Aino Acid Catabolism
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Transcript of Aino Acid Catabolism
8/6/2019 Aino Acid Catabolism
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There are multiple transaminase enzymes which vary in
substrate specificity.
Some show preference for particular amino acids or classes of amino acids as amino group donors, and/or for
particular E-k eto acid acceptors.
H
R1 C COO-
+ R2 C COO-
NH3
+
O
Transaminase
H
R1 C COO
-
+ R2 C COO
-
O NH3
+
Transaminases
(aminotransferases)catalyze thereversible reactionat right.
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Example of a Transaminase reaction:
Aspartate donates its amino group, becoming theE-keto acid oxaloacetate.
E-Ketoglutarate accepts the amino group,
becoming the amino acid glutamate.
aspartate E-ketoglutarate oxaloacetate glutamate
Aminotrans erase (Transaminase)
COO
CH2
CH2
C
COO
O
COO
CH2
HC
COO
NH3+
COO
CH2
CH2
HC
COO
NH3+
COO
CH2
C
COO
O+ +
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In another example, alanine becomes pyruvate as
the amino group is transferred to E-ketoglutarate.
alanine E-ketoglutarate pyruvate glutamate
Aminotransferase (Transaminase)
COO
CH2
CH2
C
COO
O
CH3
HC
COO
NH3+
COO
CH2
CH2
HC
COO
NH3+
CH3
C
COO
O+ +
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Transaminases equilibrate amino groups amongavailableE-keto acids.
This permits synthesis of non-essential amino acids,using amino groups from other amino acids & carbon
skeletons synthesized in a cell.
Thus a balance of different amino acids is maintained,as proteins of varied amino acid contents aresynthesized.
Although the amino N of one amino acid can be usedto synthesize another amino acid, N must be
obtained in the diet as amino acids ( proteins).
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Essential amino acids must be consumed in the diet.
Mammalian cells lack enzymes to synthesize their carbon skeletons (E-keto acids). These include:
Isoleucine, leucine, & valine
Lysine
Threonine
Tryptophan
Phenylalanine (Tyr can be made from Phe.)
Methionine (Cys can be made from Met.)
Histidine (Essential for infants.)
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The prosthetic group of Transaminase is
pyridoxal phosphate (PLP), a derivative of
vitamin B6.
pyridoxal phosphate (PLP)
NH
C
O
P
O
O
O
OH
CH3
C
H O
H2
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In the resting state, the aldehyde group of pyridoxal phosphate is in a Schiff base linkage to the I-aminogroup of an enzyme lysine side-chain.
NH
C
O
P
O
O
O
O
CH3
HC
H2
N
(CH2)4
Enz
H
+
RHC COO
NH2
Enzyme (Lys)-PLP Schiff base
Amino acid
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The amino group remains on what is now pyridoxamine
phosphate (PMP).
A different E-keto acid reacts with PMP and the processreverses, to complete the reaction.
N
H
C
O
P
OO
O
OH
CH3
CH2
NH2
H2
R C COO
O
Enz sNH2
Pyridoxamine phosphate (PMP)
E-keto acid
What was anamino acidleaves as anE-k eto acid.
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Several other enzymes that catalyze metabolism or synthesis of amino acids also utilize PLP as prostheticgroup, and have mechanisms involving a Schiff base
linkage of the amino group to PLP.
NH
C
O
P
OO
O
O
CH3
HC
H2
N
HC
H+
R COO
Enz sNH2
Amino acid- hi base (aldimine)
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Chime Exercise
Two neighboring students or student groups shouldteam up, each displaying one of the following:
Transaminase with PLP in Schiff base linkage tothe active site lysine residue.
Transaminase in the PMP form, with glutarate, ananalog of E-ketoglutarate, at the active site.
Students should then show and ex plain the structuredisplayed by them to the neighboring student or student group.
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In addition to equilibrating amino groups among
available E-keto acids, transaminases funnel amino
groups from excess dietary amino acids to those amino
acids (e.g., glutamate) that can be deaminated.
Carbon sk eletons of deaminated amino acids can be
catabolized for energy, or used to synthesize glucose or
fatty acids for energy storage.
Only a few amino acids are deaminated directly.
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It is one of the few enzymes that can use NAD+ or NADP+
as e acceptor.
Oxidation at the E-carbon is followed by hydrolysis,releasing NH4
+.
OOCH
2CH
2C C COO
O
+ NH4+
NAD(P)+
NAD(P)H
OOC
H2C
H2C C COO
NH3+
Hglutamate
E-ketoglutarate
Glutamate ehydrogenase
H2O
Glutamate
Dehydrogenase
catalyzes a major reaction that effects
net removal of Nfrom the aminoacid pool.
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Summarized above:The role of transaminases in funneling amino N toglutamate, which is deaminated via lutamateDehydrogenase, producing NH4
+.
