Download Amino Acid Catabolism: N

Molecular Biochemistry II
Amino Acid Catabolism: N
Copyright © 1999-2008 by Joyce J. Diwan.
All rights reserved.
H
-
R1 C COO
+
R2
+
NH3
Transaminases
(aminotransferases)
catalyze the
reversible reaction
at right.
-
C COO
O
Transaminase
H
-
R1 C COO
O
+
R2
-
C COO
+
NH3
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 a-keto acid acceptors.
COO
COO
COO
CH2
COO
CH2
CH2
CH2
CH2
CH2
HC
NH3+
COO
+
C
O
COO
C
O
+
COO
HC
NH3+
COO
aspartate a-ketoglutarate oxaloacetate glutamate
Aminotransferase (Transaminase)
Example of a Transaminase reaction:
 Aspartate donates its amino group, becoming the
a-keto acid oxaloacetate.
 a-Ketoglutarate accepts the amino group,
becoming the amino acid glutamate.
CH3
HC
COO
COO
CH2
CH2
CH2
NH3+
COO
alanine
+
C
CH3
O
COO
a-ketoglutarate
C
CH2
O
COO
+
HC
NH3+
COO
pyruvate glutamate
Aminotransferase (Transaminase)
In another example, alanine becomes pyruvate as
the amino group is transferred to a-ketoglutarate.
Transaminases equilibrate amino groups among
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).
Essential amino acids must be consumed in the diet.
Mammalian cells lack enzymes to synthesize their
carbon 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.)
H
O
O
P
O
O
C
H2
C
OH
O

N
H
CH3
pyridoxal phosphate (PLP)
The prosthetic group of Transaminase is
pyridoxal phosphate (PLP), a derivative of
vitamin B6.
R
H
C
COO
Enz
(CH2)4
NH2
Amino acid
N+
HC
O
O
H2
C
P
O
H
O
O

N
H
CH3
Enzyme (Lys)-PLP Schiff base
In the resting state, the aldehyde group of pyridoxal
phosphate is in a Schiff base linkage to the e-amino
group of an enzyme lysine side-chain.
EnzLysNH2
R
H
C
COO
N+
The a-amino group
of a substrate amino
acid displaces the
enzyme lysine, to
form a Schiff base
linkage to PLP.
HC
O
O
H2
C
P
O
H
O
O

N
H
CH3
Amino acid-PLP Shiff base (aldimine)
The active site lysine extracts H+, promoting
tautomerization, followed by reprotonation & hydrolysis.
O
EnzLysNH2
H2
C
P
O
What was an
amino acid
leaves as an
a-keto acid.
CH2
O
O
NH2
R
C
COO
a-keto acid
OH
O

N
CH3
H
Pyridoxamine phosphate (PMP)
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.
EnzLysNH2
R
H
C
COO
N+
HC
O
O
H2
C
P
O
H
O
O

N
H
CH3
Amino acid-PLP Shiff base (aldimine)
Several other enzymes that catalyze metabolism or
synthesis of amino acids also utilize PLP as prosthetic
group, and have mechanisms involving a Schiff base
linkage of the amino group to PLP.
Chime Exercise
Two neighboring students or student groups should
team up, each displaying one of the following:
 Transaminase with PLP in Schiff base linkage to
the active site lysine residue.
 Transaminase in the PMP form, 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 or
student group.
In addition to equilibrating amino groups among
available a-keto acids, transaminases funnel amino
groups from excess dietary amino acids to those amino
acids (e.g., glutamate) that can be deaminated.
Carbon skeletons 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.
Glutamate
Dehydrogenase
catalyzes a major
reaction that effects
net removal of N
from the amino
acid pool.
H2

