AMİNOASİTLERİN OKSİDASYONU

AMİNOASİTLERİN
OKSİDASYONU
AMİNOASİTLERİN
METABOLİZMASI
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AZOT METABOLİZMASI*
ÜRE DÖNGÜSÜ*
KARBON İSKELETLERİNİN
METABOLİZMASI
AZOT METABOLİZMASI
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Aminoasit katabolizması tüm vücutta
gerçekleşen geniş bir süreç olan azot
metabolizmasının bir bölümüdür.
Azot vücuda besinlerde var olan
bileşikler şeklinde girer ve vücudu
üre,amonyak şeklinde terkeder.
Besinsel protein vücut için en önemli
olan aaleri içerir.
Besinsel protein
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Proteinlerin sindirimi midede başlar ve ince
barsakta sonlanır
Serbest aaler barsak epitel hücrelerinden
emilir
Bir kısmı karaciğere gelerek metabolize edilir
Bir kısmı genel dolaşıma verilir
Overview
of Amino
Acid
Catabolis
m
Metabolic Circumstances of
Amino Acid Oxidation
Amino acids undergo oxidative catabolism
under three circumstances:
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Protein amino-acid residues from normal turnover
are recycled to generate energy and molecular
components
Dietary amino acids that exceed body’s protein
synthesis needs are degraded
Proteins in the body are broken down to supply
amino acids for catabolism when carbohydrates
are in short supply (starvation, diabetes mellitus),
The Amino
Group is
Removed
From All
Amino Acids
First
Fates of Nitrogen in
Organisms
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Plants conserve almost all the nitrogen
Many aquatic vertebrates release ammonia to their
environment
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Many terrestrial vertebrates and sharks excrete nitrogen
in the form of urea
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Urea is far less toxic that ammonia
Urea has very high solubility
Some animals, such as birds and reptiles excrete
nitrogen as uric acid
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Passive diffusion from epithelial cells
Active transport via gills
Uric acid is rather insoluble
Excretion as paste allows to conserve water
Humans and great apes excrete both urea (from amino
acids) and uric acid (from purines)
Excretory
Forms of
Nitrogen
Enzymatic Transamination
• Typically, -ketoglutarate
accepts amino groups
• L-Glutamine acts as a
temporary storage of nitrogen
• L-Glutamine can donate the
amino group when needed for
amino acid biosynthesis
• All aminotransferases rely on
the pyridoxal phosphate
cofactor
Structure of Pyridoxal
Phosphate and Pyridoxamine
Phosphate
• Intermediate, enzymebound carrier of
amino groups
• Aldehyde form can
react reversibly with
amino groups
• Aminated form can
react reversibly with
carbonyl groups
Pyridoxal Phosphate is
Covalently Linked to the
Enzyme at Rest
• The linkage is made via an
nucleophilic attack of the
amino group an active-site
lysine side chain
• After dehydration, a Schiff
base linkage is formed
• The covalent complex is
called internal aldimine
because the Schiff base
connects PLP to the enzyme
Internal Aldimine in Aspartate
Aminotransferase (Lys258purple)
Chemistry of the Amino Group
Removal by the Internal
Aldimine
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The external aldimine of PLP is a good
electron sink, allowing removal of -hydrogen
PLP Also Catalyzes
Racemization of Amino Acids
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The external aldimine of PLP is
a good electron sink, allowing
removal of -hydrogen
PLP Also Catalyzes
Decarboxylation of
Amino Acids
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The external aldimine of PLP
is a good electron sink,
allowing removal of carboxylate
Ammonia in
Transported in
the Bloodstream
Safely as
Glutamate
• Un-needed glutamine
is processed in
intestines, kidneys
and liver
Glutamate can Donate
Ammonia to Pyruvate
to Make Alanine
• Vigorously working muscles
operate nearly anaerobically
and rely on glycolysis for
energy
• Glycolysis yields pyruvate that
muscles cannot metabolize
aerobically; if not eliminated
lactic acid will build up
• This pyruvate can be
converted to alanine for
transport into liver
Excess Glutamate is Metabolized
in the Mitochondria of
Hepatocytes
The Glutamate