Amino acid E-ketoglutarate NADH + NH4+
E-keto acid glutamate NAD+
+ H2O
Transaminase Glutamate Dehydrogenase
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Some other pathways for deamination of amino acids:1. Serine Dehydratase catalyzes:
serine pyruvate + NH4+
2. Peroxisomal L- and D-amino acid oxidases catalyze:
amino acid + FAD + H2O E-k eto acid + NH4
+ + FADH2
FADH2 + O2 FAD + H2O2
Catalase catalyzes: 2H2O22 H2O + O2
HO CH2
HC COO
NH3+
C COO
OH2O NH4+
C COO
NH3+
H2C H3C
H2O
serine aminoacrylate pyruvate
Serine Dehydratase
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Most terrestrial land animals convert excess nitrogen tourea, 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 lutamate 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.
H2N C
O
NH2
urea
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Carbamoyl PhosphateSynthase (Type I) catalyzes
a 3-step reaction, with
carbonyl phosphate and
carbamate intermediates.Ammonia is the N input.
The reaction, which
involves cleavage of 2 ~P bonds of ATP, is essentially
irreversible.
H2N C OPO32
O
H2N C O
O
HO C
O
OPO32
HCO3
ATP
NH3
ADP
ATP
Pi
ADP
carbonyl phosphate
carbamate
carbamoyl phosphate
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Alternate forms of Carbamoyl Phosphate
Synthase (Types II & III)
initially generate ammonia
by hydrolysis of glutamine.
The type II enzyme includes
a long internal tunnel
through which ammonia &
reaction intermediates such
as carbamate pass from one
active site to another.
H2N C OPO32
O
H2N C O
O
HO C
O
OPO32
HCO3
ATP
NH3
ADP
ATP
Pi
ADP
carbonyl phosphate
carbamate
carbamoyl phosphate
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Carbamoyl Phosphate Synthase is the committed step
of the Urea Cycle, and is subject to regulation.
H2N C OPO
3
2
O
HCO3
+ NH3 + 2 ATP
+ 2 ADP + Pi
Carbamoyl hosphateynthase
carbamoyl phosphate
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Carbamoyl Phosphate Synthase has an absoluterequirement for an allosteric activator N -acetylglutamate.
This derivative of glutamate is synthesized fromacetyl-CoA & glutamate when cellular [glutamate] is high,signaling an excess of free amino acids due to protein breakdown or dietary intake.
H3N+
C COO
CH2
CH2
COO
H
glutamate (Glu)
N
H
C COO
CH2
CH2
COO
H
CH3C
O
N -acet lgluta ate
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H2N C OPO32
O
CH2
CH2
CH2
HC
COO
NH3+
NH3+
CH2
CH2
CH2
HC
COO
NH3+
NH
CO NH2
COO
CH2
HC
COO
NH2
CH2
CH2
CH2
HC
COO
NH3+
NH
C NH2+
COO
CH2
HC
COO
HN
AMP + PPi
ATP
CH2
CH2
CH2
HC
COO
NH3+
NH
C
NH2+
H2N
COO
HC
CH
COO
C NH2H2N
O H2O
Pi
ornithine
urea
citrulline
aspartate
arginino-succinate
umarate
arginine
carbamoyl phosphate
rea Cycle
1
2
3
4
Urea Cycle
Enzymes in
mitochondria:
1. Ornithine
Trans-
carbamylase
Enzymes in
cytosol:
2. Arginino-
Succinate
Synthase
3. Arginino-
succinase
4. Arginase.
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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.
cytosol
mitochon rial matri
carbamoyl phosphatei
ornithine citrulline
ornithine citrullineurea aspartate
arginine argininosuccinate
fumarate
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A complete K rebs Cycle functions only withinmitochondria.
But cytosolic isozymes of some Krebs Cycle enzymes
are involved in regenerating aspartate from fumarate.
cytosol
mitoc o rial matri
carbamoyl phosphate
i
ornithine citrulline
ornithine citrullineurea aspartate
arginine argininosuccinate
fumarate
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Fumarate is converted to oxaloacetate via Krebs Cycle
enzymes Fumarase & Malate Dehydrogenase.Oxaloacetate is converted to aspartate viatransamination (e.g., from glutamate).
Aspartate then reenters Urea Cycle, carrying an amino
group derived from another amino acid.
aspartate E-ketoglutarate oxaloacetate glutamate
Aminotrans erase (Transaminase)
COO
CH2
CH2
C
COO
O
COO
CH2
HC
COO
NH3+
COO
CH2
CH2
HC
COO
NH3+
COO
CH2
C
COO
O+ +
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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.
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Postulated mechanisms for toxicity of high [ammonia]:
1. High [NH3] would driveG
lutamine Synthase:glutamate + ATP + NH3 glutamine + ADP + Pi
This would deplete glutamate ± a neurotransmitter &
precursor for synthesis of the neurotransmitter ABA.
