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