Biochem Exam IV

Chapters 22 and 23

What is nitrogen fixation?

the chemical process of converting inert atmospheric nitrogen into useable forms (e.g. NH3, NO2, NO3)

What is nitrification?

The chemical process of converting NH3 to NO3

Nitrogen fixation in diazotrophs (nitrogen fixing bacteria) to carried out by what?

The nitrogenase complex

What is required for the nitrogenase complex to work?

Uses energy from ATP binding (2 ATP) and hydrolysis to transfer electrons from an iron sulfur center to an FeMo cofactor to lower the activation energy. Needs 6 electrons to reduce N2 to ammonia.

What are the components within the nitrogenase complex?

dinitrogenase reductase and dinitrogenase

How does the nitrogenase complex reduce N2?

1. Pyruvate passes e- to dinitrogenase reductase via ferredoxin

2. Reductase transfers 8 e- to dinitrogenase one e- at a time

   1. energy intensive

   2. ATP binding and hydrolysis brings iron-sulfur center closer to P cluster

3. Dinitrogenase catalyzes two reactions with the use of FeMo cofactor

   1. reduce N2 to 2 NH3 (6 e-)

   2. reduce 2 H+ to H2 (2 e-)

How is ammonia incorporated into biomolecules?

reduced nitrogen (ammonia) is assimilated into glutamate and glutamine.

What is glutamate dehydrogenase?

The process of converting glutamate to α-ketoglutarate. Produces NADH and NH4 in the process.

How is glutamate converted to glutamine?

Glutamate + ATP (enzyme → glutamine synthetase) → Intermediate + ADP (enzyme → glutamine synthetase) + NH4 → Glutamine + phosphate

How is glutamine synthetase regulated?

1. Cumulative feedback inhibition by: Glycine, Alanine, AMP, Tryptophan, Carbamoyl phosphate, CTP, Histidine, Glucosamine 6-phosphate. Each inhibits only a small amount; needs multiple to fully inhibit glutamine synthetase.

2. Covalent modification. Reversible. Covalent attachment of AMP (adenylation) on Tyr397 by adenylyltransferase (AT) inhibits glutamine synthetase.

3. Uridylation. Protein PII is uridylated (add UMP) under low nitrogen levels, this activates glutamine synthetase. Uridylation of PII is regulated by uridylyltransferase (needs high levels of ATP and α-ketoglutarate, inhibited by glutamine)

Glutamate and glutamine are needed for why nitrogen-containing biomolecules?

1. Amino Acids (proteins)

2. Neurotransmitters (epi)

3. Heme

4. Nucleic acids (DNA/RNA)

5. Cofactors (NADH)

What are common reactions for the biosynthesis of nitrogen containing biomolecules?

• Transamination reactions

  • PLP dependent, reversible reactions

• One carbon transfers using tetrahydrofolate (THF)

  • i.e. breakdown of serine to glycine

  • THF: reduced vitamin B9

  • required for amino acids and nucleotides (i.e. breakdown of serine to glycine)

• Methyl transfers using S-adenosylmethionine (SAM)

  • needed for neurotransmitters and phosphocreatine

  • destabilizing the sulfonium ion allows for the transfer of methyl group

• Transfer of nitrogen of γ-amino group from glutamine

Transfer of amino groups from glutamine is an important reaction in the biosynthesis of____?

• amino acids

• phosphocreatine

• nucleotides

What are sources of amino acids?

• breakdown of proteins

• dietary intake

• de novo biosynthesis

What are the essential amino acids?

PVT. Tim Hall

Where does synthesis of AA occur?

In the liver

Amino acids are derived from the intermediates of what metabolic processes?

• Glycolysis

  • 3-phosphoglycerate → serine → glycine and cysteine

  • phosphoenolpyruvate → tyrosine

  • pyruvate → alanine

• Pentose phosphate pathway

  • erythrose 4-phosphate → tyrosine

• TCA

  • Oxaloacetate → aspartate → asparagine

  • α-ketoglutarate → glutamate → proline → arginine

How is alanine synthesized?

From pyruvate derived by glycolysis. Pyruvate and glutamate converted in a trans-amination reaction via alanine α-transferase (ALT) to alanine and an α-ketoglutarate.

Glutamate derived from α-ketoglutarate is a source for _____?

Glutamine, proline, and arginine.

α-ketoglutarate → glutamate

• Transamination

• Glutamate Dehydrogenase

glutamate → glutamine (glutamine synthetase), proline (cyclic), arginine (urea cycle)

Proline is a cyclic form of ____?

glutamate

How is glutamate used to make proline?

1. Transfer phosphate to make an acyl (glutamate kinase) (ATP needed)

2. Reduction (γ-glutamyl phosphate reductase) (NADH needed)

3. Cyclization (nonenzymatic)

4. Reduction (pyrroline carboxylate reductase) (NADH needed)

Proline Produced!