catabolism
TRANSCRIPT
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Amino Acid Catabolism: N
Copyright 1999-2008 by Joyce J. Diwan.
All rights reserved.
Molecular Biochemistry II
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There are multiple transaminase enzymes which vary in
substrate specificity.
Some show preference for particular amino acidsor
classes of amino acids as amino group donors, and/or for
particular -keto acid acceptors.
H
R1 C COO- + R2 C COO
-
NH3+ O
Transaminase
H
R1 C COO
-
+ R2 C COO
-
O NH3+
Transaminases(aminotransferases)
catalyze the
reversible reaction
at right.
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Example of a Transaminasereaction:
Aspartate donates its amino group, becoming the
a-keto acid oxaloacetate.
a-Ketoglutarate accepts the amino group,
becoming the amino acid glutamate.
aspartate a-ketoglutarate oxaloacetate glutamate
Aminotransferase (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 a-ketoglutarate.
alanine a-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 groupsamong
available a-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 are
synthesized.
Although the amino N of one amino acid can be used
to synthesize another amino acid, N must be
obtained in the diet as amino acids(proteins).
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Essential amino acidsmust be consumed in the diet.
Mammalian cells lack enzymes to synthesize theircarbon skeletons (a-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
CO
P
OO
O
OH
CH3
CH O
H2
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In the resting state, the aldehyde group of pyridoxal
phosphate is in a Schiff baselinkage to the e-amino
group of an enzyme lysine side-chain.
NH
C
O
P
OO
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 active site lysine extracts H+, promoting
tautomerization, followed by reprotonation & hydrolysis.
NH
CO
P
OO
O
O
CH3
HC
H2
N
HC
H
+
R COOEnzLysNH2
Amino acid-PLP Shiff base (aldimine)
The a-amino group
of a substrate amino
aciddisplaces the
enzyme lysine, toform a Schiff baselinkage to PLP.
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The amino group remains on what is now pyridoxamine
phosphate(PMP).
A different a-keto acid reacts with PMP and the process
reverses, to complete the reaction.
NH
CO
P
OO
O
OH
CH3
CH2
NH2
H2
R C COO
O
EnzLysNH2
Pyridoxamine phosphate (PMP)
a-keto acid
What was anamino acid
leaves as an
-keto acid.
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Several other enzymesthat catalyze metabolism or
synthesis of amino acids also utilize PLPas prosthetic
group, and have mechanisms involving a Schiff base
linkage of the amino group to PLP.
NH
CO
P
OO
O
O
CH3
HC
H2
N
HC
H
+
R COO
EnzLysNH2
Amino acid-PLP Shiff base (aldimine)
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Chime Exercise
Two neighboring students or student groups should
team up, each displaying one of the following:
Transaminase with PLPin Schiff base linkage to
the active site lysine residue.
Transaminase in the PMPform, with glutarate, an
analog of a-ketoglutarate, at the active site.
Students should then show and explain the structure
displayed by them to the neighboring student orstudent group.
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It is one of the few enzymes that can use NAD+or NADP+as eacceptor.
Oxidation at the a-carbon is followed by hydrolysis,releasing NH4
+.
OOC
H2C
H2C C COO
O
+ NH4+
NAD(P)+
NAD(P)H
OOC
H2C
H2C C COO
NH3+
Hglutamate
a-ketoglutarate
Glutamate Dehydrogenase
H2OGlutamateDehydrogenase
catalyzes a major
reaction that effects
net removal of Nfrom the amino
acid pool.
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Summarized above:The role of transaminases in funneling amino N to
glutamate, which is deaminated via Glutamate
Dehydrogenase, producing NH4+.
Amino acid -ketoglutarate NADH + NH4+
-keto acid glutamate NAD+ + H2O
Transaminase Glutamate Dehydrogenase
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Some other pathways for deamination of amino acids:
1. Serine Dehydratasecatalyzes:
serine pyruvate + NH4+
2. PeroxisomalL- and D-amino acid oxidases catalyze:
amino acid + FAD + H2O-keto acid + NH4
++ FADH2
FADH2+ O
2 FAD + H
2O
2
Catalasecatalyzes: 2
H2O2 2 H2O + O2
HO CH2HC 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 to
urea, prior to excreting it.
Urea is less toxic than ammonia.
The Urea Cycleoccurs mainly in liver.
The 2 nitrogen atoms of urea enter the Urea Cycle as
NH3(produced mainly via Glutamate Dehydrogenase)and as the aminoN of aspartate.
The NH3and 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 Phosphate
Synthase(Type I) catalyzes
a 3-step reaction, with
carbonyl phosphateand
carbamateintermediates.
Ammoniais the N input.
The reaction, which
involves cleavage of 2 ~Pbonds of ATP, is essentially
irreversible.H2N C OPO3
2
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 Synthaseis the committed step
of the Urea Cycle, and is subject to regulation.
H2N C OPO32
O
HCO3 + NH3 + 2ATP
+ 2ADP + Pi
Carbamoyl PhosphateSynthase
carbamoyl phosphate
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Carbamoyl Phosphate Synthasehas an absoluterequirement for an allostericactivatorN-acetylglutamate.