Dehydrogenase
Reaction
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Two-electron oxidation
of glutamate followed
by hydrolysis
Net process is oxidative
deamination of
glutamate
Occurs in mitochondrial
matrix in mammals
Can use either NAD+ or
NADP+ as electron
acceptor
Ammonia is Re-captured via
Synthesis of Carbamoyl
Phosphate
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This is the first nitrogen-acquiring reaction
Fate of Individual Amino Acids
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Seven to acetyl-CoA
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Six to pyruvate
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Ile, Met, Thr, Val
Two to fumarate
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Arg, Glu, Gln, His, Pro
Four to succinyl-CoA
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Ala, Cys, Gly, Ser, Thr, Trp
Five to -ketoglutarate
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Leu, Ile, Thr, Lys, Phe, Tyr, Trp
Phe, Tyr
Two to oxaloacetate
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Asp, Asn
Summary
of Amino
Acid
Catabolism
Aminoasitlerin Yıkımı(1)
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C,H,O,N atomlarını içeren moleküllerdir
İçerdikleri N depolanamaz ve hücrenin
ihtiyacından fazla olan aaler hemen
yıkılırlar
alfa-amino grupları transaminasyon ve
oksidatif deaminasyonla uzaklaştırılarak
alfa-ketoasit ve NH3 oluşturulur
Amino Acid Oxidation
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Animals are unable to convert fatty acids into
carbohydrate.
During times of starvation, amino acids are
used to replenish TCA cycle intermediates
and as precursors for gluconeogenesis.
In addition, organisms with a diet high in
proteins can catabolize excess amino acids as
fuel.
Unlike carbohydrates or lipids, amino acids
are not stored. They are either used or
burned.
For animals, amino acids (in the form of
polypeptides) represent their major source of
nitrogen, and amino acids are used to make
use nitrogenous compounds.
Amino Acid Oxidation
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There is a key difference between
amino acids and the other 2 types of
oxidizable molecules: the nitrogen.
The first step in their degradation is
usually the removal of the amino group.
This is carried out by
aminotransferases (a.k.a.
transaminases), which catalyze the
transfer from an amino acid to an keto acid (usually -ketoglutarate):
amino acid + -Kg –> -keto acid +
Glu
The coenzyme pyridoxal phosphate is
Pyridoxal phosphate
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Catalyzes many reactions with amino acids
functional end = aldehyde attached to
hydroxypyridine derivative
Mechanism:
1.
2.
3.
4.
5.
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formation of a Schiff's base with substrate amino group
(stabilized by internal H-bond)
abstraction of H+ from -C (stabilized by resonance; N of
pyridoxine acts as e- sink)
donation of H+ to C-4' shifts Schiff's base (C=N) to -C
resolution depends upon enzyme
eventually the Schiff's base will be hydrolyzed to
regenerate an amine and a carbonyl
Can also catalyze decarboxylation of amino acids
(lose CO2 instead of H+ in step 2; otherwise
similar).
Amino Acid Oxidation
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The amino groups are thus collected in the
form of Glu. They can be removed by
glutamate dehydrogenase (in mito):
Glu + NAD(P)+  -iminoglutarate +
NAD(P)H
The imine is then spontaneously hydrolyzed:
-iminoglutarate + H2O –> -Kg + NH4+
The combined action of transaminases and
Glu DH to remove amino groups as ammonia
is sometimes called transdeamination.
The logic
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The amino groups are harvested from the
various amino acids that are in excess and
collected as Glu.
Thus, glutamate serves as a universal Ncarrier.
For example, Glu can serve as an indicator of
intracellular N supply, as well as a donor of
amino groups.
If there is an excess of amino groups in the
system, then Glu DH removes them as
ammonia. (You do not need a separate
enzyme for each a.a.)
Then the carbon skeletons can be attacked.
But what does the cell do with
the excess ammonia?
