21' Amino acid metabolism: nitrogen fixation, transamination and NH3 transport

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21. Amino acid metabolism nitrogen fixation,
transamination and NH3 transport
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Nitrogen cycles between oxidized reduced forms
in the biosphere
degradation (animals microorganisms)
synthesis (microorganisms, plants animals)
(Rhizobium some other bacteria)
nitrification (Nitrosomonas other soil
bacteria)
nitrification (Nitrobacter other soil
bacteria)
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In the industrial Haber process, N2 is reduced to
NH3 by H2 at high temperature and pressure with
an iron oxide catalyst
The reaction is exothermic by 92.4 kJ/mol at
standard temperature pressure, but has a very
high activation energy
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The roots of leguminous plants have nodules that
contain N2-fixing bacteria
Bacteroids (rod-like bacteria) containing
nitrogenase live inside the nodule cells
nodule cell nucleus
bacteroids
Nitrogenase is very sensitive to O2. It is
protected in the nodules by leghemoglobin, a heme
protein with a strong affinity for O2.
Leghemoglobin is produced by the plant, but
carries O2 for reduction by the bacterial
respiratory chain, keeping the O2 concentration
low.
(This electron micrograph is colorized
artificially.)
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Nitrogenase from Azobacter vinelandii has
iron-sulfur and iron-molybdenum centers
FeMo protein
Azotobacter are free-living, aerobic soil
bacteria.
Mo-Fe-S cluster (Mo7Fe9S)
8Fe7S cluster
4Fe4S cluster
Fe protein
Mg ADP (2)
Two ATP-binding sites, structurally homologous to
G-proteins
1n2c.pdb
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The Fe-Mo cofactor contains homocitrate
Cys residue of the protein
The mechanism of the N2-fixation reaction is not
known, but intermediates in which partially
reduced derivatives of N2 replace one of the O
atoms bound to the Mo have been proposed.
Homocitrate (3-hydroxy-3-carboxyadipic acid)
I wont expect you to remember this structure
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Nitrogenase uses 8 electrons and 16 ATP to
reduce N2 2 H to 2 NH4 H2
The Fe protein transfers one electron at a time
to the Fe-Mo protein.
The ATP stoichiometry is uncertain. Only 8 ATP
are needed under some conditions.
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The first step in catabolism of most amino acids
is transamination
?-keto acid
amino acid
?-ketoglutarate
glutamate
The main function of transamination is to funnel
amino groups into a small number of amino acids,
particularly Glu Asp. Some amino transferases
(transaminases) are specific for
a-ketoglutarate and Glu others use oxaloacetate
and Asp.
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Transaminases use pyridoxal phosphate as a
prosthetic group
Pyridoxal phosphate forms a Schiff-base
(aldimine) bond to a lysine residue of the
enzyme. This reaction is readily reversible.
pyridoxal phosphate
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Pyridoxal phosphate transfers the amino group by
shuttling between aldehyde and amine forms
amino acid 1
?-keto acid 1
CH2NH3
pyridoxal phosphate (on enzyme)
pyridoxamine phosphate (on enzyme)
amino acid 2
?-keto acid 2
Both steps occur with the coenzyme bound
non-covalently to the enzyme. This is a classic
ping-pong enzyme mechanism.
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The positive charge of the pyridoxine ring
facilitates interconversions of Schiff-base
intermediates
amino acid
Schiff base
H
H
pyridoxal phosphate
?-keto acid
H2O
H
pyridoxamine phosphate
Schiff base
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The active site has additional residues that
could facilitate proton binding and release
Arg 222
Lys 258
Asn 194
Schiff base formed from PLP 2-methyl-Asp
aspartate aminotransferase
Asp 222
1ajs.pdb
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Related enzymes use pyridoxal phosphate to
catalyze amino acid racemizations and
decarboxylations
Schiff base
H2O
amino acid
amine
Schiff base
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Amino acid decarboxylases generate amines that
serve as neurotransmitters
5-hydroxy-Trp
Glu
dihydroxy-Phe (DOPA)
CO2
CO2
CO2
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The amino groups of glutamic acid and glutamine
can be released as ammonia in liver mitochondria
cellular protein
ingested protein
transaminases
a-keto acids
amino acids
?-keto-glutarate
Glu
glutamate dehydrogenase
NADH or NADPH H
H2O
NAD or NADP
But ammonia is toxic, particularly to neural
tissue. Terrestrial organisms must prevent it
from accumulating.
Gln from muscle other tissue
NH4
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Ammonia is incorporated into many biological
molecules through glutamine and glutamate
(2)
H2O
Glu
(3)
a-keto-glutarate
(1)
a-keto-glutarate
Glutamate dehydrogenase (1) and glutamine
synthetase (2) are found in all organisms.
Reaction (3) occurs in plants bacteria, but not
animals.
Glu
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Glutamine serves as a donor of amine groups for
synthesis of many other molecules
In most terrestrial animals, Gln also carries
ammonia in the blood to the liver kidneys for
excretion as urea.
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Glutamine synthetase catalyzes formation of
glutamine from glutamate and NH4
glutamine synthetase
Glu
Gln
The reaction proceeds through an enzyme-bound
?-glutamylphosphate intermediate
glutaminase
In terrestrial animals, Gln carries ammonia in
the blood to the liver kidneys, where it is
hydrolyzed for excretion as urea.
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Glutamine synthetase is inhibited allosterically
by many of the end-products
carbamoyl-phosphate
Gln
Glu
glutamine synthetase
glucosamine-6-P
ATP ADP NH4 Pi
alanine
glycine
histidine
The inhibitory effect of all the products acting
together is greater than the sum of their
individual effects.
tryptophan
AMP
CTP
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E. coli glutamine synthetase also is controlled
by covalent modification
Gln
ATP PPi
adenylylation
deadenylylation
ADP Pi
a-keto-glutarate
adenyl group
The regulation by Gln and a-ketoglutarate
involves similar covalent modifications
(uridylylation) of the enzymes that add or remove
the adenyl group to glutamine synthetase.
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Bacterial glutamine synthetase has 12 identical
subunits
views of the Salmonella typhimurium enzyme
parallel and perpendicular to the 6-fold symmetry
axis
2gls.pdb
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Alanine carries amino groups from muscle to the
liver for excretion
muscle protein
blood
liver
muscle
glucose
glucose
urea
amino acids
NH4
Glu
Glu
pyruvate
pyruvate
a-keto- glutarate
a-keto- glutarate
Ala
Ala