OOC C
glutamate
H2
C
NH3+
C
H
H2O
COO
NAD(P)+
NAD(P)H
O
H2 H2

OOC C C
a-ketoglutarate
C
COO + NH4+
Glutamate Dehydrogenase
It is one of the few enzymes that can use NAD+ or NADP+
as e acceptor.
Oxidation at the a-carbon is followed by hydrolysis,
releasing NH4+.
Amino acid
a-ketoglutarate
a-keto acid
glutamate
Transaminase
+
NADH + NH4
+
NAD + H2O
Glutamate
Dehydrogenase
Summarized above:
The role of transaminases in funneling amino N to
glutamate, which is deaminated via Glutamate
Dehydrogenase, producing NH4+.
+
H2O NH4
H2O
HO
CH2
H
C
COO
NH3+
serine
H2C
C
COO
O
H3C
C
COO
NH3+
aminoacrylate
pyruvate
Serine Dehydratase
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 
a-keto acid + NH4+ + FADH2
FADH2 + O2  FAD + H2O2
Catalase catalyzes: 2H2O2  2 H2O + O2
O
H 2N
C
NH2
urea
Most terrestrial land animals convert excess nitrogen to
urea, 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 Glutamate 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.
HCO3
Carbamoyl Phosphate
Synthase (Type I) catalyzes
a 3-step reaction, with
carbonyl phosphate and
carbamate intermediates.
ATP
ADP
O
HO
carbonyl phosphate
NH3
Pi
O
Ammonia is the N input.
The reaction, which
involves cleavage of 2 ~P
bonds of ATP, is essentially
irreversible.
OPO32
C
H2N
C
O
carbamate
ATP
ADP
O
H2N
C
OPO32
carbamoyl phosphate
HCO3
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.
ATP
ADP
O
HO
OPO32
C
carbonyl phosphate
NH3
Pi
O
H2N
C
O
carbamate
ATP
ADP
O
H2N
C
OPO32
carbamoyl phosphate
HCO3 + NH3 + 2 ATP
O
H2N
C
Carbamoyl Phosphate
Synthase
OPO32 + 2 ADP + Pi
carbamoyl phosphate
Carbamoyl Phosphate Synthase is the committed step
of the Urea Cycle, and is subject to regulation.
glutamate (Glu)
N-acetylglutamate
H
H3N+
C
O
COO
H3C
C
H
N
H
C
COO
CH2
CH2
CH2
CH2
COO
COO
Carbamoyl Phosphate Synthase has an absolute
requirement for an allosteric activator N-acetylglutamate.
This derivative of glutamate is synthesized from
acetyl-CoA & glutamate when cellular [glutamate] is high,
signaling an excess of free amino acids due to protein
breakdown or dietary intake.
O
NH3+
Urea Cycle
C
CH2
NH3+
NH
ornithine
4
O
H2O
citrulline
CH2
Pi
CH2
COO
C
urea
NH2
3
COO
CH2
HC
CH2
HC
HC
H
N
COO
NH
arginine
CH
NH3+
COO
NH2
COO
aspartate
CH2
C
CH2
HC

NH2+
H2N
CH2
2
ATP
AMP + PPi
COO
H2N
COO
NH3+
HC
Urea Cycle
COO
NH2
CH2
1
CH2
HC
OPO 32
carbamoyl
phosphate
CH2
Enzymes in
mitochondria:
1. Ornithine
Transcarbamylase
Enzymes in
cytosol:
2. ArgininoSuccinate
Synthase
3. Argininosuccinase
4. Arginase.
H2N
C
O
COO
fumarate
C
NH2+
NH
CH2
CH2
argininosuccinate
CH2
HC
NH3+
COO
cytosol
mitochondrial matrix
carbamoyl phosphate
Pi
ornithine
citrulline
ornithine
citrulline
aspartate
argininosuccinate
urea
arginine
fumarate
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
mitochondrial matrix
carbamoyl phosphate
Pi
ornithine
citrulline
ornithine
citrulline
aspartate
argininosuccinate
urea
arginine
fumarate
A complete Krebs Cycle functions only within
mitochondria.
But cytosolic isozymes of some Krebs Cycle enzymes
are involved in regenerating aspartate from fumarate.
COO
COO
COO
CH2
COO
CH2
CH2
CH2
CH2
CH2
HC
NH3+
COO
+
C
O
COO
C
O
COO
+
HC
NH3+
COO
aspartate a-ketoglutarate oxaloacetate glutamate
Aminotransferase (Transaminase)
Fumarate is converted to oxaloacetate via Krebs Cycle
enzymes Fumarase & Malate Dehydrogenase.
Oxaloacetate is converted to aspartate via
transamination (e.g., from glutamate).
Aspartate then reenters Urea Cycle, carrying an amino
group derived from another amino acid.
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.
Postulated mechanisms for toxicity of high [ammonia]:
1. High [NH3] would drive Glutamine Synthase:
glutamate + ATP + NH3  glutamine + ADP + Pi
This would deplete glutamate – a neurotransmitter &
precursor for synthesis of the neurotransmitter GABA.
2. Depletion of glutamate & high ammonia level would
drive Glutamate Dehydrogenase reaction to reverse:
glutamate + NAD(P)+  a-ketoglutarate +
NAD(P)H + NH4+
The resulting depletion of a-ketoglutarate, an essential
Krebs Cycle intermediate, could impair energy
metabolism in the brain.
Treatment of deficiency of Urea Cycle enzymes
(depends on which enzyme is deficient):
 limiting protein intake to the amount barely
adequate to supply amino acids for growth, while
adding to the diet the a-keto acid analogs of
essential amino acids.
 Liver transplantation has also been used, since
liver is the organ that carries out Urea Cycle.
cytosol
The complete
Urea Cycle is
significantly only
in liver.
mitochondrial matrix
carbamoyl phosphate
Pi
ornithine
citrulline
However some
ornithine
citrulline
urea
aspartate
enzymes of the
arginine
argininosuccinate
pathway are in
fumarate
other cells and
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.
arginine (Arg)
H
H3N+
C
COO
CH2
CH3
CH2
H2N
CH2
N
CH2
NH2+
NH

