Transcript BIO 322_Rec_4part2_Spring 2013

Amino Acid Oxidation
and
the Production of Urea
L EH N IN GER C H . 1 8
BIO 3 2 2 R EC ITATION 4 - 2 / SPR IN G 2 0 1 3
In animals, amino acids undergo oxidative degradation in three
different metabolic circumstances:
1.
During the normal synthesis and degradation of cellular
proteins (protein turnover), some amino acids that are
released from protein breakdown and are not needed for
new protein synthesis undergo oxidative degradation.
2.
When a diet is rich in protein and the ingested amino acids
exceed the body's needs for protein synthes, the surplus is
catabolized, amino acids cannot be stored.
3.
During starvation or in uncontrolled diabetes mellitus, when
carbohydrates are either unavailable or not properly utilized,
cellular proteins are used as fuel.
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• Amino acids lose their
amino groups to form αketo acids  the carbon
skeletons of AA.
• Seperation α amino group
from carbon skeleton
• α-keto acids undergo
oxidation to CO2 and H2O,
or 3C-4C units that can
be converted to glucose
via gluconeogenesis
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Metabolic Fates of Amino Groups
• Nitrogen is abundant in atmosphere but inert
for use. Only few organisms can convert
nitrogen gas in to useful form ammonia, so
amino groups are controlled carefully.
• AA from diet are the source of most amino
groups. Most of them metabolised in the liver.
• As a result : Some generated ammonia is recycled
and used for biosynthesis. Excess is excreted
directly or converted to urea depending on the
organism.
• Excess ammonia generated in extrahepatic
tissues travel to the liver in the form of amino
groups for the conversion to excretory form.
• Glutamine and glutamate – collecting point of
amino groups
• In hepatocytes, amino groups are transferred to α
keto glutarate to form glutamate. Then glutamate
enters into mitochondria to give the amino group
and form ammonia as ammonium ion.
• Skelatal muscle – excess amino
groups are transferred to pyruvate to
form alanine – another important
transport molecule of amino groups to
liver
• Excess ammonia in other tissue are converted to
amide nitrogen of glutamine.
 Glutamine and glutamate conc. is higher than
other AA.
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Dietary Protein Is Enzymatically Degraded to AA
Degradation of ingested proteins to
AA in gastrointestinal tract.
Entry of dietary protein into the
stomach
Stimulation of gastric mucosa to
secrete gastrin
Stimulates the secretion of HCL from
parietal cells and pepsinogen by chief
cells of the gastric glands.
• Gastric juice : pH 1-2.5 – antiseptic, denaturing agent, increased
vulnerability to enzymatic hydrolysis for proteins
• Pepsinogen – pepsin at low pH – hydrolyzes peptide bonds on the
amino terminal side of aromatic AA Phe, Trp,Tyr – smaller 5peptide.
Dietary Protein Is Enzymatically Degraded to AA
Low pH in small intestine causes secretin
secretion into blood
Pancreas secretes bicarbonate to small
intestine  neutralize pH to 7.
(Panceatic secretions pass into SI via
pancreatic duct)
Arrival of AA at Duodenum (Upper SI) causes
cholecystokinin secretion to blood, that
causes pancreatic enzymes secretion that work
best around pH 7 or 8.
• Trypsinogen, chymotrypsinogen and procarboxypeptidases A and B are secreted
from exocrine cells of pancreas.
• Trypsinogen to trypsin via enteropeptidase secreted from SI. Free trypsin further
catalyzes trypsinogen to trypsin, also activates chymotrypsinogen,
procarboxypeptidases and proelastase.
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Why this elaborate mechanism for getting active digestive enzymes into the
gastrointestinal tract?
• Inactive precursors protects exocrine cells from proteolytic attack.
• Pancreas protects itself from digestion via pancreatic trypsin inhibitor
• İnhibit premature production of active proteolytic enzymes within
pancreatic cells.
• Pepsin, trypsin and chymotrypsin have different AA specifities.
• Degradation of small peptides is completed by other intestinal peptidases
such as carboxypeptidase A and B and another aminopeptidase.
• Resulting free AA to liver, keratin and some plant protein due to cellulose
masking cannot be fully digested.
Acute pancreatitis – active forms of zymogens inside pancreatic cells and
attack pancreatic tissue itself  pain, organ damage
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Pyridoxal Phosphate Participates in the Transfer of αketoglutarate
• First step in catabolism of L-amino acids in liver is
promoted by enzymes called aminotransferases and
transaminases.
• Transamination reactions, the α amino group is
transferred to the α carbon of α- ketoglutarate, leaving
behind a α- keto acid.
• Aim is to collect amino groups from many different AA in
the form of L-glutamate.
• Glutamate functions as amino group donor biosynthesis or
excretion pathways.
• Aminotransferases
• Many aminotransferases – specific for α-ketoglutarate but
differ in specificity for L-amino acid  named for the amino
group donor.
• Amino transferase rxns are reversible.
• Prosthetic group – PLP (pyridoxal phosphate) – coenzyme form
of pyridoxine or Vitamin B6
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Pyridoxal Phosphate
• PLP- Coenzyme of glycogen
phosphorylase – not usual role. Primary
role is in the metabolism of molecues with
amino groups.
• PLP- Intermediate carrier of amino groups
at the active site of aminotransferases.
• PLP- Aldehyde form (pyridoxal phosphate)
can accept amino groups and aminated
form is called pyridoxamine phosphate,
can donate its amino group to α-keto acid
• PLP is bound to active site of the enzyme
through aldimine (Schiff base) linkage to
the ε-amino group of a Lysine residue.
