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21.

Amino acid metabolism: nitrogen fixation,


transamination and NH3 transport
amino acids &
Nitrogen cycles between oxidized other organic
& reduced forms in the biosphere compounds

synthesis degradation
reduction
(microorganisms, (animals &
(plants & some plants & animals) microorganisms)
anaerobic
bacteria)

denitrification nitrogen
fixation
nitrate NO3- N2 NH4+ ammonium
(anaerobic (Rhizobium &
bacteria) some other
bacteria)

nitrification nitrification
NO2-
(Nitrobacter & (Nitrosomonas &
other soil bacteria) nitrite other soil bacteria)

more oxidized more reduced


In the industrial Haber process, N2 is reduced to NH3 by H2
at high temperature and pressure with an iron oxide catalyst

N2 3H2

The reaction is exothermic


by + 92.4 kJ/mol at standard
200 oC temperature & pressure, but
200 Atm has a very high activation
energy
FeO catalyst

2 NH3
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.) 2 m


Nitrogenase from Azobacter vinelandii has
iron-sulfur and iron-molybdenum centers
FeMo protein

Azotobacter are
free-living, aerobic
soil bacteria.

Mo-Fe-S cluster
(Mo:7Fe:9S)

8Fe:7S cluster

Fe protein 4Fe:4S cluster

Mg ADP (2)
Two ATP-binding sites, structurally
homologous to G-proteins
1n2c.pdb
Cys residue
S-CH2CH
of the protein
The Fe-Mo cofactor
contains homocitrate

The mechanism of the N2-


fixation reaction is not known,
but intermediates in which
partially reduced derivatives of Fe
N2 replace one of the O atoms S
bound to the Mo have been
proposed. Mo
O
C

Homocitrate (3-hydroxy-3-
carboxyadipic acid)

I wont expect you to


remember this structure
Nitrogenase uses 8
4 CoA-SH + 4 CO2 +
electrons and ~16 ATP
4 pyruvate 4 acetyl-CoA
to reduce N2 + 2 H+ to
2 NH4+ + H2 8 e-
8 ferredoxin 8 ferredoxin
(oxidized) (reduced)
8 e-
The Fe protein
transfers one
electron at a time to 8 Fe protein 8 Fe protein
the Fe-Mo protein. (reduced) (oxidized)

~16 ADP
~16 ATP + 16 Pi
8 e-

Fe-Mo protein Fe-Mo protein


(oxidized) (reduced)
8 e-
The ATP stoichiometry
is uncertain. Only 8 2 NH4+ N2
ATP are needed under
some conditions. H2 2 H+
The first step in catabolism of most amino acids is transamination

CO2- CO2-
+
H3N-C-H C=O
amino acid R R -keto
acid

CO2- CO2-
C=O +
H3N C H
CH2 CH2
CH2 CH2

-ketoglutarate CO2- CO2- 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
-ketoglutarate and Glu; others use oxaloacetate and Asp.
Transaminases use pyridoxal phosphate as a prosthetic group

CH2O- P H2O CH2O- P

Enz Lys NH2 + O=CH NH + Enz Lys N=CH NH +

HO CH3 H2O HO CH3

pyridoxal phosphate Pyridoxal phosphate forms a


Schiff-base (aldimine) bond to a
lysine residue of the enzyme.
This reaction is readily reversible.

CH2O- P CH2OH

+
H3NCH2 NH
+ HOCH2 NH + Cl -

HO CH3 HO CH3
pyridoxamine phosphate pyridoxine hydrochloride (vitamin B6)
Pyridoxal phosphate transfers the amino group
by shuttling between aldehyde and amine forms

H
amino -
O2C C R1 -
O2C C R1 -keto
acid 1 NH3+ acid 1
O

CH=O CH2NH3+

HO CH2O- P HO CH2O- P

pyridoxal pyridoxamine
phosphate CH CH3 N phosphate
3 N
H+ H+
(on enzyme) (on enzyme)

H
amino -
O2C C R2 -
O2C C R2 -keto
acid 2 NH3+ O acid 2

Both steps occur with the coenzyme bound non-covalently to the


enzyme. This is a classic ping-pong enzyme mechanism.
amino
The positive charge of the pyridoxine ring facilitates
acid interconversions of Schiff-base intermediates
H
H H2O H
- Schiff
O2C C R H+ -
- O2C C R base
NH2 O2C C R
.. NH N
CH=O H+ CH-OH CH

HO CH2O- P HO CH2O- P HO CH2O- P

pyridoxal
CH3 N CH3 N CH3 N
H+ phosphate H+ H+

H+

-keto -
acid O2C C R H2O -
O2C C R -
O2C C R
O N
+
H
N
CH2NH2 CH2 CH

CH2O- P HO CH2O- P
HO HO CH2O- P

CH3 N pyridoxamine CH3 N N


Schiff CH3
H+ phosphate H+ H
base
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
Related enzymes use pyridoxal phosphate to catalyze
amino acid racemizations and decarboxylations
H
H Schiff
-
O2C C R base
-
O2C C R H2O N CO2
amino NH2
acid CH