C
C
O
NH2
C
O
creatine
NH2
The amino acid arginine, in addition to being a constituent
of proteins and an intermediate of the Urea Cycle, is
precursor for synthesis of creatine & the signal molecule
nitric oxide.
NH2
C
NH2
NH
+
C
NADPH
NADP+
CH2
O2
CH
C
OH
CH2
O2
arginine
+
H3 N
CH
+
NO
CH2
H2O
CH2
CH2
COO
O
NH
1/2 NADPH 1/2 NADP+
CH2
H 2O
CH2
H3N
NH
N
CH2
CH2
+
NH2
NH2
COO
hydroxyarginine
+
H 3N
CH
COO
citrulline
Nitric Oxide Synthase
Synthesis of the radical species nitric oxide (·NO) from
arginine is catalyzed Nitric Oxide Synthase, a distant
relative of cytochrome P450.
Different isoforms of Nitric Oxide Synthase (e.g., eNOS
expressed in endothelial cells and nNOS in neuronal cells)
are subject to differing regulation.
 ·NO is a short-lived signal molecule with diverse roles
in different cell types, including regulation of smooth
muscle contraction, gene transcription, metabolism, and
neurotransmission.
Many of the regulatory effects of ·NO arise 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 ·NO observed under some
conditions are attributed to its non-enzymatic reaction
with superoxide (O2·) to form the strong oxidant
peroxynitrite (ONOO).
+
+
H3N CH2
CH2
CH2
CH2
H3N
CH2
CH2
NH
CH2
NH3+
CH2
putrescine
CH2
CH2
CH2
NH3+
spermidine
 Polyamines include putrescine,
spermidine, spermine.
 Ornithine is a major precursor for
synthesis of polyamines.
Conversion of ornithine to putrescine is
catalyzed by Ornithine Decarboxylase.
H
H3N+
C
COO
CH2
CH2
CH2
 NH3
ornithine
+
+
H3N CH2
CH2
CH2
CH2
H3N
CH2
CH2
NH
CH2
NH3+
CH2
putrescine
CH2
CH2
CH2
NH3+
spermidine
 The cationic polyamines have diverse roles in cell
growth & proliferation.
Disruption of polyamine synthesis or metabolism leads
to disease in animals & humans.
H
H3N+
There is no tRNA for
citrulline & this amino acid
is not incorporated
translationally into proteins.
C
H
COO
H3N+
C
CH2
CH2
CH2
CH2
CH2
CH2
NH
NH
COO

C
NH2
NH2
arginine
C
NH2
O
citrulline
However, Ca++-activated Peptidylarginine Deiminases
convert arginine residues within proteins to citrulline as
a post-translational modification.
H
H3N+
Substitution of citrulline,
which lacks arginine's
positive charge, may alter
structure & properties such as
binding affinities of a protein.
C
H
COO
H3N+
C
CH2
CH2
CH2
CH2
CH2
CH2
NH
NH
COO

C
NH2
C
NH2
NH2
O
E.g., citrullination of certain
arginine
citrulline
proteins, including keratin
intermediate filament proteins,
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 diseases such as rheumatoid
arthritis and multiple sclerosis.