Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432

Biochemistry

Spring 2009

Lecture Notes: 18 February

© R. Paselk 2006
 
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Nitrogen Fixation

Last time we looked at the fixation of nitrogen as ammonia by Nitrogenase. So what do we do with the ammonian?

Glutamate dehydrogenase. Once ammonia has been formed the nitrogen can be incorporated via one of two major pathways. The first is familiar - Glutamate dehydrogenase found in mitochondria (or bacteria). Note that the equilibria will allow the reaction to go either direction, depending on substrate concentrations. (You may also note the enzyme will accept either NADH or NADPH, the only enzyme known to be non-specific!)

Structural diagram of the reaction of Glutamate DH

In animals ammonia is quite toxic, so its concentration is kept quite low, and glutamate DH is not a major source of nitrogen incorporation.

Glutamine Synthetase. Glutamine is a major storage form of nitrogen and a source of nitrogen in various synthetic pathways. It has the advantage over glutamate dehydrogenase of a much lower Km - that is it can incorporate ammonia into biomolecules at much lower concentrations. Glutamine is made from glutamate and ammonia with energy supplied by ATP:

Structural diagram of the reaction sequence of Glutamine Synthase

Glutamine's central position in nitrogen metabolism makes control of its biosynthesis essential.

Degredation of Intracellular Proteins

All cell proteins are turned over, with half-lives commonly ranging from minutes to hours. Generally the proteins used for basic cell operations ("housekeeping proteins, e.g. glycolytic enzymes) have relatively long half-lives, while those involved in adaptation (e.g. gluconeogenesis, ketone body synthesis) have short half-lives. Two well known systems are used by eukaryotic cells to degrade proteins:

  1. The lysosomal system.
  2. The ubiquitin/proteosome system.

The lysosomal system is generally non-selective in well nourished cells, but becomes more selective with starvation. Thus proteins with the KFERQ sequence (lys-phe-glu-arg-gln) are preferentially degraded, shortening theri half-lives.

The second system is specific to eukaryotes and is based on the degredation of proteins which have had the protein ubiquitin covalently attached to them. Ubiquitin itself is a highly conserved protein (i.e. identical in humans and fruit flys), possibly because it is recognized by a number of other proteins. Ubiquitin is attached to a "condemned" protein in a three stage process:

  1. It is conjugated to a thiol group on ubiquitin activating enzyme via the C-terminal carboxyl group to give a thioester bond. As this is an unstable linkage (high energy) ATP energy is required (ATP right arrow AMP + PPi via a two step reaction involving a ubiquitin-adenylate intermediate).
  2. The ubiquitin is then transferred to a second protein, ubiquitin-conjugating enzyme.
  3. Finally the activated ubiquitin is transferred to the side-chain amino group of a lysine on the target protein by ubiquitin-protein ligase.

The ubiquinated protein is then broken down by the proteosome, a large 28 subunit, 6182 aa, 700 kD, barrel -shapped, protein. The proteosome breaks proteins down to octapeptides, which are released to the cytosol for further degredation. The ubiquitin is recycled, not degraded.

Nucleotide Metabolism

Nucleoside = nitrogenous base + ribose

Nucleotide = nitrogenous base + ribose + phosphate

We have already seen how important the nucleotides etc. are to life as the monomer residues in nucleic acids (DNA & RNA), energy transfer cofactors, components of essential cofactors (e.g. NADH, CoA), and secondary messengers (e.g. cAMP) etc.

Today we want to look at their anabolism and catabolism.

Recall the basic structures:

Structural diagrams for the Purine and Pyrimidine rings

Purines are synthesized via IMP:

Structural diagram of IMP

Pyrimidines are synthesized via UMP:

Structural diagram of UMP

the other nucleotides are then obtained by modification of IMP and UMP.

The two initial nucleotides are biosynthesized by two quite different strategies:

Structural diagram showing the origins of the atoms in the Purine ring

Structural diagram showing the origins of the atoms in the Pyrimidine ring

 The strategies also differ for subsequent modification. Thus for the purines the monophosphte is modified and then phosphorylated:

Flow diagram for the conversion of IMP to GTP & ATP

while for the pyrimidines the monophophate is first phosphorylated, and then modified:

Orotate + PRPP right arrow OMP right arrow CO2 + UMP right arrow UDP right arrow UTP

dTMP is derived from dUMP which in turn is modified from dUTP.

Purine Biosynthesis

Purines are synthesized in a multi-step process outlined in the Purine Biosynthesis Pathway in your Chem 431 packet.

Note the strategy: Start with PRPP (phosphoribosyl pyrophosphate synthesized from ribose-5-P, then add atoms one at a time to build ring system with the exception of a three atom group from glycine.

Recall the origins of the purine ring system:

Structural diagram showing the origins of the atoms in the Purine ring

Structural diagram of the reactions converting IMP to AMP

Structural diagram of the reactions converting IMP to GMP

 


Pathway Diagrams

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Last modified 18 February 2008