HO CH2O- P
H C R

CH=O N
CH3 N
H+ CH
HO CH2O- P
HO CH2O- P
H
CH3 N H C R
H+ N
N CH3
H
CH
amine
HO CH2O- P
H
H2O
H C R H+
CH3 N
NH2 H+ Schiff
base
Amino acid decarboxylases generate
amines that serve as neurotransmitters

-
CO2- CO2-
CO2 +
H3N C H +
H3N C H
+
H3 N C H
CH2 CH2
CH2 5-hydroxy-Trp
Glu dihydroxy-Phe
CH2 (DOPA) HO NH
- OH
CO2 OH
CO2 CO2
CO2
NH3+ NH3+ NH3+
CH2 CH2 CH2
CH2 CH2 CH2
CH2
HO NH
- OH
CO2
-aminobutyrate OH
(GABA) dopamine serotonin

Also: Histidine histamine + CO2


cellular The amino groups of glutamic acid and
protein
glutamine can be released as ammonia
in liver mitochondria
ingested
protein
transaminases
CO2- CO2-
C=O -keto
amino H3N C H
+
acids
acids R R
CO2- CO2-
C=O +
H3N C H
CH2 CH2
-keto-
glutarate CH2 CH2 Glu
glutamate
CO2 - dehydrogenase - CO2-
CO2 +
H3 N C H
NADH or CH2
NADPH + H+ NAD+ or H2O CH2
NADP+
CONH2
But ammonia is toxic,
particularly to neural tissue. Gln from
NH4+ NH4+ muscle &
Terrestrial organisms must
prevent it from accumulating. other tissue
Ammonia is incorporated into many biological
molecules through glutamine and glutamate

CO2- CO2- CO2-


NH4+ NH4+ + ATP ADP + Pi
O=C +
H3N-C-H +
H3N-C-H
CH2 CH2 CH2
CH2 CH2 (2) CH2 Gln
H2O
NADH NAD+ or
CO2- or NADP CO2- Glu C-NH2
O
NADPH
(3)
-keto-
(1)
glutarate

CO2- CO2-
+
H3N-C-H O=C -keto-
Glutamate dehydrogenase (1) glutarate
CH2 CH2
and glutamine synthetase (2)
are found in all organisms. CH2 CH2
Reaction (3) occurs in plants & Glu
bacteria, but not animals. CO2- CO2-
Glutamine serves as a donor of amine groups
for synthesis of many other molecules

carbamoyl-phosphate

glucosamine-6-P
-
CO2
+
H3N-C-H alanine
CH2
CH2 glycine

C-NH2
O histidine

tryptophan
In most terrestrial animals, Gln also
carries ammonia in the blood to the AMP
CTP
liver & kidneys for excretion as urea.
Glutamine synthetase catalyzes formation
of glutamine from glutamate and NH4+
glutamine
synthetase
- CO2-
CO2 CO2-
+
+ H3N-C-H +
H3N-C-H H3N-C-H
CH2
CH2 ATP ADP NH4+ Pi CH2
CH2
CH2 CH2
C-O- P
CO2 - O C-NH2
Glu O Gln
The reaction proceeds through an enzyme-
bound -glutamylphosphate intermediate

NH4+ H2O
glutaminase
In terrestrial animals, Gln carries ammonia in the blood to the
liver & kidneys, where it is hydrolyzed for excretion as urea.
Glutamine synthetase is inhibited
allosterically by many of the end-products
carbamoyl-
Glu Gln phosphate
glutamine
synthetase glucosamine-
CO2-
ATP ADP CO2- 6-P
+
H3N-C-H NH4+ Pi + alanine
H3N-C-H
CH2 CH2
CH2 CH2 glycine
X X X X X X X X
CO2- C-NH2
O histidine

The inhibitory tryptophan


effect of all the
products acting CTP AMP
together is
greater than the
sum of their
individual effects.
E. coli glutamine synthetase also is
controlled by covalent modification

Gln
ATP PPi

adenylylation
glutamine glutamine
synthetase --OH synthetase --O-AMP
(active) deadenylylation (inactive)

ADP Pi
-keto-
adenyl group
glutarate
O
enz O P OCH2 O Adenine
O

The regulation by Gln and -ketoglutarate involves similar covalent modifications


(uridylylation) of the enzymes that add or remove the adenyl group to glutamine synthetase.
Bacterial glutamine
synthetase has 12
identical subunits

views of the Salmonella


typhimurium enzyme
parallel and perpendicular
to the 6-fold symmetry axis

2gls.pdb
Alanine carries amino groups from
muscle to the liver for excretion

muscle
muscle blood liver O
protein
H2N-C-NH2
glucose glucose
urea
amino acids
NH4+

CO2 - CO2-
Glu
C=O C=O Glu

CH3 pyruvate CH3


pyruvate

CO2- CO2-
-keto- +
H3N C H +
H3N C H
Ala -keto-
glutarate
CH3 CH3 glutarate
Ala

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