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If it is not a liver cell, it must excrete it,
but not as NH3. It uses glutamine
synthetase to incorporate it as Gln:
Glu + NH3 + ATP –> Gln + ADP + Pi
The carboxylate of Glu is first activated
by forming an acid anhydride with
phosphate
(You will see this mechanism again!)
But what does the cell do with
the excess ammonia?
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The Gln can then be excreted to the
circulatory system and reach the liver, where
it is hydrolyzed by glutaminase: Gln + H2O
–> Glu + NH4+
What to do with the carbon
skeletons
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All 20 of the amino acids can be catabolized to
either acetyl-CoA or TCA cycle intermediates.
They can be classified into 5 categories based
upon the end product:
1.
2.
3.
4.
5.
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-ketoglutarate: Glu, Gln, Pro, Arg, (His)
oxaloacetate: Asp, Asn
Succinyl-CoA: Val, Ile, Met
Pyruvate: Ala, Ser, Gly, Thr, (Cys)
Acetoacetyl-CoA: (Lys, Trp, Phe, Tyr), Leu
Note that this is an oversimplification, since
several amino acids have more than one end
product.
(We will discuss in detail all but the ones in parentheses.)
Aminoasitlerin Yıkımı(1)
NH3
İDRARLA ATILIR
ÜRE DÖNGÜSÜNE* GİRER
*Azotun vücuttan uzaklaştırılması için kullanılan en
önemli mekanizma üre döngüsüdür.
Transaminasyon
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Bir çok aminoasitin yıkımındaki ilk aşama
α-amino gruplarının α-ketoglutarata aktarılmasıdır
Ürünler:α-keto asit ve glutamat
α-ketoglutaratın aa metabolizmasındaki tek rolü glutamat şekline
geçerek diğer aalerden amino gruplarını toplamaktır
Glutamat:oksidatif deaminasyon veya
esansiyel olmayan aalerin sentezinde –NH3 vericisi
olarak
Transaminasyon
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Her aminotransferaz bir ya da birkaç amino
grubu vericisine özgündür
aminotransferazlar özgün amino grup
vericisine göre adlandırılırlar(örn/alanin)
amino grubunun alıcısı her zaman
α-ketoglutarattır
Transaminasyon
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En önemli iki aminotransferaz reaksiyonu
ALT ve AST tarafından katalizlenir
ALT veya GPT
alaninin -NH3 grubunu α-ketoglutarata aktarır
piruvat ve glutamat oluşur
Transaminasyon
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AST veya GOT
glutamatın amino grubunu okzaloasetata
transfer eder
aspartat oluşur
aspartat bir azot kaynağı olarak üre
döngüsüne girer
Transaminasyon
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Tüm aminotransferazların koenzimi
piridoksal fosfattır
aminotransferazlar,bir aminoasitin -NH3
grubunu piridoksale transfer ederek
piridoksamin fosfat oluştururlar
piridoksamin fosfat bir α-ketoasit ile
reaksiyona girerek bir aa oluştururken kendisi
de orijinal aldehit formuna yeniden dönüşür
Oksidatif Deaminasyon
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Oksidatif deaminasyon amino grubunu
serbest NH3 şeklinde açığa çıkarır
Glutamat,hızlı bir oksidatif deaminasyona
giren tek aa dir
Enzim: glutamat dehidrogenaz
Koenzim: NAD
NADP
Oksidatif Deaminasyon
glutamat dehidrogenaz
Degradation of Amino Acids
1.
2.
3.
4.
Discuss in general terms the use
of amino acids for the synthesis of
nitrogen-containing compounds
Discuss the various functions of
glutamine
Discuss the roles of
transamination
Discuss the glutamate
dehydrogenase (GDH) &
regulation
Amino Acid Degradation
Degradation : → removal & disposal of amino group
→ utilization of the carbon skeleton for
energy and gluconeogenesis
ALA & GLN → non-toxic vehicles for transport of NH4+
from the periphery to the liver for AA catabolism
Most nitrogenous waste is disposed of as → urea. N is
also disposed of as NH4+, uric acid & creatinine
.