This derivative of glutamate is synthesized from
acetyl-CoA & glutamate when cellular [glutamate] is high,
signaling an excess of free amino acidsdue to protein
breakdown or dietary intake.
H3N+
C COO
CH2
CH2
COO
H
glutamate(Glu)
NH
C COO
CH2
CH2
COO
H
CH3C
O
N-acetylglutamate
O CO NH
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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
fumarate
arginine
carbamoylphosphate
Urea Cycle
1
2
3
4
Urea Cycle
Enzymes in
mitochondria:1. Ornithine
Trans-
carbamylase
Enzymes incytosol:
2. Arginino-
Succinate
Synthase
3. Arginino-
succinase
4. Arginase.
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For each cycle, citrullinemust leave the mitochondria,and ornithinemust enter the mitochondrial matrix.
An ornithine/citrulline transporter in the inner
mitochondrial membrane facilitates transmembrane
fluxes of citrulline & ornithine.
cytosol
mitochondrial matrix
carbamoyl phosphate
Pi
ornithine citrulline
ornithine citrulline
urea aspartate
arginine argininosuccinate
fumarate
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A complete Krebs Cyclefunctions only within
mitochondria.
But cytosolic isozymes of some Krebs Cycle enzymes
are involved in regenerating aspartatefrom fumarate.
cytosol
mitochondrial matrix
carbamoyl phosphatePi
ornithine citrulline
ornithine citrullineurea aspartate
arginine argininosuccinate
fumarate
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Fumarateis converted to oxaloacetatevia Krebs Cycle
enzymes Fumarase & Malate Dehydrogenase.Oxaloacetateis converted to aspartatevia
transamination (e.g., from glutamate).
Aspartate then reenters Urea Cycle, carrying an amino
group derived from another amino acid.
aspartate a-ketoglutarate oxaloacetate glutamateAminotransferase (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 deficiencyof 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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Treatmentof deficiency of Urea Cycle enzymes
(depends on which enzyme is deficient):
limiting protein intaketo the amount barely
adequate to supply amino acids for growth, while
adding to the diet the a-keto acid analogs ofessential amino acids.
Liver transplantationhas also been used, sinceliver is the organ that carries out Urea Cycle.
l
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tissues where they generate arginine& ornithine, which
are precursors for other important molecules.
E.g., Argininosuccinate Synthase, which catalyzes
synthesis 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
Piornithine citrulline
ornithine citrullineurea aspartate
arginine argininosuccinate
fumarate
The complete
Urea Cycleis
significantly onlyin liver.
However some
enzymesof thepathway are in
other cellsand
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NH2 NH2 NH2
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Synthesis of the radical species nitric oxide (NO) from
arginineis catalyzed Nitric Oxide Synthase, a distant
relative of cytochrome P450.
Different isoformsof Nitric Oxide Synthase (e.g., eNOS
expressed in endothelial cells and nNOSin 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
1/2NADPH 1/2NADP+
+ NO
Nitric Oxide Synthase
arginine hydroxyarginine citrulline
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NOis a short-lived signalmoleculewith diverse roles
in different cell types, including regulation of smoothmuscle contraction, gene transcription, metabolism, and
neurotransmission.
Many of the regulatory effectsof NOarise from its
activation of a soluble cytosolic Guanylate Cyclase
enzyme that catalyzes synthesis of cyclic-GMP
(analogous in structure to cyclic-AMP).
Cytotoxic effects of NOobserved under someconditions are attributed to its non-enzymatic reaction
with superoxide (O2) to form the strong oxidant
peroxynitrite(ONOO).
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Polyaminesinclude putrescine,spermidine, spermine.
Ornithineis a major precursorfor
synthesis of polyamines.
Conversion of ornithine to putrescine is
catalyzed by Ornithine Decarboxylase.
+H3N CH2 CH2 CH2 CH2 NH3
+
+H3N CH2 CH2 CH2 NH CH2 CH2 CH2 CH2 NH3
+
putrescine
spermidine
H3N+ C COO
CH2
CH2
CH2
NH3
H
ornithine
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Thecationic polyamineshave diverse roles in cell
growth & proliferation.
Disruption of polyamine synthesis or metabolism leadsto disease in animals & humans.
+H3N CH2 CH2 CH2 CH2 NH3
+
+H3N CH2 CH2 CH2 NH CH2 CH2 CH2 CH2 NH3
+
putrescine
spermidine
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However, Ca++-activated Peptidylarginine Deiminasesconvert arginineresidues within proteins to citrullineas
a 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
arginine citrulline
There is no tRNA for
citrulline& this amino acid
is not incorporated
translationally into proteins.
H H
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is essential to terminal differentiation of skin cells.
Excessive protein citrullination, with production of
antibodies against citrullinated proteins, is found to be
a factor in the autoimmune diseasessuch as rheumatoid
arthritis and multiple sclerosis.
H3N+ C COO
CH2
CH2
CH2
NH
C
NH2
NH2
H
H3N+ C COO
CH2
CH2
CH2
NH
C NH2
H
O
arginine citrulline
Substitution of citrulline,
which lacksarginine's
positive charge, may alterstructure & properties such as
binding affinities of a protein.
E.g., citrullinationof certainproteins, including keratin
intermediate filament proteins,