Nitrogen Assimilation

1
Nitrogen Assimilation

• How is NH3 incorporated into organic molecules?
• Glutamate Dehydrogenase vs Glutamate Synthase
• Properties of Glutamine Synthetase
• Regulation of Glutamine Synthetase
• Glutamine as a major nitrogen donor

2
The only inorganic nitrogen source for mammals is
NH3
3 Gateways to Biological Molecules
a-Ketoglutarate
Glutamate
Aspartate
Of the 3, the most versatile is glutamine
3
Glutamate Dehydrogenase
?-Kg NH3 NAD(P)H 2H
Glutamate NAD(P)
H2O
Bacteria and Plants
Make glutamate, assimilate NH3
Glutamate is major solute in the bacteria
Provide NH3 to urea cycle, provide ?-Kg to Krebs
Animals
High Km for NH3 limits forward
Mitochondrial location
(-) by ATP, GTP
() by ADP, GDP
4
Glutamate Synthase (Bacteria Only)
?-Kg glutamine NADPH H
2 glutamates NADP
Glutamine is the nitrogen donor
Reaction is a reductive amination
5
Glutamine Synthetase is a Primary
Regulatory Point in Nitrogen Metabolism
Properties of Bacterial Enzyme
12 identical 50,000 mwt subunits
6
Combined Mwt of 600,000
6
Hexagonal stacked rings
SIDE VIEW
8 allosteric sites on each subunit
One covalent site (Tyr 397)
TOP VIEW
Regulation is cumulative
6
Glutamine Synthetase
(Biosynthesis - anabolic)
?-Ketoglutarate
(Degradation catabolic)
Take Home Shutting down the enzyme favors using
glutamate as an energy substrate.
Not shutting down the enzyme keeps the cell in
a biosynthetic mode.
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Covalent Modification by Adenylylation
O
C
N
H
C
O
O
2
C
H
C
H
2
2
C
H
C
H
2
2


C
N
H
H
C
N
H
H



A
D
P





P
N
H

A
T
P

i
3
3
3
C
O
O
C
O
O
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Allosteric Effectors
Each inhibits Glutamine Synthetase (Favors
boosting cell energy or shutting down a
pathway requiring glutamine )
AMP
(Low energy state exists, oxidize ?-Kg)
CTP
(End product of pyrimidine synthesis)
Histidine
Glycine
Amino acids that are allosteric effectors
Tryptophan
Alanine
Glucosamine
(Glutamine sufficient for amino sugar synthesis)
(Glutamine sufficient for pyrimidine synthesis)
Carbamoyl-PO4
9
Covalent Regulation
Adenylylation of Tyrosine 397 on EACH of the 12
subunits
Adenyl group attached
Adenylylation group attached and process
For example
An acyl group attached via an acylation reaction
is
ACYLACYLATION
OR
ACYLYLATION
(PRONOUNCED ACIL-LIL-ATION)
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Covalent Regulation of Glutamine Synthetase
2 Transferases
Each puts on and takes off groups
AT (Adenylyltransferase - adenylylates GS)
UT (Uridylyltransferase - uridylylates PII)
One Regulatory Protein
PII
Two States
AT-PII
Adenylates
AT-PII-UMP
Deadenylylates
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Rules of Engagement

• Transferases catalyze adenylylation
  (uridylylation) and deadenylylation
  (deuridylylation reactions
• Adenylylation shuts GS down cumulatively
  Deadenylylation turns GS back on cumulatively
• AT requires PII to adenylylate
• AT requires PII-UMP to deadenylylate

12
Active
GS
ATP
Less Active
UMP
Useless
Uridylylates
Adenylylates
(2 activities on same enzyme)
Deadenylylates
Deuridylylates
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Activates GS
ATP
(-) glutamine
() ?-Kg
Inactivates GS
Responsive to cells nitrogen requirements