Transamination
Transamination
15 N AA  free exchange
among AA (except THR & LYS)
(not true of the carbon portion)
Enzyme = transaminase or
aminotransferase
Quantitatively most important
reaction of AA metabolism
Involved in:
Synthesis NEAA
Degradation most AA
Redistribution
Transamination Reaction
1.
2.
3.
4.
5.
6.
There are many transaminases
Coenzyme is pyridoxal PO4 (PPal) formed
from vitamin B6
All AA except THR &v LYS can undergo
transamination with α ketoglutarate
Equilibrium of reaction is close to 1
therefore reaction direction depends on the
[reactants] which are directed by other
cellular processes
Directionality  removal/addition of
products of AA pool
Urea Synthesis provides direction by
withdrawing amino groups from the AA pool
 increase deamination and AA
catabolism
Transaminases – Clinical Use
Transaminases – Clinical Use
1.
2.
3.
4.
ASP & ALA transaminases are the most abundant
Several are present in both cytosol and mitochondria
(isoenzymes)
ASP aminotransferase is one of the most frequently
assayed enzymes in the clinical laboratory. Its
determination in serum  diagnostic acid especially for
assessing liver disorders
Nomenclature of transaminases is confusing: same
enzyme =
aspartate – glutamate transaminase
aspartate transaminase
glutamate – oxaloacetate transaminase
SGOT (clinical literature)
Role of Transamination
(i) Redistridution of  amino groups to balance AA pool
-- dietary proteins provide a mixture of AA whose
proportions differ from AA pool required by body
 correct imbalance
(ii) AA synthesis / degradation performed in conjunction with
glutamate dehydrogenase (GDH)
GDH can remove or add amino groups to the AA pool
Most  amino groups  glutamate due to the action of
transaminases.
When there is a surplus of AAs in the pool, the  amino
groups can be funneled through glutamate and released
as NH4+
Coupling
The release of  amino groups as NH4+ is catalysed by glutamate
dehydrogenase through oxidative deamination. Since the
reaction is reversible it can also synthesize amino groups.
GDH Requirements
1. The enzyme is the principal source of NH4+ in the body. GDH is a
mitochondrial enzyme located in matrix, present in liver cells and
most tissues.
2. Important for three reasons:
(i) Link between TCA cycle & metabolism of AA ( keto acids are
TCA cycle intermediates).
(ii) In mammals, only reaction in which an inorganic molecule (NH4+)
can be fixed to a C skeleton. Therefore essential AA could be
provided in the diet as  keto acids and the amino groups as NH4+
because NH4+  glutamate  other AA by transamination.
(iii) GDH is the major AA oxidative pathway and the major source of
NH4+
Also provides directionality to transamination/GDH. In vivo,  [GLU] ,
NAD+ & removal of NH4+ drive deamination of glutamate. With
excess NH4+ (bacterial metabolism in intestine), glutamate can be
formed.
Glutamate Dehydrogenase
1. Driving Force: necessity to maintain low levels of ammonia
which is toxic. Therefore Transaminase + GDH mediates
α amine  NH3  urea
2. Glutamate: link between transamination and Urea
synthesis
Transamination  funnels amino groups through
glutamate & a single dehydogenase suffices therefore
activity of GDH is key
Regulation of GDH
Regulation of GDH: allosteric control through diverse substances.
Major:
(i) energy  is there enough? If not oxidize AA
(ii) AA load  surplus? Therefore degrade (even when energy is
high)
Energy:
a) GTP (&  ATP) inhibit GDH. When GTP (TCA cycle) & ATP
(glycolysis / oxid. phosphorylation) are , energy index cell 
therefore GDH 
b) Conversely ADP and GDP , energy  therefore GDH active in
order to produce Keto acids  TCA cycle to produce ATP/GTP
c)  NAD H inhibits GDH
AA LOAD:
Excess AA: override inhibition caused with  energy therefore AA
themselves can  GDH activity.