchial asthma and other
allergic reactions. In addition, histamine stimulates
secretion of both pepsin and hydrochloric acid by the
stomach, and is useful in the study of gastric activity
Serotonin

5-hydroxytryptamine (5-HT), also known as serotonin, results from
hydroxylation of tryptophan by a tetrahydrobiopterin-dependent
enzyme, hydroxylase and decarboxylation by a pyridoxal phosphatecontaining decarboxylase. 5-HT is a neurotransmitter in the brain
and causes contraction of smooth muscle of arterioles and
bronchioles.
polyamines
Figure 9-12
Polyamines are important in cell proliferation
and tissue growth. They are growth factors for
cultured mammalian cells and bacteria. Since
polyamines bear multiple positive charges that
can interact with polyanions such as DNA and
RNA, and thus can stimulate synthesis of
nucleic acid and protein.