2. Depletion of glutamate & high ammonia level would
drive Glutamate Dehydrogenase reaction to reverse:
glutamate + NAD(P)+ E-k etoglutarate +
NAD(P)H + NH4+
The resulting depletion of E-ketoglutarate, an essential
Krebs Cycle intermediate, could impair energy
metabolism in the brain.
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Treatment of deficiency of Urea Cycle enzymes(depends on which enzyme is deficient):
limiting protein inta
k e to the amount barelyadequate to supply amino acids for growth, while
adding to the diet the E-keto acid analogs of essential amino acids.
Liver transplantation has also been used, sinceliver is the organ that carries out Urea Cycle.
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tissues where they generate arginine & ornithine, which
are precursors for other important molecules.E.g., Argininosuccinate Synthase, which catalyzessynthesis of the precursor to arginine, is in most tissues.
Mitochondrial Arginase II, distinct from the cytosolic
Urea Cycle Arginase, cleaves arginine to yield ornithine.
cytosol
mitochondrial matrix
carbamoyl phosphate
Pi
ornithine citrulline
ornithine citrulline
urea aspartatearginine argininosuccinate
fumarate
The complete
Urea Cycle issignificantly onlyin liver.
However some
enzymes of the pathway are inother cells and
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The amino acid arginine, in addition to being a constituentof proteins and an intermediate of the Urea Cycle, is precursor for synthesis of creatine & the signal moleculenitric oxide.
H3
H2
H2
H2
H
C
NH2
NH2
H
a e (
H2N C N
NH2+
CH2
CH3
C
O
O
creatine
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Synthesis of the radical species nitric oxide (·NO) fromarginine is catalyzedNitric Oxide Synthase, a distantrelative of cytochrome P450.
Different isoforms of Nitric Oxide Synthase (e.g., eNOS
ex pressed in endothelial cells and nNOS in neuronal cells)
are subject to differing regulation.
+H3N CH COO
CH2
CH2
CH2
NH
C
NH2
NH2+
NADPH NADP+
O2 H2O O2 H2O
+H3N CH COO
CH2
CH2
CH2
NH
C
NH2
N OH
+H3N CH COO
CH2
CH2
CH2
NH
C
NH2
O
1/2 NADPH 1/2 NADP+
+ NO
Nitric Oxide Synthasearginine hydroxyarginine citrulline
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·NO is a short-lived signal molecule with diverse rolesin different cell types, including regulation of smoothmuscle contraction, gene transcription, metabolism, andneurotransmission.
Many of the regulatory effects of ·NO arise from itsactivation of a soluble cytosolic Guanylate Cyclase
enzyme that catalyzes synthesis of cyclic-GMP
(analogous in structure to cyclic-AMP).
Cytotoxic effects of ·NO observed under someconditions are attributed to its non-enzymatic reactionwith superoxide (O2·
) to form the strong oxidantperoxynitrite (ONOO).
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Polyamines include putrescine,spermidine, spermine.
Ornithine is a major precursor for synthesis of polyamines.
Conversion of ornithine to putrescine iscatalyzed by Ornithine Decarboxylase.
+
H3N CH2 CH2 CH2 CH2 NH3+
+H3N CH2 CH2
CH2 NH CH2CH2 CH2
CH2 NH3+
putr escine
s per idine
H3N+
C COO
CH2
CH2
CH2
NH3
H
ornithine
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The cationic polyamines have diverse roles in cellgrowth & proliferation.
Disruption of polyamine synthesis or metabolism leadsto disease in animals & humans.
+
3 2 2 2 2 3+
+3 2 2 2 2 2 2 2 3
+
putrescine
s per i ine
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However, Ca++-activated PeptidylarginineDeiminasesconvert arginine residues within proteins to citrulline asa post-translational modification.
H3N+
C COO
CH2
CH2
CH2
NH
C
NH2
NH2
H
H3N+
C COO
CH2
CH2
CH2
NH
C NH2
H
O
arginin itrullin
Th r is no tRNA for citrulline & this amino acid
is not incorpor at dtr anslationally into prot ins.
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is essential to terminal differentiation of sk in cells.
Excessive protein citrullination, with production of antibodies against citrullinated proteins, is found to bea factor in the autoimmune diseases such as rheumatoidarthritis and multiple sclerosis.
H N C COO
CH
CH
CH
NH
C
NH
NH
H
H N C COO
CH
CH
CH
NH
C NH
H
O
ar i i citr lli
S bstit tio of citrulline,
which lack s ar i i 'spositive charge, may alt r str ct r & prop rti s s ch as bi di affi iti s of a prot i .
E. ., citrullination of certai protei s, i cl di k eratin
i termediate filament proteins,