biochemistry 432/832
DESCRIPTION
Biochemistry 432/832. February 12, 2002 Chapter 26 Nitrogen Acquisition. Announcements: -. Outline. 26.1 The Two Major Pathways of N Acquisition 26.2 The Fate of Ammonium 26.3 Glutamine Synthetase 26.4 Amino Acid Biosynthesis 26.5 Metabolic Degradation of Amino Acids. - PowerPoint PPT PresentationTRANSCRIPT
Biochemistry 432/832
February 12, 2002February 12, 2002
Chapter 26Chapter 26
Nitrogen Acquisition
Announcements:
-
Outline
• 26.1 The Two Major Pathways of N Acquisition
• 26.2 The Fate of Ammonium
• 26.3 Glutamine Synthetase
• 26.4 Amino Acid Biosynthesis
• 26.5 Metabolic Degradation of Amino Acids
Major Pathways for N Acquisition• All biological compounds contain N in a reduced
form
• The principal inorganic forms of N are in an oxidized state
• Thus, N acquisition must involve reduction of the oxidized forms (N2 and NO3
-) to NH4+
• Nearly all of this is in microorganisms and green plants
• Animals gain N through diet.
The nitrogen cycleThe nitrogen cycle
Overview of N AcquisitionNitrogen assimilation and nitrogen fixation
• Nitrate assimilation occurs in two steps: 2e- reduction of nitrate to nitrite and 6e- reduction of nitrite to ammonium
• Nitrate assimilation accounts for 99% of N acquisition by the biosphere
• Nitrogen fixation involves reduction of N2 in prokaryotes by nitrogenase
Nitrate AssimilationElectrons are transferred from NADH to nitrate
• Pathway involves -SH of enzyme, FAD, cytochrome b and MoCo - all protein-bound
• Nitrate reductases are big - 210-270 kDa
• MoCo required both for reductase activity and for assembly of enzyme subunits to active dimer
Novel prosthetic groups used in N acquisitionNovel prosthetic groups used in N acquisition
Molybdopterin
Siroheme
Mo-containing enzymesMo-containing enzymes
Molybdopterin
Mo is the heaviest element used by eukaryotes
Two classes of molybdoenzymes
1) Molybdopterin-dependent enzymes
Nitrate reductase
Formate dehydrogenase
Aldehyde oxidase
Xanthine dehydrogenase
Sulfate oxidase
2) Nitrogenase
Nitrite ReductaseLight drives reduction of ferredoxins and electrons flow to 4Fe-4S and siroheme and
then to nitrite
• Nitrite is reduced to ammonium while still bound to siroheme
• In higher plants, nitrite reductase is in chloroplasts, but nitrate reductase is cytosolic
Enzymology of N fixationOnly occurs in certain prokaryotes
• Rhizobia fix nitrogen in symbiotic association with plants
• Rhizobia fix N for the plant and plant provides Rhizobia with carbon substrates
• All nitrogen fixing systems are very similar
• They require nitrogenase, a reductant (reduced ferredoxin), ATP, O-free conditions and regulatory controls (ADP inhibits reaction and NH4
+ inhibit
expression of nif genes)
Nitrogenase ComplexTwo protein components: nitrogenase reductase and
nitrogenase • Nitrogenase reductase is a 60 kDa homodimer with a
single 4Fe-4S cluster • Very oxygen-sensitive • Binds MgATP • 4ATP required per pair of electrons transferred
• Reduction of N2 to 2NH3 + H2 requires 4 pairs of electrons, so 16 ATP are consumed per N2
Why should nitrogenase need ATP???
• N2 reduction to ammonia is thermodynamically favorable
• However, the activation barrier for breaking the N-N triple bond is enormous
• 16 ATP provide the needed activation energy
To break the triple bond, energy input in To break the triple bond, energy input in necessarynecessary
NitrogenaseA 220 kDa heterotetramer
• Each molecule of enzyme contains 2 Mo, 32 Fe, 30 equivalents of acid-labile sulfide (FeS clusters, etc.)
• Four 4Fe-4S clusters plus two FeMoCo, an iron-molybdenum cofactor
• Nitrogenase is slow - 12 e- pairs per second, i.e., only three molecules of N2 per second
Structures of Structures of two types of two types of metal clusters in metal clusters in nitrogenase:nitrogenase:
The P-clusterThe P-cluster
FeMoCoFeMoCo
The nitrogenase reactionThe nitrogenase reaction
Accumulation
of electrons
Nitrogenase Nitrogenase reductasereductase
Complex between Complex between nitrogenase reductase nitrogenase reductase and nitrogenaseand nitrogenase
Regulation of Regulation of nitrogen nitrogen fixationfixation
ADP inhibitsADP inhibits
NH4+ represses NH4+ represses expressionexpression
ADP-ribosylation ADP-ribosylation inhibitsinhibits
The Fate of AmmoniumThree major reactions in all cells
• Carbamoyl-phosphate synthetase– two ATP required - one to activate bicarbonate,
one to phosphorylate carbamate
• Glutamate dehydrogenase – reductive amination of alpha-ketoglutarate to
form glutamate
• Glutamine synthetase – ATP-dependent amidation of gamma-carboxyl
of glutamate to glutamine
The glutamate dehydrogenase reactionThe glutamate dehydrogenase reaction
The glutamine synthetase reactionThe glutamine synthetase reaction
Ammonium AssimilationTwo principal pathways
• Principal route: GDH/GS in organisms rich in N
• both steps assimilate N
• Secondary route: GS/GOGAT in organisms confronting N limitation
• GOGAT is glutamate synthase or glutamate:oxo-glutarate amino transferase
The glutamate dehydrogenase/glutamine The glutamate dehydrogenase/glutamine synthase pathwaysynthase pathway
One each
Two N fixing steps - one inefficient
The glutamate synthase reactionThe glutamate synthase reaction
The glutamine synthase/GOGAT pathwayThe glutamine synthase/GOGAT pathway
One NADPH
Two ATP
One N fixing step - inefficient but expensive
Glutamine SynthetaseA Case Study in Regulation
• GS in E. coli is regulated in three ways:– Feedback inhibition– Covalent modification (interconverts between
inactive and active forms)– Regulation of gene expression and protein
synthesis - - control the amount of GS in cells
The glutamine synthetase reactionThe glutamine synthetase reaction
Glutamine synthetase structure
stack of two hexagons
Allosteric Regulationof Glutamine Synthetase
• Nine different feedback inhibitors: Gly, Ala, Ser, His, Trp, CTP, AMP, carbamoyl-P and glucosamine-6-P
• Gly, Ala, Ser are indicators of amino acid metabolism in cells
• Other six are end products of biochemical pathways
• This effectively controls glutamine’s contributions to metabolism
Allosteric regulation of glutamine synthase activity by feedback inhibition
Covalent Modificationof Glutamine Synthetase
• Each subunit is adenylylated at Tyr-397
• Adenylylation inactivates GS
• Adenylyl transferase catalyzes both the adenylylation and deadenylylation
• PII (regulatory protein) controls both activities
• AT:PIIA catalyzes adenylylation
• AT:PIID catalyzes deadenylylation
-ketoglutarate and Gln also affect
Covalent modification of glutamine synthase - Covalent modification of glutamine synthase - adenylylation of Tyr397adenylylation of Tyr397