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Glutamate Releases Its Amino Group as Ammonia in the liver
• Amino groups from many AA are collected in the liver
in the form of amino group of L glutamate.
• Amino groups from glutamate must be removed to
prepare them for excretion.
• In hepatocytes: Glutamate to mitochondria – oxidative
deamination by glutamate dehdyrogenase.
•
Present in mitochondrial matrix, only enzyme that can
both use NAD or NADP as the acceptor.
• Combined action of aminotransferase and glutamate
dehydrogenase – TRANSDEAMINATION
• α-ketoglutarate from glutamate deamination can be
used in TCA cycle for glucose synthesis.
• Glutamate dehydrogenrase: Positive modulator ADP,
negative modulator GTP
•
The metabolic rationale has not been elucidated yet.
• Mutations at at GTP binding side causes
hyperinsulism-hyperammonemia syndrome,
characterized by elevated levels of ammonia in the
blood stream and hypoglycemia.
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Glutamine Transports Ammonia in
the Bloodstream
• FROM EXTRAHEPATIC TISSUES
• Ammonia is toxic to cell.
• The free ammonia produced in tissues is
combined with glutamate to yield
glutamine by action of glutamine
synthetase.
• Requires ATP. Two step reaction
• Glutamate and ATP react to form ADP and
gamma-glutamyl phosphate intermediate,
which then reacts with ammonia to produce
glutamine and inorganic phosphate.
• Glutamine is non toxic – Higher
concentrations in blood.
• Intestine, liver and kidneys
• Amide nitrogen is released as ammounium ion
in the mitochondria.
• Glutaminase catalyzes glutamine to glutamate
and ammonia in liver Mitochondria Ammmonia is converted to urea.
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Alanine Transports Ammonia from
Skeletal Muscle to the Liver
FROM MUSCLE- Muscle amino groups collected as
glutamate. Glutamate may be converted to glutamine
for transport to liver
OR
• Glutamate transfers amino group to pyruvate by
alanine amino transferase.
• Alanine goes to blood, travels to liver.
• In cytosol of hepatocytes, alanine amino transferase
transfers the amino group of alanine to α ketoglutarate forming pyruvate and glutamate.
• Glutamate goes to mitochondria releases ammonia or
can undergo transamination with oxaloacetate to form
aspartate, another nitrogen donor in urea synthesis.
 Lactate from muscle or erythrocytes to blood, targeted
to liver, converted back to glucose – back to muscle
(Cori cycle). Energetic burden of gluconeogenesis is
imposed on liver rather than muscle
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Nitrogen
Excretion
and The
Urea Cycle
• In liver mitoch. whatever the source ammonia
generated + product of mitochonrial respiration,
carbondioxide as bicarbonate, produce carbamoyl
phosphate in the matrix by ATP dependent
Carbamoyl phosphate synthetase I
1.
Carbamoyl phosphate donates it s carbamoyl
group to ornithine to form citrulline, with
release of Pi. (Ornithine transcarbamoylase) –
Citrulline to cytosol
 Ornithine resembles oxaloacetate of TCA cycle
beucase it accepts material at each turn of the
cycle.
2. Condensation of amino group of aspartate and
ureido (carbonyl) of citrulline forms
arginosuccinate. (Arginosuccinate synthetase) –
requires ATP, citrullyl-AMP intermediate
3. Arginosuccinate is then cleaved to form arginine
and fumarate Arginosuccinase. Fumarate goes
back to mitochondria and joins TCA cycle. – Only
reversible step in urea cycle
4. Cytosolic arginase cleaved arginine to yield urea
and ornithine. Ornithine back to mitochonria for
another round of urea cycle.
 Mitochondrial and cytosolic enzymes are clustered
– The citrulline passed directly to the active site
of arginosuccinate synthetase  14
chanelling. Only
urea is released to general cytosolic pool.
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The Citric Acid and Urea Cycles Can Be Linked
•
Krebs Bicycle - TCA + UREA
interconnected. Each can
operate independently
•
TCA enzymes – fumarase
and malate dehydrogenase
have cytosolic isozymes
 Fumarate to malate in
cytosol can go back to
mitochondria as malate
•
Aspartate formed in
mitochondria via
transamination between
oxaloacetate and glutamate
can be transported back to
cytosol where it serves as a
nitrogen donor in the rxn
catalyzed by
arginosuccinate synthetase.
•
These rxns make up
aspartate-arginosuccinate
shunt – provide metabolic
links
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The Activity of the Urea Cycle Is Regulated at Two Levels
•
When dietary intake is primarly protein- excess AA – excess urea
•
Prolonged starvation – excess urea due to breakdown of muscle
protein
•
All urea cycle enzymes are synthesized at higher rates in starvation
and high protein diet.
•
Shorter time scale regulation of first enzyme, carbomyl phosphate
synthetase I is allosterically activated by N-acetylglutamate, which
is synthesized from acetyl-coA and glutamate by N-acetylglutamate
synthase.
•
Arginine, an intermediate of urea cycle is also the activator of Nacetylglutamate synthase.
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• Humans are incapable of synthesizing half of the 20 common
amino acids  essential amino acids.
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•
7 amino acids
broken down to
acetyl-CoA
•
5 alfa-ketoglutarate
•
4 to succinyl-CoA
•
2 to fumarate
•
2 to oxaloacetate
•
6 to pyruvate 
acetyl-CoA or
oxaloacetate
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