Transcript Nitrogen Assimilation

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
3 Gateways to
Biological Molecules
CO2 ATP
Carbamoyl
phosphate
Arginine
Urea
NH3
Aspartate
Asparagine
None
The only inorganic
nitrogen source for
mammals is NH3
a-Ketoglutarate
Glutamate
Other
amino
acids
Of the 3, the most versatile is glutamine
Glutamate
Glutamine
Purine nucleotides,
Cytidine nucleotides
Amino sugars,
Tryptophan, Histidine
Pyrimidine nucleotides
Glutamate Dehydrogenase
a-Kg + NH3 + NAD(P)H + 2H+
Glutamate + NAD(P)+
+ H2O
Bacteria and Plants
Animals
Make glutamate, assimilate NH3
Glutamate is major solute in the
bacteria
Provide NH3 to urea cycle,
provide a-Kg to Krebs
High Km for NH3 limits forward
Mitochondrial location
(-) by ATP, GTP
(+) by ADP, GDP
Glutamate Synthase (Bacteria Only)
a-Kg + glutamine + NADPH + H+
2 glutamates + NADP+
Glutamine is the nitrogen donor
Reaction is a reductive amination
Glutamine Synthetase is a Primary Regulatory
Point in Nitrogen Metabolism
Properties of Bacterial Enzyme
12 identical 50,000 mwt subunits
Combined Mwt of 600,000
6
6
Hexagonal stacked rings
SIDE VIEW
Each subunits subject to allosteric
regulation
8 allosteric sites on each subunit
One covalent site (Tyr 397)
Regulation is cumulative
TOP VIEW
Glutamine Synthetase
Glutamate + NH4+ + ATP
Glutamine + ADP + Pi
(Biosynthesis - anabolic)
a-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.
Covalent Modification by Adenylylation
Adenylylated tyrosine residue
Enzyme
O
Tyr
H
COO
CH2
CH2
+
C NH3
COO
+
NH3 +
ATP
O
P
O
O
H
CH2
O
Adenine
OH OH
O
C NH2
CH2
CH2
+
C NH3 + ADP + Pi
COO
Allosteric Effectors
Each inhibits Glutamine Synthetase (Favors boosting cell energy
or shutting down a pathway requiring glutamine )
AMP
(Low energy state exists, oxidize a-Kg)
CTP
(End product of pyrimidine synthesis)
Histidine
Glycine
Tryptophan
Alanine
Amino acids that are allosteric effectors
Glucosamine (Glutamine sufficient for amino sugar synthesis)
Carbamoyl-PO4 (Glutamine sufficient for pyrimidine synthesis)
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)
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
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
Active
GS*
ATP
AT
PII
AMP
AT
PII UMP
AMP
GS
Less Active
UTP
PPi
PII
UMP
AT = Useless
PII UMP
UT UR
AT PII = Adenylylates
AT PII UMP = Deadenylylates
Uridylate removing
Enzyme
UT = Uridylylates
(2 activities on
same enzyme)
UR = Deuridylylates
Activates
GS
ATP
(-) glutamine
(+) a-Kg
UTP
UR
UT
PII
Inactivates
GS
PII
Deadenylylates
PPi
PII UMP
UT
UR
PII UMP
UMP
Leads to adenylylation
Responsive to cell’s
nitrogen requirements